DNA replication commences at eukaryotic replication origins following assembly and activation of bidirectional CMG helicases. Once activated, CMG unwinds the parental DNA duplex and DNA polymerase α-primase initiates synthesis on both template strands. By utilizing an origin-dependent replication system using purified yeast proteins, we have mapped start sites for leading-strand replication. Synthesis is mostly initiated outside the origin sequence. Strikingly, rightward leading strands are primed left of the origin and vice versa. We show that each leading strand is established from a lagging-strand primer synthesized by the replisome on the opposite side of the origin. Preventing elongation of primers synthesized left of the origin blocked rightward leading strands, demonstrating that replisomes are interdependent for leading-strand synthesis establishment. The mechanism we reveal negates the need for dedicated leading-strand priming and necessitates a crucial role for the lagging-strand polymerase Pol δ in connecting the nascent leading strand with the advancing replisome.  •S. cerevisiae DNA replication originsMapping of leading-strand start sites at two •Leading-strand synthesis is established from “lagging-strand” primers•Pol δ likely plays a key role in establishing all nascent leading strands•Replisomes remain interdependent until new leading strands are established Aria and Yeeles describe the mechanism by which leading-strand replication is established at eukaryotic DNA replication origins. “Lagging-strand” primers, synthesized by replisomes on opposite sides of the origin, are elongated back across the origin by Pol δ until the 3ʹ ends are coupled to Pol ε at the advancing replication forks. Bidirectional DNA replication is initiated from specific regions of the genome, termed origins. In eukaryotes, assembly of the DNA replication machinery (replisome) begins in the G1 phase of the cell cycle when the ATP-dependent motor component of the replicative helicase, the hexameric Mcm2–7 complex (MCM), is loaded at origins by the origin recognition complex (ORC), Cdc6 and Cdt1 . The MCMin vivo studies in budding yeast have reached conflicting conclusions regarding the location of leading-strand start sites relative to an origin. For example, one study concluded that the ARS1 origin contains a single leading-strand start site (Once sufficient single-stranded DNA (ssDNA) has been exposed at origins, synthesis of leading and lagging strands is initiated by the DNA polymerase α-primase complex (Pol α). Lagging-strand synthesis requires repeated cycles of Pol α-dependent priming and subsequent primer extension by Pol δ. Pol α first synthesizes 7–12 nucleotide (nt) RNA primers before transferring them to the DNA polymerase domain, where further extension to about 20–25 nt takes place . Evidencart site . The sitart site , and theart site . Howeverart site . Consequin vivo   C. Subseq (PIP is very similar to the 35% rate reduction that we observed previously when PCNA was omitted from reactions (PIP contributes significantly to leading-strand synthesis.At early time points (6 and 9 min), Pol εeriments E and S1Aeriments . In reacents  . Moreoveeactions . These fPIP in leading-strand replication, we created two additional Pol ε mutants: one in which catalysis was disrupted by a point mutation in the polymerase active site (Pol εCat) (PIP/Cat). Replication with Pol εPIP/Cat proceeded at a similar rate to Pol εCat   and a muPol εCat F. In con. We reasoned that initiation via pathway 1 should be refractory to challenge by this mutant, whereas it should block initiation via pathway 2 , a potent block to replicative polymerases, 16 nt to the left of the ACS in the lagging-strand template A. The CPBased on our previous work , preventS. cerevisiae origins. Synthesis is predominantly initiated outside the origin sequence; Left leading strands are started to the right, and Right leading strands are started to the left .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Joseph Yeeles  containing integrated expression constructs; or from Escherichia coli RosettaTM 2(DE3) cells (Novagen) (genotype: F– ompT hsdSB(rB– mB–) gal dcm (DE3) pRARE2 (CamR)) transformed with plasmids for protein overexpression. Plasmids details are reported in the Proteins were purified from The ARS306 templates used in this study were modified from the plasmid ZN3  using stTo generate linear replication templates all plasmids were purified with CsCl gradients, linearized at unique restriction sites and processed as previously indicated . ExperimCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGTTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTCAACGCCTTTTACCTTGTCCTTTGATGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAACTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGAGCATACGACTACCGCCGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTGCGGCCGCTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTAGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGACGTCAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTGCTGAGGACTTAAGTGCTGAGGGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTGCGGCCGCTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTAGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGACGTCAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCTGGAACACCAAAGACCAATTGAGATTGTACAGTCCACGCAATAGGAACATCTTGAGGCAAAGCAGATTTGACGTAGTCATTATAGTGTTGCAAATTAGCCCCCAATCCCAATAGTTCGAGGGCAGTCCAAGACTGAATTTGCACAGCACCGGTCGTATGAGCTCGCATGTTGGGAAAGCGGCTGCCAAGGCTGGAAATCTCTTTGCAGTTTTTCAGTTGGTCCTTCATCAGTGAAGAAAATGACTGAACCGTAAGCCTCATCTCTGCAAGACTCTGGTCTCTTATAAAGCAGTTGGCATTGCGCTCGCAACAGCATCCCATATCCTTTTGTGTGTATCACCAACGATAATGACAGCGCGATTCACTTGTGAGTTAAAAGCTGTTGGCGTATTCTTGAGAATAACGTGTACAGTTCTCTTTACATCATCCAAACCGACACCTTGTGGTAATTCGGGCTTCAAATTGTAGATGGTACGACGGTTTGTAATAGCGTTTAAGTAGTTTCCAGTTGGGGACATTTCTTTGGCTTGGAGGTCTGGTGTTCTTGATTTTGATGGTGTATATAGCTTTAAAAAACCAAAAATGATCAACCTTTATATCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACpol2 gene was modified in yJF1 (PIP) and D640A (Pol εCat) were obtained by site directed mutagenesis, and subsequently sub-cloned into pRS304 (Pol2 + Dpb4-CBP) (The endogenous  in yJF1  strain t in yJF1  as templpb4-CBP) . The Polpb4-CBP)  was mutaWith the exception of the Pol ε and Pol δ mutants (see details below), all proteins were purified as described previously . A list Pol ε mutants  were expressed and purified in S. cerevisiae essentially as for wild-type Pol ε . Protein elution was carried out with 10 CV of Buffer T + 200 mM NaCl, supplemented with 2 mM EDTA and 2 mM EGTA. Eluted protein was diluted 1:2 in Buffer T lacking NaCl to reduce the salt concentration tot 100 mM, and then loaded on MonoQ column . The column was washed with Buffer T containing 100 mM NaCl and Pol δCat was eluted with 20 CV of 100-600 mM NaCl. The peak fraction was dialysed against storage buffer .Pol δ protein , with so2; 50 mM potassium glutamate; 0.5 mM ATP; 60 μM dATP; 60 μM dGTP. Protein mix containing PCNA and Pol ε or Pol εPIP  was added to RPA-bound template. The two protein-DNA mixtures were split and increasing concentrations of RFC were added. The reactions were incubated at 30°C for 5 minutes and DNA synthesis started by adding nucleotide mix (60 μM of each dTTP and dCTP and 1 μCi [α-32P]-dCTP). After 12 minutes at 30°C, the reactions were stopped with stop solution  and separated on 0.7% alkaline agarose gels.Primer extension reactions were essentially performed as described previously but using M13-mp18 ssDNA (NEB), rather than ϕX174 . Singula2; 0.1 mg/ml BSA; 3 mM ATP; 5 nM DNA template; 75 nM Cdt1-Mcm2-7; 40 nM Cdc6; 20 nM ORC; 25 nM DDK. After a 10 min incubation S-CDK was added to 100 nM and incubation prolonged for a further 5 min. The loading reaction was 4-fold diluted into replication buffer containing the following components, reported in their final concentrations: 25 mM HEPES-KOH, pH 7.6; 250 mM potassium glutamate; 0.01% NP-40-S; 1 mM DTT; 10 mM Mg(OAc)2; 0.1 mg/ml BSA; 3 mM ATP; 200 μM CTP; 200 μM GTP; 200 μM UTP; 30 μM dATP; 30 μM dCTP; 30 μM dGTP; 30 μM dTTP; 1 μCi [α-32P]-dCTP. Samples were pre-warmed at 30°C for 1 minute and reactions were initiated by addition of replication proteins to the following concentrations: 30 nM Dpb11; 100 nM GINS; 30 nM Cdc45; 10 nM Mcm10; 20 nM Ctf4; 20 nM Csm3/Tof1; 20 nM RFC; 20 nM PCNA; 20 nM Pol ε (or mutants); 10 nM Pol δ (or Pol δCat); 100 nM RPA; 20 nM Pol α; 10 nM Topoisomerase I; 10 nM Mrc1; 12.5 nM Sld3/7; 30 nM Sld2. The samples were incubated at 30°C for the indicated times and were processed and imaged as described  through ∼400 μL Sephacryl-S400 High Resolution  columns, previously equilibrated in 25 mM HEPES-KOH, pH 7.6; 100 mM potassium glutamate; 0.01% NP-40-S; 1 mM DTT; 10 mM Mg(OAc)2; 40 mM KCl. MCM loading and phosphorylation was performed by adding  3 mM ATP; 0.1 mg/ml BSA; 75 nM Cdt1-Mcm2-7; 40 nM Cdc6; 25 nM DDK. After 10 minutes at 24°C, S-CDK was added to 25 mM and incubation continued for 5 minutes. The nucleotide components were added  and the mix was pre-warmed at 30°C for 1 minute. Reactions were initiated by addition of replication proteins as described for standard replication reactions. Nhp6 and FACT were also added to final concentrations of 400 nM and 40 nM respectively. For the experiments on the ARS1 template the DDK concentration was 50 nM, the Sld3/7 concentration was 25 nM, the GINS concentration was 200 nM, the Mcm10 concentration was 5 nM and the RPA concentration was 60 nM. After quenching by addition of an equal volume of 50 mM EDTA, proteins were removed from the replication mix by proteinase K  – SDS (0.1%) treatment, for 15 minutes at 37°C, followed by phenol-chloroform-isoamyl alcohol extraction (Sigma-Aldrich). Unincorporated nucleotide was removed using Illustra G-50 columns . Samples were analyzed as described  in Cut Smart buffer, for 30 minutes at 37°C. Digests were stopped by adding EDTA to final concentration of 50 mM, followed by deprotinization with proteinase K - SDS treatment and phenol-chloroform extraction as described above. Sample aliquots were analyzed on 1% alkaline and 0.8% native agarose gels, where required. The remaining digested products were ethanol precipitated, washed with 70% ethanol, air-dried and resuspended in 10 mM Tris-HCl, pH 8; 1 mM EDTA. For the RNase HII experiments, digestion products were further treated with RNase HII enzyme (NEB) for 1 hour at 37°C. The reactions were stopped with 50 mM EDTA and processed as described above for the restriction digests. For polyacrylamide gel analysis an equal volume of 2x loading dye  was added to the samples. Samples were incubated for 3 minutes at 95°C, promptly transferred to ice, before being applied to a 40 cm x 20 cm denaturing 4% polyacrylamide (Bis-Acrylamide 19:1 – Fisher scientific), 7 M Urea, in 1x Tris-Borate-EDTA buffer (TBE) gel. Gels were run for 170 minutes at constant 40 Watt.Native agarose gels and acrylamide gels were dried directly onto 3MM chromatography paper . Alkaline agarose gels were fixed with two 15 min incubations at 4°C in 5% trichloroacetic acid solution before drying on 3MM chromatography paper . For quantification, gels were exposed on BAS-IP MS Storage Phosphor Screens  and imaged on a Typhoon phophorimager . Gels were also autoradiographed using Amersham Hyperfilm MP  for presentation.Quantification and data analysis were performed with ImageJ software and Prism 7. For pulse-chase experiments to determine maximum leading-strand synthesis rates  and S1 lFor the initiation site mapping experiments lanes profiles were generated in ImageJ. The migration positions of the replication products were converted to product lengths using standard curves generated from the molecular weight markers. To generate the standard curve the migration position of the marker bands were plot against the Log10 of their length and data were fit to a second order polynomial. Signal from the no enzyme lanes was subtracted from the profiles and data were normalized by dividing by the maximum peak value for a given profile. Initiation site positions relative to the 5ʹ end of the ACS were then derived by subtracting the distance between the enzyme cleavage site and the 5ʹ end of the ACS.


CRISPR–Cas9 enables recombination between homologous chromosome arms at predefined sites and also underscores the need for caution when applying CRISPR technologies in translational medicine. Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms—often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention.CRISPR–Cas9–based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in  CRISPR–Cas9–based genome editing has revolutionized genetic research, triggering the development of a plethora of technologies and applications that provide unprecedented control over genes in a growing list of model species , 6, 7, 8Drosophila. Although these findings expand the tool-box of CRISPR-based genome manipulation in research, they also raise concerns about the use of gene editing in therapeutic settings.Here, we set out to examine the occurrence and frequency of genetic exchanges between homologous chromosome arms initiated by Cas9-induced DSBs. We show that Cas9-triggered DSBs induce germline-transmitted recombination between homologous chromosome arms in up to 39% of the CRISPR events in Drosophila , is in-frame with the downstream ORF and resides within the unique 20-nt gRNA target, starting 4 nt upstream of the PAM sequence. Activation of CIGAR is achieved by Cas9-induced DNA cleavage within the STOPT codon. The induced DSBs will then be mended by NHEJ-mediated repair concomitantly severing or eliminating the STOPT codon. The resulting indel leads to repositioning of the upstream ATGs relative to the ORF, that is, causing one of the ATGs to be “shifted in-frame” with the ORF.NHEJ is a major repair mechanism triggered by CRISPR–Cas9–induced DSBs in osophila , 30. Levosophila . CIGAR cery cell , (ii) a ery cell , and /+), we found that in some of the F1 animals, the CRISPR target sequence of the CIGARmCherry reporter became located 5′ of the eGFP ORF and vice versa. Sequence analysis of 84 animals from different crosses revealed a total of 26 animals in which the sequences on one side of the DSB had been exchanged ; molecular analysis of the target site. Deleted bp shown as: –. Inserted/exchanged bp shown as: N. Bold, grey sequence numbers show un-rearranged (un-CRISPRed) CIGAR reporters. To facilitate the detection of recombination, the first 4 nt of the shifter of CIGAReGFP (CGGC) and CIGARmCherry (CCCC) are highlighted. The PAM site is shown in bold letters. In addition, we highlight the PCR setup used to amplify the shifter sequences of CIGAReGFP (green) or CIGARmCherry (red). The fwd primer anneals within the ubiquitin promoter , and the reverse primers are either specific for eGFP or mCherry, respectively. The used primers for CIGAReGFP: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGAReGFP-rev: GAACTTCAGGGTCAGCTTGC. The used primers for CIGARmCherry: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG.Shown