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A human okazaki fragment12/27/2023 Received: AugAccepted: OctoPublished: November 6, 2015Ĭopyright: © 2015 Becker et al. PLoS Genet 11(11):Įditor: Sue Jinks-Robertson, Duke University, UNITED STATES (2015) Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing. These findings expand the known functions of PRR and reveal their importance in promoting the viability of cells lacking a known tumor suppressor.Ĭitation: Becker JR, Pons C, Nguyen HD, Costanzo M, Boone C, Myers CL, et al. Specifically, we report a requirement for PRR in cells lacking RAD27, the yeast homolog of the tumor suppressor FEN1. Our findings indicate that in addition to previously described roles in rescuing DNA synthesis defects, PRR is also required in response to aberrant DNA processing. In this study, we utilized a non-biased genome wide genetic screen to systematically identify conditions under which PRR is required. Postreplicative repair (PRR) pathways are two such mechanisms with well described functions in promoting the completion of replication under adverse conditions. Not surprisingly, evolution has selected for mechanisms to mitigate the effects of defective replication and avoid the most harmful outcomes. Furthermore, there is strong evidence to suggest that defects in DNA replication are prominent contributors to mutation and genome instability, a hallmark of cancer. This fundamentally important mechanism has been highly conserved throughout eukaryotic evolution, allowing us to use the relatively simple and genetically tractable Saccharomyces cerevisiae as a model to better understand DNA replication in human cells. Genome duplication via the process of DNA replication is a prerequisite for cell division and underlies the propagation of all living organisms. Our findings demonstrate that PCNA ubiquitination at K164 in response to replication stress is not limited to DNA synthesis defects but extends to DNA processing during lagging strand replication. This suggests a model in which unprocessed flaps may directly participate in PRR signaling. Furthermore, we demonstrate that alternative flap processing by overexpression of catalytically active exonuclease 1 eliminates PCNA ubiquitination. Accordingly, ablation of PCNA ubiquitination increased S phase checkpoint activation, indicated by hyperphosphorylation of the Rad53 kinase. We further provide evidence that K164 ubiquitination suppresses replication stress resulting from defective flap processing during Okazaki fragment maturation. Notably, only rad27Δ cells exhibited a decline in cell viability upon elimination of PRR pathways, whereas elg1Δ mutants were not affected. We observed chronic ubiquitination of PCNA at K164 in both rad27Δ and elg1Δ mutants. Rad27 is the primary flap endonuclease that processes 5’ flaps generated during lagging strand replication, whereas Elg1 has been implicated in unloading PCNA from chromatin. The SGA signature of the K164R allele showed a striking correlation with profiles of mutants deficient in various aspects of lagging strand replication, including rad27Δ and elg1Δ. cerevisiae temperature-sensitive alleles. To experimentally address this question, we performed synthetic genetic array (SGA) analysis with a ubiquitination-deficient K164 to arginine (K164R) mutant of PCNA against a library of S. However, it has remained elusive as to whether cells engage PRR in response to replication defects that do not directly impair DNA synthesis. Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine (K)164 triggers postreplicative repair (PRR) to fill single-stranded gaps that result from stalled DNA polymerases.
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