In eukaryotic cells, DNA replication proceeds with continuous synthesis of leading-strand DNA and discontinuous synthesis of lagging-strand DNA. Here we describe a method, eSPAN (enrichment and sequencing of protein-associated nascent DNA), which reveals the genome-wide association of proteins with leading and lagging strands of DNA replication forks. Using this approach in budding yeast, we confirm the strand specificities of DNA polymerases delta and epsilon and show that the PCNA clamp is enriched at lagging strands compared with leading-strand replication. Surprisingly, at stalled forks, PCNA is unloaded specifically from lagging strands. PCNA unloading depends onthe Elg1-containing alternative RFC complex, ubiquitination of PCNA, and the checkpoint kinases Mec1 and Rad53. Cells deficient in PCNA unloading exhibit increased chromosome breaks. Our studies provide a tool for studying replication-related processes and reveal a mechanism whereby checkpoint kinases regulate strand-specific unloading ofPCNA from stalled replication forks to maintain genome stability.
Bibliographical noteFunding Information:
We thank Dr. Oscar Aparicio and Dr. Grant Brown for yeast strains and plasmids. We thank Dr. Jayme Dahlin, Dr. David MacAlpine, Dr. Robin M. Ricke, Dr. Bruce Horazdovsky, Dr. Rentian Wu, and Dr. Zhenkun Lou for critical reading of this manuscript. This work was supported by NIH grants (GM72719, GM81838 to Z.Z.). A.C. is supported by the Swedish Cancer Society. G.F. and T.O. are supported by PO1 DK068055. S.J. is a Kempe Foundation scholarship recipient, and Z.Z. is a scholar of the Leukemia and Lymphoma Society.
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