are the readable sequences (n = 84) for recombination on the X chromosome . Such individuals were crossed to yw animals, and their offspring were scored for recombination events ; molecular analysis of the target site. Deleted bp shown as: –. Inserted/exchanged bp shown as: N. Bold, grey sequence numbers show un-rearranged (un-CRISPRed) CIGAR reporters. To facilitate the detection of recombination, the first 4 nt of the shifter of CIGAReGFP (CGGC) and CIGARmCherry (CCCC) are highlighted. The PAM site is shown in bold letters. In addition, we highlight the PCR setup used to amplify the shifter sequences of CIGAReGFP (green) or CIGARmCherry (red). The fwd primer anneals within the ubiquitin promoter , and the reverse primers are either specific for eGFP or mCherry, respectively. The used primers for CIGAReGFP: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGAReGFP-rev: GAACTTCAGGGTCAGCTTGC. The used primers for CIGARmCherry: CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG.Shown are the readable sequences (n = 156) for recombination on the fourth chromosome  differed. However, the recombination events could only be demonstrated by sequence analysis of the immediate vicinity of the CRISPR site; more distant phenotypic markers were not present on these chromosomes. Therefore, we could not rule out that, at least in some cases, other mechanisms, such as gene conversion, were responsible for the observed sequence exchange between the two CIGAR reporters in trans .w+-marked CIGARmCherry,102F, w+ and the recessive viable mutation svspa-pol close to the tip of the right arm on chromosome 4. We selected the Cas9 target site, targeted with sgRNA-3, in the 3′UTR of the toy gene residing about 18 kb downstream of the CIGARmCherry,102F, w+ transgene insertion site. To induce recombination at the target site 1021,y+, svspa-pol embryos with active Cas9-sgRNA RNP complexes containing recombinant Cas9 and in vitro–translated sgRNA-3 1021,y+, svspa-pol flies to visually detect recombination events in the offspring , the frequency of CRISPR-mediated targeted recombination in this experiment is estimated to be ∼1.1%. This frequency is at least four orders of magnitude higher than that of the rare spontaneous recombination rate predicted for chromosome 4 , 41w+-mamosome 4 .Recombination between two visible markers.Table S4 mCherry 102F, w+/Dp(1021) y+, svspa-pol injected with Cas9.List of recombinants recovered from yw; CIGARTable S5 mini-white gene (+w), used as transgene reporter, is juxtaposed to heterochromatic regions via chromosomal rearrangements or translocations 1021,y+, svspa-pol animals exhibiting PEV were crossed to yw; ciD,svspa-pol/svΔ122 animals. Without exception, the tested PEV chromosomes were unable to complement the svΔ122 mutation, indicating that they must have lost distal parts of chromosome 4.The remaining 36 (out of 57) putative recombinants recovered from the above experiment were homozygous lethal and exhibited position effect variegation PEV;  in the aocations , 44, 45.d to PEV . We, theromosome . To assey+ marker on the short left arm of chromosome 4 and the w+-marked CIGARmCherry,102F transgene inserted at 102F of the right arm 1021, y+, svspa-pol were injected with RNPs containing Cas9 protein complexed with in vitro–translated sgRNA-3. The RNPs induce DSBs 3′ of toy located distal of CIGARmCherry,102F but should not have any influence on the recombination between the y+ and the w+ markers. G0 animals were crossed with yw animals to score recombination events between the y+ and the w+ marker  on the fourth chromosome , we repeated the experiment using the same experimental conditions, but assessed recombination between a + marker . We scre+ marker , TR wereIn summary, our experiments involving chromosome 4 revealed that germ-line–transmitted recombinants can be recovered at frequencies ranging from about 1.1% (recombination between visible markers) to 26% . These percentages represent the number of recombinants recovered from the total number of animals analyzed. When only the detected Cas9-mediated events are taken into consideration, the frequency of recombination was even 39%.Drosophila, however, the first meiotic divisions occur at much later timepoints , likelysgRNAs are provided via transgenes (nanos (nos) promoter and the Cas9 transgene contains the nos 3′UTR recapitulating germline-specific nos expression, transcript localization, and translational control -EcoRI fragment from pCaSpR3-Up2-RX polyA was subcloned into a pBluescript (pBS) vector between the EcoRI and XhoI sites using blunt-end ligation. The EcoRI-ubi-Acc65I fragment from the resulting plasmid was subcloned into pEPattB , a unique gRNA target sequence (referred to as sgRNA-1), the linker sequence, and the eGFP gene were designed (as shown below), synthesized by GenScript, and delivered ligated into the pUC57-Kan vector (pCIGAR-D0).To create the The pCIGAR-D0 insert:5′-KpnI_CAACATGGTGCAACATGGTGCAACATGGTGCGGCGACAGCAGA ACGTAGCGGGACGATAGGCTGCAGATCCTTGGCGCGCCTTCAGGAGGCGGTGCTACTGCTGGCGCTGGTGGAGCCGGTGGACCTGCGGGGTTAATTGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCATAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTC_EcoRI-3′.KpnI and EcoRI and ligated into pre–double-digested pKB342 vector in line with a tubulin 3′ trailer, transformed, and purified, resulting in pKB342_CIGAR. To remove the CIGAR sequence including the tubulin trailer, pKB342_CIGAR was then digested with KpnI and XbaI, and the fragment was ligated into a KpnI- and XbaI-digested pUbiattB vector containing an ubiquitin-p63E promoter (see above). This resulted in the final product referred to as pUbiattB-CIGAReGFP.The synthetic sequence was excised with pUbiattB-CIGAReGFP were used for the construction of the pUbiattB-CIGARmCherry. The only difference was in the design of the target site; the gRNA (sgRNA-2) has a different unique target sequence and contains the mCherry gene as the fluorescent marker. The insert ligated into the pUC57-Kan vector was ordered from GenScript.The same sequential digestions and ligation as for the making of the CIGARmCherry insert:The 5′-KpnI_CAACATGGTGCAACATGGTGCAACATGGTGCCCCGAGACAAGCACCTGACGGGACGATAGGCTGCAGATCCTTGGCGCGCCTTCAGGAGGCGGTGCTACTGCTGGCGCTGGTGGAGCCGGTGGACCTGCGGGGTTAATTGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTTAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA_EcoRI-3′.pCFD5 vector was a gift from Fillip Port   following the protocol described in the supplementary methods of pCFD5 cloning protocol.The Addgene) . pCFD5 ipCFD5 internal tRNA multi-gRNA Scaffold:5′-GTCGGGGCTTTGAGTGTGTGTAGACATCAAGCATCGGTGGTTCAGTGGTAGAATGCTCGCCTGCCACGCGGGCGGCCCGGGTTCGATTCCCGGCCGATGCAGGGTCTTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT-3′.sgRNA-1 and sgRNA-2 were inserted into the pCFD5 vector using primer (5′-GCGGCCCGGGTTCGATTCCCGGCCGATGCACGGCGACAGCAGAACGTAGCGTTTTAGAGCTAGAAATAGCAAG-3′) for sgRNA-1 and (5′-ATTTTAACTTGCTATTTCTAGCTCTAAAACGTCAGGTGCTTGTCTCGGGGTGCACCAGCCGGGAATCGAACCC-3′) for sgRNA-2.Through the process of Gibson cloning, both our sgRNAs were performed as described in reference IVT of sgRNA-3:IVT oligo used specific for CTGTTGATAAGCACGCAATCGTTTTAGAGCTAGAAATAGC-3′5′-GAAATTAATACGACTCACTATAGG.sgRNA-R used for template PCR:IVT oligo used AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3′.5′-sgRNA primer and the sgRNA-R are shown in bold letters.Complementary sequences of the specific sgRNA . This corresponds roughly to a 1:2 ratio (SpCas9: sgRNA-3). SpCas9 has about 5× the molecular weight