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.
Highlights
The accurate copying of a cellular genome and subsequent transmission of genetic material to two daughter cells occurs on a microscopic scale, but is a prodigious task
Our findings indicate that in addition to previously described roles in rescuing DNA synthesis defects, postreplicative repair (PRR) is required in response to aberrant DNA processing
We report a requirement for PRR in cells lacking RAD27, the yeast homolog of the tumor suppressor FEN1
Summary
The accurate copying of a cellular genome and subsequent transmission of genetic material to two daughter cells occurs on a microscopic scale, but is a prodigious task. Considering the difficulty of accomplishing this fundamental process for living cells, it is hardly surprising that evolution has selected for a complex and multi-layered system of checkpoints and redundancies that promote its completion under sub-optimal conditions [1,2]. Many of these processes are regulated by post-translational modification of proteins that act as molecular switches to regulate downstream responses. SUMO modification of chromatin-bound PCNA, or sumoylation, occurs during an unperturbed S phase at K164 and–to a lesser extent– at K127 [3]. Sumoylation acts primarily to recruit the helicase/anti-recombinase suppressor of rad six 2 (Srs2), which prevents illegitimate recombination at replication forks [4,5,6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
