Becker Jordan R, Pons Carles, Nguyen Hai Dang, Costanzo Michael, Boone Charles, Myers Chad L, Bielinsky Anja-Katrin
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America.
PLoS Genet. 2015 Nov 6;11(11):e1005659. doi: 10.1371/journal.pgen.1005659. eCollection 2015 Nov.
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.
复制夹增殖细胞核抗原（PCNA）在保守残基赖氨酸（K）164处的泛素化触发复制后修复（PRR），以填补由停滞的DNA聚合酶导致的单链间隙。然而，细胞是否因不直接损害DNA合成的复制缺陷而参与PRR仍不清楚。为了通过实验解决这个问题，我们对酿酒酵母温度敏感等位基因文库，使用PCNA的泛素化缺陷型K164精氨酸（K164R）突变体进行了合成基因阵列（SGA）分析。K164R等位基因的SGA特征与后随链复制各方面缺陷的突变体谱，包括rad27Δ和elg1Δ，显示出显著相关性。Rad27是处理后随链复制过程中产生的5' 瓣片的主要瓣片内切酶，而Elg1与从染色质上卸载PCNA有关。我们在rad27Δ和elg1Δ突变体中均观察到PCNA在K164处的慢性泛素化。值得注意的是，仅rad27Δ细胞在消除PRR途径后细胞活力下降，而elg1Δ突变体不受影响。我们进一步提供证据表明，K164泛素化抑制了冈崎片段成熟过程中瓣片处理缺陷导致的复制应激。因此PCNA泛素化的缺失增加了由Rad53激酶的过度磷酸化所表明的S期检查点激活。此外，我们证明通过催化活性核酸外切酶1的过表达进行的替代瓣片处理消除了PCNA泛素化。这提示了一个模型，其中未处理的瓣片可能直接参与PRR信号传导。我们的研究结果表明，响应复制应激时PCNA在K164处的泛素化不仅限于DNA合成缺陷，还扩展到后随链复制过程中的DNA处理。