of the sgRNA.The concentration of SpCas9 injected was about 800 ng/μl SpCas9  and about 300 ng/μl of sgRNA, ddH2O, and 10× incubation buffer NEB and mix thoroughly. Gently add the SpCas9 and mix again thoroughly by pipetting up and down. It is mandatory to add SpCas9 as the last ingredient because low salt concentrations may cause SpCas9 to precipitate : add cipitate .sgRNA .X μl 1 μl 10× NEB incubation buffer.2O (RNase free) to a total of 10 μl (X + Y = 6.5 μl).Y μl ddH2.5 μl SpCas9 .Mix gently and incubate the mix at 37°C for 2 min.Load the mix onto a column (Ultrafree-MC-HV 0.45 μm [Ref: UFC30HV00]).g.Spin for 1 min in a table-top centrifuge @14,000Reincubate the flow-through at 37°C for 2 min.Let the mix equilibrate at RT for ∼30 min before injection. Never put the mix back on ice.Drosophila Stock Center (see the Acknowledgments section).Crosses were done at 25°C. Unless noted otherwise, fly lines were obtained from the Bloomington ZH-attP 5D [X chromosome] or ZH-attP 102F [fourth chromosome]) were generated by phiC31 integrase-mediated transgenesis ; CIGAR-fwd: CAACAAAGTTGGCGTCGATA and CIGARmCherry-rev: AAGCGCATGAACTCCTTGATG , respectively. PCR settings were as follows: 95°C, 5 min; 35 cycles of 95°C, 25 s; 60°C, 25 s; and 72°C, 30 s); final elongation of 72°C, 10 s. The same PCR setting was used to analyze the shifter sequence of the CIGAR reporters by single fly PCR.Transgenic CIGAR fly lines  was inserted into the attP 40 site .The The images were taken on Axio Zoom V16 (Zeiss) and were processed in Adobe Photoshop or Adobe Illustrator.CIGAReGFP or CIGARmCherry construct on the fourth chromosome at position 102F ). To test if CRISPR-Cas9–mediated DSBs also result in recombination, the fourth chromosome, nos-Cas9/Y; U6:3-sgRNACIGAR/+; CIGAReGFP,102F, w+/CIGARmCherry,102F,w+ G0 males were crossed to yw females /+; CIGAReGFP,102F, w+/CIGARmCherry,102F, w+ G0 females to yw males). The offspring of such crosses were first scored at the larval stage to identify animals with either an activated CIGAReGFP,102F or CIGARmCherry,102F reporter. This preselection was made to ascertain that DSBs occurred in both constructs enhancing the likelihood of detecting recombination events on the fourth chromosome in case they occur. 17 vials  containing GFP and mCherry-positive larvae were selected for further analysis. A total of 172 y,w+ F1 animals harboring either a CIGAReGFP,102F or CIGARmCherry,102F reporter but lacking the sgRNA plasmid U6:pCFD5 (to avoid mosaic flies) from 13 of these crosses were randomly picked right after hatching. Single fly PCR of the target as well as part of the fluorophore region for the CIGAR reporters was performed for these 172 animals (Tables S2 and S3). PCR products and readable sequences were obtained in 156 cases.To determine if we could also induce CRISPR/Cas9–mediated recombination on the fourth chromosome, we first generated flies harboring either a ion 102F . These aFor the recombination experiments using the CIGAR transgenes on chromosome 4, we estimate the recombination frequency to be 26%: 41 recombinants identified/156 flies analyzed × 100  located downstream of the w+ marker near the tip of chromosome 4  and is visible if the mutation is homozygous or over a null allele of sv such as svΔ122 .To confirm that indeed recombination between sister chromatids occurs after CRISPR–Cas9–induced DSBs, we tested if we could induce recombination between two visible markers that are separated by about 100 kb  and S3. mosome 4 . The Casion site . The rouyw; CIGARmCherry,102F, w+/Dp1021,y+, svspa-pol embryos were injected with recombinant Cas9 protein complexed with in vitro–translated sgRNA-3 targeting the 3′UTR of the toy gene. From the eclosing G0 animals, a total of 135 crosses were set up. Either five G0 females (29 crosses) or single G0 males (106 crosses) were crossed with homozygous Dp1021,y+, svspa-pol flies. F1 offspring were either yw; CIGARmCherry,102F, w+/Dp1021,y+, svspa-pol  or homozygous Dp1021,y+, svspa-pol animals  1021,y+, svspa-pol and would be scored as having rough, red eyes . The second recombination event (TR-B) that could occur would be yw; Dp1021,y+, sv+/Dp1021,y+, svspa-pol flies having white and smooth eyes . From a total of 8,604 scored F1 animals, 253 putative recombinant flies were recovered: 216 had rough red eyes  and 37 animals had white and smooth eyes (five independent G0 crosses). Notably, in one cross, 31 w−, y+sv+ animals were present in one tube corresponding to about 25–30% of the offspring. 70 of the 253 putative recombinants  were backcrossed to homozygous Dp1021,y+, svspa-pol flies to establish stocks. 61 of the 70 putative recombinants were recovered from independent crosses . 13 of the 70 crosses remained without offspring. Most importantly, from 21 putative recombinants, we could generate homozygous viable lines. 17 of the 21 lines showed the y, w+, svspa-pol phenotype, whereas four lines were w, y+, sv+ marking all 21 lines as true recombinants. The remaining 36 putative recombinants were homozygous lethal .spa-pol) . Recombiy, w+, svspa-pol phenotype, the other was phenotypically w, y+, sv+, indicating that we may have recovered both chromosomes from the same recombination event. A third recombined animal from the same cross showed no CRISPR mark and thus can be counted as independent recombination event.We then investigated these animals by PCR specific for the CRISPR target site. Sequence analysis revealed that with the exception of two animals all CRISPR sites were without any indel . Interestingly, two recombinants from the same cross  showed the same CRISPR mark (6-bp deletion). However, they had the complementary phenotype: Whereas one fly showed the y+ marker on the short, left arm of chromosome 4 and the w+-marked CIGARmCherry,102F inserted at 102F 1021,y+, svspa-pol) were injected with Cas9 protein complexed with in vitro–translated sgRNA-3. This again would induce DSBs 3′ of toy located distal of CIGARmCherry,102F but should not have any influence on the recombination between the y+ and the w+ markers. G0 animals were this time crossed with yw animals to be able to score crossover events between the y+ and the w+ marker , we backcrossed such animals again against yw flies. Spontaneous recombination between y+ and w+ could be excluded as the above-mentioned backcross would have only revealed phenotypically y+w+ or yw flies. Instead, nondisjunction could be confirmed in all 11 cases because the backcross revealed phenotypically y+w+, yw, y+w, and yw+ flies. We determined the non-disjunction rate for the fourth chromosome to be 1 in 1,200, which is similar to the one observed for the X chromosome (To assess if CRISPR–Cas9–induced DSBs in general would enhance the frequency of recombination away from the Cas9 target site, we repeated the experiment and assessed the recombination frequency between a + marker . As experomosome .


