title: Genetic Interactions Implicating Postreplicative Repair in Okazaki Fragment Processing.
abstract: 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. However, it has remained elusive as to whether cells engage PRR in response to replication defects that do not directly impair DNA synthesis. 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. cerevisiae temperature-sensitive alleles. 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. 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. We observed chronic ubiquitination of PCNA at K164 in both rad27Δ and elg1Δ mutants. Notably, only rad27Δ cells exhibited a decline in cell viability upon elimination of PRR pathways, whereas elg1Δ mutants were not affected. We further provide evidence that K164 ubiquitination suppresses replication stress resulting from defective flap processing during Okazaki fragment maturation. Accordingly, ablation of PCNA ubiquitination increased S phase checkpoint activation, indicated by hyperphosphorylation of the Rad53 kinase. Furthermore, we demonstrate that alternative flap processing by overexpression of catalytically active exonuclease 1 eliminates PCNA ubiquitination. This suggests a model in which unprocessed flaps may directly participate in PRR signaling. 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.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Jordan R
authors.initials: JR
authors.lastname: Becker
authors.affiliation: Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Carles
authors.initials: C
authors.lastname: Pons
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Hai Dang
authors.initials: HD
authors.lastname: Nguyen
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Costanzo
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Charles
authors.initials: C
authors.lastname: Boone
authors.affiliation: Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Chad L
authors.initials: CL
authors.lastname: Myers
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Anja-Katrin
authors.initials: AK
authors.lastname: Bielinsky
mesh_terms: D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D005090:Exodeoxyribonucleases / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D054875:Ubiquitination
chemical_list: C022262:Okazaki fragments; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I