The replisome must overcome DNA damage to ensure complete chromosome replication. Here, we describe the earliest events in this process by reconstituting collisions between a eukaryotic replisome, assembled with purified proteins, and DNA damage. Lagging-strand lesions are bypassed without delay, leaving daughter-strand gaps roughly the size of an Okazaki fragment. In contrast, leading-strand polymerase stalling significantly impacts replication fork progression. We reveal that the core replisome itself can bypass leading-strand damage by re-priming synthesis beyond it. Surprisingly, this restart activity is rare, mainly due to inefficient leading-strand re-priming, rather than single-stranded DNA exposure or primer extension. We find several unanticipated mechanistic distinctions between leading- and lagging-strand priming that we propose control the replisome’s initial response to DNA damage. Notably, leading-strand restart was specifically stimulated by RPA depletion, which can occur under conditions of replication stress. Our results have implications for pathway choice at stalled forks and priming at DNA replication origins.  •Reconstitution of collisions between a eukaryotic replisome and DNA damage•Leading-strand damage specifically causes fork stalling and uncoupling•The eukaryotic replisome can re-initiate leading-strands downstream of DNA damage•Multiple mechanistic differences exist between leading- and lagging-strand priming To study the earliest events following DNA polymerase stalling during replication, Taylor and Yeeles reconstituted collisions between a yeast replisome assembled with purified proteins and template DNA damage. Surprisingly, re-priming of leading-strand synthesis beyond damage is inefficient but is promoted by RPA depletion, whereas lagging-strand priming occurs robustly after damage. Accurate and efficient DNA replication is vital to ensure faithful and timely transmission of genetic information. This task is accomplished by a complex and highly regulated molecular machine known as the replisome. Replisomes frequently encounter a wide variety of obstacles to their progression, including template DNA damage, DNA secondary structures, and the transcription machinery . ConsideEscherichia coli found that, following UV irradiation of nucleotide excision repair (NER)-defective cells, replication continued and nascent DNA was synthesized as small fragments interspersed with single-stranded DNA (ssDNA) gaps (E. coli proteins (E. coli replisome has the inherent capacity to “skip” over multiple leading-strand lesions via re-priming (Early studies in NA) gaps . Based oproteins . Howeverproteins . Mechani-priming .Experiments in eukaryotic cells also found that replication was discontinuous following UV irradiation . More rein vivo following treatment of cells with DNA-damaging agents  by which it might operate, how efficiently it occurs, or how it might be regulated. In this study, we sought to address these outstanding questions by analyzing the response of a reconstituted core eukaryotic replisome to site-specific DNA damage.More generally, how the in vivo rate, DNA polymerase epsilon (Pol ε) catalyzes the bulk of leading-strand synthesis in conjunction with PCNA, DNA polymerase delta (Pol δ) is required for complete lagging-strand synthesis, and Mrc1 and Csm3/Tof1 are critical for maximum replication rates , which is one of the primary lesions generated by UV irradiation, at a specific location in plasmid DNA B and devLAG)  with a similar efficiency to those synthesized from an undamaged template  C. The retemplate D, nativents. LAG E, as pronts. LAG G illustrin vitro studies  by linearizing plasmids with AhdI  required for the chase. Compared to an undamaged template, replication forks were almost universally delayed for several min by CPDLEAD  composed of stalled leading strands, Okazaki fragments, and a distinct population migrating at the expected position for leading-strand restart  were only visible after AvrII digestion in reactions lacking RPA but containing Pol α in step 2  , ∼60 bp ynthesis . It is ain vivo occurs following treatment with DNA damaging agents, but it had not been possible to exclude other indirect effects of genotoxins or the involvement of DNA repair complexes and the transcription machinery. Because fork progression can be rapidly and efficiently rescued by artificially mimicking leading-strand “re-priming” with an oligonucleotide, we propose that the underlying basis for prolonged fork stalling and continued uncoupled synthesis may simply be a failure to re-prime the leading-strand template. If correct, then observations of stalled, slow-moving, and uncoupled replication forks in vivo , isolated stalled forks should be rapidly rescued by forks from neighboring origins. We propose that this may be the principal mechanism by which stalled forks are rescued when DNA damage is infrequently encountered, which would generate daughter-strand gaps for processing by DDT pathways.In striking contrast to E. coli , our resin vivo. It is notable that some eukaryotes encode an additional primase, PrimPol   promote  PrimPol . PrimPol PrimPol .in vitro raises the possibility that re-priming might fulfill a more significant role under conditions of catastrophic global fork arrest, which can have a dramatic effect on free RPA levels in vivo .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Joseph Yeeles  modified to overexpress proteins of interest as detailed in the Key Resources table by transforming with linearized plasmids using standard genetic procedures; or Escherichia coli Rosetta™ 2(DE3) cells (Novagen) (genotype: F– ompT hsdSB(rB– mB–) gal dcm (DE3) pRARE2 (CamR)) transformed with plasmids for overexpression of proteins of interest as detailed in the Proteins were purified from Escherichia coli are listed in the ORC – ORC, carrying a CBP-TEV tag on the Orc1 subunit, was purified using affinity purification with Calmodulin Sepharose 4B , followed by gel filtration through a Superdex 200 column.Cdc6 – GST-tagged Cdc6 was bound to Glutathione Sepharose 4B  and released by cleavage with GST-tagged 3C protease. The eluted protein was further purified using a Bio-Gel HT hydroxyapatite column (Bio-Rad).Cdt1-Mcm2-7 – Cdt1-Mcm2-7 with a CBP-TEV tag on the N terminus of Mcm3 was purified using a Calmodulin Sepharose 4B column, followed by Superdex 200  gel filtration.DDK – DDK carrying a CBP tag on Dbf4 was bound to and eluted from Calmodulin Sepharose 4B resin. The eluted protein was dephosphorylated with lambda protein phosphatase and was then further purified by gel filtration (Superdex 200).S-CDK – The S-CDK complex carrying a N-terminal CBP-TEV tag on Clb5 was bound to Calmodulin Affinity Resin (Agilent). Protein was eluted by cleavage with Tobacco Etch Virus protease and was further purified by gel filtration (Superdex 200).Cdc45 – Cdc45 carrying a double internal FLAG tag was immunoprecipitated using Anti-FLAG M2 affinity gel (Sigma). The resulting eluate was further purified using a Bio-Gel HT hydroxyapatite column.Sld3/7 – Sld3/7 carrying a C-terminal TCP tag on Sld3 was first bound to IgG Sepharose 6 Fast Flow resin  and the protein eluted by cleavage with Tobacco Etch Virus protease. The His-tagged protease was removed by passing the eluate over Ni-NTA resin (QIAGEN) and the flow through was further purified through a Superdex 200 gel filtration column.Sld2 – 3xFLAG-tagged Sld2  was precipitated from cell lysate with ammonium sulfate, resuspended in buffer, and then immunoprecipitated with Anti-FLAG M2 affinity gel. Protein was eluted with 3xFLAG peptide (Sigma) and the peptide was removed by binding and eluting the protein from a HiTrap SP HP column .Dpb11 – Dpb11-3xFLAG was purified using Anti-FLAG M2 affinity gel followed by MonoS  chromatography.Pol ε – Pol ε, tagged with a C-terminal CBP tag on Dpb4, was purified sequentially over Calmodulin Sepharose 4B, HiTrap Heparin HP  and Superdex 200 columns.GINS – The GINS complex, modified with a 6xHis tag at the N terminus of Psf3, was bound to Ni-NTA and eluted with increasing imidazole concentration. Eluted protein was further purified over MonoQ  and Superdex 200 columns, followed a second Ni-NTA column.Pol α – Pol alpha – primase, modified with an N-terminal CBP tag on Pri1 was purified sequentially over Calmodulin Sepharose 4B, MonoQ and Superdex 200 columns.Mcm10 – 6xHis-Mcm10 was purified over Ni-NTA followed by two rounds of MonoS  chromatography.Ctf4 – CBP-TEV-Ctf4 was purified by Calmodulin Sepharose 4B chromatography, followed by MonoQ and Superdex 200 gel filtration columns.Mrc1 – Mrc1-2xFLAG was purified by FLAG immunoprecipitation followed by MonoQ chromatography.Topo I – CBP-TEV-Topo I was bound to Calmodulin Sepharose 4B. Protein was eluted with Tobacco Etch Virus protease. His-tagged protease was removed by passing the eluate over a TALON column (Clontech) and the flow through was concentrated and separated through a Superdex 200 column.Csm3/Tof1 – Csm3/Tof1 carrying a N-terminal CBP-TEV tag on Csm3 was bound to Calmodulin Sepharose 4B. Protein was eluted with Tobacco Etch Virus protease. His-tagged protease was removed by passing the eluate over a TALON column and the flow through was concentrated and separated through a Superdex 200 column.RFC – The RFC complex containing an N-terminal CBP tag on Rfc3 was purified by sequential chromatography over Calmodulin Sepharose 4B, MonoS and Superdex 200 columns.PCNA – Native PCNA was purified following overexpression in Escherichia coli. Nucleic acids were precipitated from the cell lysate with polymin P and then proteins were selectively precipitated with ammonium sulfate. Precipitated material was resuspended in buffer and then applied to HiTrap SP HP and HiTrap Heparin HP columns assembled in tandem. The flow through containing PCNA was further purified over HiTrap DEAE Fast Flow  and MonoQ columns.Pol δ – Pol δ with a C-terminal TEV-CBP tag on Pol32 was purified over Calmodulin Sepharose 4B, HiTrap Heparin HP and Superdex 200 columns.RPA – Native RPA was purified following overexpression in Escherichia coli. Protein was purified over HiTrap Blue HP , ssDNA cellulose (Sigma) and MonoQ columns.Isw1a – Isw1a carrying a C-terminal 3xFLAG tag on Ioc3 was immunoprecipitated from cell lysate using Anti-FLAG M2 affinity gel. Protein was eluted with 3xFLAG peptide and was further purified by MonoQ chromatography.Histones – Native histones were purified following overexpression in Escherichia coli by HiTrap Heparin HP and Superdex 200 chromatography.Nap1 – GST-Nap1 was bound to Glutathione Sepharose 4B and eluted from the resin with GST-3C protease. Eluted protein was further purified on a MonoQ column.FACT – His-tagged FACT was purified by TALON and MonoQ chromatography.Nhp6 – Native Nhp6 was purified following overexpression in Escherichia coli. Proteins were precipitated from the cell lysate with trichloroacetic acid. The precipitated protein was resuspended in buffer and further purified over a HiTrap SP HP column.Proteins were purified as described previously . All yeaThe DNA replication template was produced by direct synthesis of a modified version of the genomic DNA surrounding the early-firing origin ARS306 and subsequent cloning and modification in pBluescript II KS(–). The complete sequence of the resulting two plasmids used for replication of damaged DNA introduced at two different sites is given below.Saccharomyces Genome Database. Modifications were made to (1) introduce restriction enzyme sites to facilitate cloning of three different fragments of this region into pBluescript II KS(–) ; (2) introduce several unique diagnostic restriction enzyme sites in proximity to both sites of DNA damage cloning, used in a variety of different assays; (3) introduce two closely spaced restriction enzymes for cloning of cassettes containing BbvCI nicking endonuclease sites for the subsequent cloning of DNA damage at two different locations approximately 3 kb (PstI and BamHI) or 4.5 kb (SphI and SpeI) left of ARS306; (4) mutate any sequences outside of the origin with a strong ORC binding consensus, to facilitate tight origin specificity of replication initiation; (5) introduce other unique restriction enzyme sites for other purposes, including for asymmetric template linearization with respect the origin. The modified sequence was synthesized by Invitrogen GeneArt Gene Synthesis in three segments flanked by (1) KpnI and SalI; (2) SalI and EagI; (3) EagI and SacI. These segments were cloned using these restriction sites into the multiple cloning site of pBluescript II KS(–). The resulting plasmids were then digested with either PstI and BamHI, or SphI and SpeI, for introduction of cassettes for the subsequent cloning of DNA damage at two different positions.A region of genomic DNA on chromosome III spanning approximately 7.5 kb left and 0.2 kb right of ARS306 was chosen from the ˆ indicates position of top strand nicking), was designed: CCˆTCAGCACTTAAGTCCˆTCAGC. This sequence and its reverse complement were incorporated into oligonucleotides flanked by overhangs compatible with the combination of either PstI and BamHI . To achieve this, the following cassette for cloning DNA damage, comprising an AflII restriction enzyme site (underlined) flanked by two Nt.BbvCI sites ) and ZraI (in the far left flank of the modified genomic DNA region) to excise approximately 1 kb DNA and the two blunt ends ligated to give a final plasmid size of approximately 9.7 kb. For the plasmid containing a cassette 4.5 kb from the origin, an additional two restriction enzyme sites, SacI and PsiI, were introduced approximately 1 kb and 1.6 kb respectively downstream of the cassette by site directed mutagenesis to facilitate characterization of the stalled fork. The final plasmids are hereafter referred to as ZN3 (plasmid with a cassette 3 kb from the origin) and ZN5SP1 (plasmid with a cassette 4.5 kb from the origin) and their sequences are given below:CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTAAAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGTTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTCAACGCCTTTTACCTTGTCCTTTGATGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAACTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGTCCTTCAATGAAACATCGTTGGCCACTAATTTGGCCAGTGCAAAGTAGAACAAATCGGCAGCCTCCCAAGAAAGCTCCTTCTTACCCTTTGCCTCAGTCAGTTCTTCAGCTTCTTCCTTGATCTTGGCATCTAACAATGCAGAGTCGTTGAATAGTCTTCTAGTATAAGATTCCTCTGGAGCGTCCTGTAGCCTTTGTTTTAGTAAAGATTCTAGCCCCACCAAACCATGCTTGAATTCACCAAAGCAAGACATGGTCTCCAAGTGGCAAAATCCAACGTTTTCTTGTTCAACGATAAACTTTAAGGCATCCGAATCACAGTCAGTAGAGATTTGTAAAAGCTTTTGGCCATTGCCAGAAGTTTCACCCTTGATCCAGATTTCATTCCTAGAACGAGAATAATAAACGCCACGACCCAATTCGATGGCCTTTGCTATAGATTTCTTCGAAGAATACACCAACCCTAGACAACGCTCATATTGGTCCACAACTAGGGTGGTATATAAACCGTCAGGACGGTCTGTACGTACTTCACCAAGCACTTCTTTGGTCAACATATCCTTGCTTAATTTCTTTATGGACACAATTTTATCTTGCGAGAATTTTTGTTTTACCATGAATTGATTGGAGAAAACACCGTTCTCTTCCACAACAACACGCTCCTTTGGTACATTCAATTGTTCAACCAAGTGTTCGGCTGTTTTAGCATCTTGGCTTGCAATGAACAGAGAAGAAACTCCGTTGTTCAAGAAGGCAATGATTTCATCATCGCTGAATTTACCACTTGGCAAGGACAAAGCCACCAATGGAACTTCTTCCTCTTTGGAGAACTGGAGAATCTCTTCATTACTCAGGCTCGAGCCATCCAAAAGTACCTGACCAACAAGTGAAACGTATTCCTTCTTACTATTCCATGAGGCCAGATCATCAATTAACGGTAGAATCGGCAAAACCATTATTCAGAAAAAAAATTTTGTAAACTATTGTATTACTATTACACAGCGCAGTTGTGCTATGATATTATAATGTATCCAGAACACACATCGGAGGTGAATATAACGTTCCATATCTATTATATACACAGTATACTACTGTTCATAGTCATATCCTTTTCTTACCTTCTATATCGAATGACTGATAATGCAACGTGAGTCACTGTGCATGGGTTTAGCAATTATTAAACTAATTTACCGGAGTCACTATTAGAGTCAGTTCGACTGCCTAGAAGAACTGCTGGTTGTCAGGATTGTGATGGGGGCATTCTGCTGTATTATGACCCATCGTATCGCAATGCTCACACCACTGTTGTCTTCCTGCCGTGGTATCGACTGGTGCAGGGGGGTCGAAAATTGGCAACGATTCCACGGCTGTTTGTGCTTGAGCCTGTTCCAACTGTTTGAACCTTTCATTAGCCTCTTCAAGTTTTTTCGTTAAGGATGCCACCTCTTCCGATGAGGAATCTTGTGGTTTTGTCAAAAATAGTTCCTTGCTCAAATTTTGGTATTCTTTACTGAGCGAATCGTTATGCATTTTCAATTGTTCGCGTTCTTTAGCCCACTTTGTCTTGTGTAACTCAAATTGGTCTTCTATGTTGCGTAATTGTTCCAGCTGTTTTTTCAGGAGCTCGACATCTTCGTTGGCACCAGTGGGTTGATTATGAGAAAGATTTCTCTCTTCGTTTTCTTTGATCTCTTCGTGTAGTTGGCTTACGACAGCAAGTAGCTGTTCATTCTCAGCGTCAAAAAACTGCTTTTGTTTGGCTTGCTGTCTGCGTTCGAGCAGACATTGTTGCTTGAGATGGTCTATCTCTTTCTCTCTTTCTTGTATTGTGGCTTCATACCTATCAAAAGTCGGTTGCACTTCTTCGAGGACCATTCTTTGGTCATCGAGTAGCCTTTTGTAGTGTAGTTGTTTCCTTTGTAGCTTTTCGATGGTCAATTGGCGATCGCGTAATTCAATTGTAACTTCGCTGCTATTGAGGTCATTCATGTGGCCATTGTCCGGTTTCCAATCGCTGGTGGTGTTGTGATTAGCCTTTCTGTCTGATGACAGGATAGAGTCCACCTCCATTCTGTCTTCTCTGTTATCGTAACCAAATTCTTGCTGTTGATGGTGATCCGATGCCTCCTGGTCCATCGACTGTTGATTACCGCTGTGCCGACTGGTGATCCGGAAACTTCTCATGGGTGTGGGGGATTTAGGATCATCCATGGGAGAGAAGCGCTTAGTGAGCCTCACAATAGATCTGTTCACGGGTATTGATAGCGGTTCCATTGTCGTTCTTCTCGAGGTTTGCCATATCGGTCCGTTCTCGATCAATGATGCGACTTTTTGCAACTGAATAAATAGTCCACTTTGAGGATACTCCGTTTGAAAATACTTCTTCCCCTAGGAATGATCCATCGTTCTTACCAATGTTGGCAAGTAAGTCTACACCAGCAAACATTCCACGCGTCGTGTCCACTGGACCCACGTATTTCAGTTGTCCGCGGCCGAAATTTGGGATTTGGTTTAAACATCCTATCTTTCTTTGATATCTATCCATGGTATATTAAGCGCATACGGCGCCAGCCACTAGTGCAGTACCAACCTCAGCACTTAAGTCCTCAGCGCAGTACCAAGCATGCCTCGTCCAAACGTTTTTGGTGTCTTGGCCAATTGCCCTTCTGAAAAATCTCACTGTCCGCAACTCATTAAAAGATACCCAAGCAAGCTACACGATAAAGAAAGGAGAAAGTTCATTACTGGAACGTACATATAGCGATACAAACGTATAGCAAAGATCTGAAATGGATACGGATAAGTTAATCTCAGAGGCTGAGTCTCATTTTTCTCAAGGAAACCATGCAGAAGCTGTTGCGAAGTTGACATCCGCAGCTCAGTCGAACCCCAATGACGAGCAAATGTCAACTATTGAATCATTAATTCAAAAAATCGCAGGATACGTCATGGACAACCGTAGTGGTGGTAGTGACGCCTCGCAAGATCGTGCTGCTGGTGGTGGTTCATCTTTTATGAACACTTTAATGGCAGACTCTAAGGGTTCTTCCCAAACGCAACTAGGAAAACTAGCTTTGTTAGCCACAGTGATGACACACTCATCAAATAAAGGTTCTTCTAACAGAGGGTTTGACGTAGGGACTGTCATGTCAATGCTAAGTGGTTCTGGCGGCGGGAGCCAAAGTATGGGTGCTTCCGGCCTGGCTGCCTTGGCTTCTCAATTCTTTAAGTCAGGTAACAATTCCCAAGGTCAGGGACAAGGTCAAGGTCAAGGTCAAGGTCAAGGACAAGGTCAAGGTCAAGGTTCTTTTACTGCTTTGGCGTCTTTGGCTTCATCTTTCATGAATTCCAACAACAATAATCAGCAAGGTCAAAATCAAAGCTCCGGTGGTTCCTCCTTTGGAGCACTGGCTTCTATGGCAAGCTCTTTTATGCATTCCAATAATAATCAGAACTCCAACAATAGTCAACAGGGCTATAACCAATCCTATCAAAACGGTAACCAAAATAGTCAAGGTTACAATAATCAACAGTACCAAGGTCGCGACGGTGGTTACCAACAACAACAGGGACAATCTGGTGGTGCTTTTTCCTCATTGGCCTCCATGGCTCAATCTTACTTAGGTGGTGGACAAACTCAATCCAACCAACAGCAATACAATCAACAAGGCCAAAACAACCAGCAGCAATACCAGCAACAAGGCCAAAACTATCAGCATCAACAACAGGGTCAGCAGCAGCAACAAGGCCACTCCAGTTCATTCTCAGCTTTGGCTTCCATGGCAAGTTCCTACCTGGGCAATAACTCCAATTCAAATTCGAGTTATGTGTACACGCAACAGGCTAATGAGTATGGTAGACCGCAACAGAATGGTCAACAGCAATCCAATGAGTACGGAAGACCGCAATACGGCGGAAACCAGAACTCCTAAGGACAGCACGAATCCTTCAATTTTTCTGGCAACTTTTCTCAACAGAACAATAACGGCGCGCCGAACCGCTACTGAACGATGATTCAGTTCGCCTTCTATCCTAAGTTTACGTATTTGCTAGCGCATATAACTTAGCGGGAAATTATTAATTGACCGGTAGGACAATTTTGTTGCACGTGATGCCTCAATCGTCTGCTTGCTTCCATAGTTAACATGAGGATCCTTTCAAAACAGAGTTGTATCTCTGCAGGATGCCCTTTTTGACGTATTGAATGGCATAATTGCACTGTCACTTTTCGCGCTGTCTCATTTTGGTGCGATGATGAAACTTTCATGAAACGTCTGTAATTTGAAACAAATAACGTAATTCTCGGGATTGGTTTTATTTAAATGACAATGTAAGAGTGGCTTTGTAAGGTATGTGTTGCTCTTAAAATATTTGGATACGACATCCAAAATCTTTTTTCCTTTAAGAGCAGGATATAAGTCGACAAGTTTCTGAAAATCAAAATGGTAGCAACAATAATGCAGACGACAACAACTGTGCTGACGACAGTCGCCGCAATGTCTACTACCTTAGCATCCCATTACATATCTTCGCAAGCTAGTTCCTCGACGAGTGTAACAACAGTAACGACAATAGCGACATCAATACGCTCTACACCGTCTAATCTACTCTTTTCTAATGTGGCGGCTCAGCCAAAATCATCTTCAGCAAGCACAATTGGGCTTTCAATCGGACTTCCCATCGGAATATTCTGTTTCGGATTACTTATCCTTTTGTGTTATTTCTACCTTAAAAGGAATTCGGTGTCCATTTCAAATCCACCCATGTCAGCTACGATTCCAAGGGAAGAGGAATATTGTCGCCGCACTAATTGGTTCTCACGGTTATTTTGGCAGAGTAAGTGTGAGGATCAGAATTCATATTCTAATCGTGATATTGAGAAGTATAACGACACCCAGTGGACCTCGGGTGATAACAAGTCTTCAAAAATACAGTACAAAATTTCCAAACCCATAATACCGCAGCATATACTGACACCTAAGAAAACGGTGAAGAACCCATATGCTTGGTCTGGTAAAAACATTTCGTTAGACCCCAAAGTGAACGAAATGGAGGAAGAGAAAGTTGTGGATGCATTCCTGTATACTAAACCACCGAATATTGTCCATATTGAATCCAGCATCCCCTCGTATAATGATTTACCTTCTCAAAAAACGGTGTCCTCAAAGAAAACTGCGTTAAAAACGAGTGAGAAATGGAGTTACGAATCTCCACTATCTCGATGGTTCTTGAGGGGTTCTACATACTTTAAGGATTATGGCTTATCAAAGACCTCTTTAAAGACCCCAACTGGGGCTCCACAACTGAAGCAAATGAAAATGCTCTCCCGGATAAGTAAGGGTTACTTCAATGAGTCAGATATAATGCCTGACGAACGATCGCCCATCTTGGAGTATAATAACACGCCTCTGGATGCAAATGACAGTGTGAATAACTTGGGTAATACCACGCCAGATTCACAAATCACATCTTATCGCAACAATAACATCGATCTAATCACGGCAAGACCCCATTCAGTGATATACGGTACTACTGCACAACAAACTTTGGAAACCAACTTCAATGATCATCATGACTGCAATAAAAGCACTGAGAAACACGAGTTGATAATACCCACCCCATCAAAACCACTAAAGAAAAGGATATAAAGAAGACAAAGTAAAATGTATCAGCATTTACAACATTTGTCACGTTCTAAACCATTGCCGCTTACTCCAAACTCCAAATATAATGGGGAGGCTTGCGTCCAATTAGGGAAGACATATACAGTTATTCAGGATTACGAGCCTAGATTGACAGACGAAATAAGAATCTCGCTGGGTGAAAAAGTTAAAATTCTGGCCACTCATACCGATGGATGGTGTCTGGTAGAAAAGTGTAATACACAAAAGGGTTCTATTCACGTCAGTGTTGACGATAAAAGATACCTCAATGAAGATAGAGGCATTGTGCCTGGTGACTGTCTCCAAGAATACGACTGATGAAAATAATATTGACGTTCGCATTTAATCTATACCTATAATTCTGTACTTATATACTGTTCCTTAATTGAAGATTTCAACATCGTTTTTGATGTAGGTCTTTTCACCTGGAGGTGCGGCTGGGCTACCGAAGACTAATTGAGCTTGTACGGTCCAAGACTCAGGGATTTTGCTTGGCAAAGCAGCTTTTATGTAACCATTGTAGTGTTGTAGGTGACCACCCAGGCCCATTGCCTCCAAGGCAACCCACGAGTTGATTTGAGCGGCACCAGAGGTATGGTCCGCGAAACTAGGGAATGCAGCTGCGTACGCTGGGAAGTCAGCCTTTAGCTTTTCAGTTACCTTGTCGTCGGTGAAGAAGATTACAGAACCAAAGGCCTCATCCCTTGCTGAAGCAGGCCTCTTTTGACCGGCAGGGCTTTCTATAGCCTTAGTCACTTCGTCCCAAACTTTTTTGTGAGTTTCACCAGTCAAGATAACAGCGCGATTTGGCTGGGAGTTGAAAGCGGTGGGTGTGGCTCGAATGATGGTTTGGACGACGGATTGGATGTCGTTGATAGTAATTTCACCAGGTAACTCCGGTTTCAAAGCGTAAATAGTACGACGAGCAGTTAAAGTTTTCAAATAAGTTGCAACAGCAGACATGATATTGGATTGCCGGAATGGCGATATGTTGATCCCGGATACTTCAGTCTACGAAAAAAGTACAAATTATGTAGTCAGTTCCTTCAGTATGGTGTCCTTATATACTGTAGTTTGGACAAGGTGCAAATGCCAAGACCCTAGCCCGAAAAGCTCGAGGCACCCCAGGATCTTCCCCTTTACGTAATTTTCACGTAAAACGCCACAGTCCGATTTTTCTCGAATAATCATTAGTAAAAGCGGTATACTGGATTATTGTACGATAACAAGGTAGAGCTTTATTACTAAGCTAAGACGTTCTTACATCAATAGTGCTGTTCGTTATTGATGTTAGGAGAAGGAGCGGGTCTGGTGAATAGTGTAAGCAGTGTTTCTGAACTTTTTCTTCGTCTAAGTCCTTGTAATGTAAGGTAAGAATGCAAGCATCTTGTTTGTAACCCGGGTGTACGTTGACGTTAGTAAGTCACAAACCCAAGCTTAACTTCTTCGTGAGGAAGGAAAGTGTTGTCTCCTACTTTTTTCAAATTTTCGAATTGTATTTATATTTATTTAGTACTTCTTGAGTTTACATATCCTTCGTAAAAATGCAACTTTTGTCGAAAAACACTTCCAAAAAAAAATAATAATGAATTTATGAAGCATACTAACGAGCGAGCACATCGCTGACCTATCATTACTTCATGAGATAAATTAAGATCTCCTCATATGCGAATTTCCTGTTCAGTGATAAACGTTGATTACGTTATTGATAAAAGTCTTTTCTTCTGGCAAGGGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACOligonucleotides for undamaged DNA  or DNA containing an abasic site  or CPD  were synthesized and purified  with 21 μL Nt.BbvCI  at 37°C for 2 h. A further 5 μL Nt.BbvCI (50 U) were then added per tube and digested for a further 1 h. Complete nicking to open circular DNA from covalently closed DNA was confirmed by agarose gel electrophoresis in the presence of ethidium bromide , contain2 with 110 U T4 DNA ligase (New England Biolabs M0202M) per μg plasmid at 16°C overnight in the dark  or AleI (for studying a lagging strand template CPD and undamaged controls), with the following exceptions. In Template linearization was performed in all cases by mixing 8-10 μg plasmid DNA in 50 μL final reaction volume containing 1X CutSmart Buffer (New England Biolabs B7204), 2.5 μL each restriction enzyme  and incubating at 37°C for 3 h. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (Sigma-Aldrich P2069) and the aqueous phase collected and DNA precipitated with 0.3 M NaCl + 2.8 volumes ice cold 100% ethanol in dry ice. The pellet was harvested, washed with room temperature 70% ethanol, harvested, air-dried and resuspended in 15-20 μL TE for use in replication assays.2 with 233 U T4 DNA ligase (New England Biolabs M0202M) per μg plasmid at 16°C overnight in the dark. The following day, SDS (to 0.1%) and proteinase K (1/100 volumes) were added and incubated at 37°C for 20 min. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (Sigma-Aldrich P2069) and the aqueous phase collected. Ligated template DNA was isolated from oligonucleotides by passing the aqueous phase over Sephacryl S-400 High Resolution matrix  columns (prepared by applying 800 μL 50% slurry to 0.8 mL Pierce Centrifuge Columns (Thermo Scientific 89868) and washing 2 × 250 μL TE by centrifuging at 700 g for 1 min). 35-40 μL was applied per column and collected by centrifuging at 700 g for 2 min. DNA was pooled, frozen, lyophilized in a vacuum concentrator and resuspended to 250-500 ng/μl in TE.Undamaged (maxi prep) and CPD-containing plasmid DNA were linearized with SapI restriction enzyme (New England Biolabs R0569), which cuts approximately 0.5 kb right of ARS306. Biotinylated oligonucleotides  , 2 M NaCl, 1 mM EDTA) then mixed with 4 μg DNA + buffer A to a total volume of 80 μl, and incubated at 25°C for 30 min, shaking at 500 rpm. The supernatant was discarded and the beads washed with 2 × 100 μL buffer B (10 mM HEPES-KOH (pH 7.6), 1 M KCl, 1 mM EDTA), 2 × 100 μL buffer C (10 mM HEPES-KOH (pH 7.6), 1 mM EDTA), resuspended in 75 μL buffer C and stored at 4°C in the dark.32P]-ATP using 10 U T4 Polynucleotide Kinase (New England Biolabs M0201S) in total volume 20 μL at 37°C for 1 h. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) (Sigma-Aldrich P2069) and the buffer exchanged and unincorporated nucleotides were removed from the aqueous phase with illustra MicroSpin G-50 columns . These markers were run as size standards in all gels and cropped from the final image for presentation.Molecular weight standards were prepared by first dephosphorylating 12.5 μg (25 μl) λ DNA-HindIII Digest (New England Biolabs) with 10 U Antarctic Phosphatase (New England Biolabs M0289S) in total volume 30 μL at 37°C for 1 h. The DNA was then purified using a QIAquick PCR Purification Kit (QIAGEN) and eluted in 30 μL water. 1 μg of this DNA was labeled with γ--dCTP, 12.5 nM Cdt1/Mcm2-7, 7.5 nM Cdc6, 3.3 nM ORC, 8.3 nM DDK, 20 nM S-CDK. In reactions omitting Pol δ, 117 mM potassium glutamate was used in all experiments , 100 mM potassium glutamate, 0.01% NP-40-S, 1 mM DTT, 10 mM Mg(OAc)eriments , except eriments F or 4 mieriments F and S5Geriments  were addFor reactions not requiring post-quenching restriction enzyme digests or native gel analysis, unincorporated nucleotides were removed from the quenched reaction mix with illustra MicroSpin G-50 columns , which also exchanged the buffer to TE. Samples were supplemented with 20 mM EDTA, and 1/10 volume alkaline loading dye  added. For all other reactions, SDS (to 0.1%) and proteinase K (1/100 volumes) were added and incubated at 37°C for 20 min. The DNA was extracted with phenol:chloroform:isoamyl alcohol 25:24:1 saturated with TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) (Sigma-Aldrich P2069) and the buffer exchanged and unincorporated nucleotides were removed from the aqueous phase with illustra MicroSpin G-50 columns . Except where indicated, samples for native gels were not digested with restriction enzymes, and mixed with native loading dye (20 mM Tris-HCl (pH 8.0), 50 mM EDTA, 10% Ficoll 400, 2% N-Lauroylsarcosine sodium salt solution) without additional processing. For restriction enzyme digests for the products of a standard 5-10 μL replication assay sample, 0.5-1 μL enzyme  was added in 1X CutSmart (New England Biolabs B7204). Enzyme (2-4 μl) and buffer volumes were scaled up for larger samples for 2D gels. Digests were performed at 37°C for 30 min, except SmaI which was incubated at 25°C. Digests were quenched with EDTA, samples split where appropriate, and added to native or alkaline loading dye.Denatured samples were analyzed in denaturing 0.6% agarose gels run at 24 V overnight in 30 mM NaOH, 2 mM EDTA. Native samples were analyzed in vertical 1% agarose gels run at 21-24 V overnight in modified 1X TAE (50 mM Tris-HCl (pH 7.9), 40 mM NaOAC, 1 mM EDTA (pH 8.0)), except for 2, 100 mM KOAc, 0.1% NP-40-S, 5% glycerol, 0.1 mg/ml BSA) on ice for 10 min. ATP (3 mM), creatine phosphate (40 mM) and creatine phosphate kinase (140 μg/ml) were added and chromatin assembly initiated by addition of DNA and transferring the reactions to 30°C for 1 h.Chromatin was assembled on 3 nM (20 ng/μl) AhdI- or AleI-linearized DNA in 40 μL total reaction volume. Nap1 (3 μM), histone octamers (370 nM) and ISW1 (30 nM) were pre-incubated in chromatin assembly buffer (25 mM HEPES-KOH (pH 7.6), 10 mM Mg(OAc)2, 40 mM KCl) and collecting by centrifuging at 700 g for 2 min. For helicase loading and phosphorylation, a volume of chromatin corresponding to the total reaction volume less the contribution from ATP, BSA, Cdt1/Mcm2-7, Cdc6, ORC and DDK was used. Chromatin replication was otherwise performed as for naked DNA templates except where noted in the figures and with the following exceptions: Sld2 was used at 30 nM; Pol δ was used at 10 nM in To prepare chromatin for replication assays, chromatin was exchanged to replication assay buffer by applying 40 μL chromatin assembly reaction to Sephacryl S-400 High Resolution matrix  columns (prepared by applying 800 μL 50% slurry to 0.8 mL Pierce Centrifuge Columns (Thermo Scientific 89868) and washing with 3 × 250 μL 25 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate, 0.01% NP-40-S, 1 mM DTT, 10 mM Mg(OAc)2 was added to 5 mM. 20 μL was collected and 1 μL 20-fold diluted MNase (100 U)  added and the mixture incubated at 37°C for 5 min. The reaction was stopped with 20 mM EGTA. 5 volumes of buffer PB  were added and the mixture applied to a QIAquick spin column, washed with buffer PE and eluted in 35 μL water. Products were analyzed by 1.5% agarose gel electrophoresis.After chromatin assembly CaCl32P]-dCTP. In 2, 0.1 mg/ml BSA, 3 mM ATP, 400 μM CTP, GTP, UTP, 30 μM dATP, dCTP, dGTP, dTTP, 66 nM α-[32P]-dCTP, 20 nM Pol ε, 5 nM Mcm10, 20 nM Ctf4, 20 nM Csm3/Tof1, 20 nM Mrc1, 20 nM RFC, 20 nM PCNA, 10 nM TopoI, 20 nM Pol α. In Helicase loading and phosphorylation was performed by incubating equal volumes of DNA-bound Dynabead slurry in buffer C (prepared as described above) with a mixture containing reaction buffer components, proteins and ATP to give a final reaction mix as used for helicase loading and phosphorylation on a standard soluble template, but without KCl. After treatment with S-CDK as described above, an appropriate volume of the mixture was collected such that it would be diluted 5-fold in the final reaction mix. Replication was initiated as described above but in the absence of α--dCTP, 20 nM Pol α and RPA (0-400 nM). RPA was first pre-bound to the template in the absence of Pol α for 10 min. Reactions were initiated by addition of Pol α and were incubated for 20 min. Samples were processed and run through alkaline agarose gels as described for soluble replication reactions.Primed template was prepared by annealing oligonucleotide JY180 (500 nM) (sequence: Denaturing gels were fixed with two changes of cold 5% trichloroacetic acid and dried onto chromatography paper (Whatman). Native gels were dried directly onto chromatography paper. Most gels were autoradiographed with Amersham Hyperfilm MP  for presentation. For quantification, gels were exposed on BAS-IP MS Storage Phosphor Screens  and screens were developed on a Typhoon phosphorimager .Replication product quantification was performed in ImageJ  after converting the .gel files to 16-Bit Tiff files using the Linearize GelData command. Lane profiles shown in In all cases, graphs of quantified data show the mean and errors (SEM) from n (stated in the figure legends) independent experimental repeats.