Effective mismatch repair depends on timely control of PCNA retention on DNA by the Elg1 complex

Abstract

Proliferating cell nuclear antigen (PCNA) is a sliding clamp that acts as a central co-ordinator for mismatch repair (MMR) as well as DNA replication. Loss of Elg1, the major subunit of the PCNA unloader complex, causes over-accumulation of PCNA on DNA and also increases mutation rate, but it has been unclear if the two effects are linked. Here we show that timely removal of PCNA from DNA by the Elg1 complex is important to prevent mutations. Although premature unloading of PCNA generally increases mutation rate, the mutator phenotype of elg1Δ is attenuated by PCNA mutants PCNA-R14E and PCNA-D150E that spontaneously fall off DNA. In contrast, the elg1Δ mutator phenotype is exacerbated by PCNA mutants that accumulate on DNA due to enhanced electrostatic PCNA–DNA interactions. Epistasis analysis suggests that PCNA over-accumulation on DNA interferes with both MMR and MMR-independent process(es). In elg1Δ, over-retained PCNA hyper-recruits the Msh2–Msh6 mismatch recognition complex through its PCNA-interacting peptide motif, causing accumulation of MMR intermediates. Our results suggest that PCNA retention controlled by the Elg1 complex is critical for efficient MMR: PCNA needs to be on DNA long enough to enable MMR, but if it is retained too long it interferes with downstream repair steps.

DNA Mismatch Repair and its Role in Huntington’s Disease

“…Thus, it is relatively straightforward to envision PCNA loaded at DNA termini as strand signals for MMR in the context of active DNA replication in dividing cells (e.g., DNA ends of Okazaki fragments). In fact, recent studies have suggested that loaded PCNA continues to direct strand-specific MMR even after the removal of strand-discontinuities, and that a physical interaction between MutS␣ and PCNA enhances the temporal window for effective strand-directed MMR [110,111]. Thus, persistence of DNA-loaded PCNA after completion of DNA synthesis can continue to provide strand-directionality "memory" to the MMR system [110].…”

Eukaryotic clamp loaders and unloaders in the maintenance of genome stability

“…This finding suggests that MMR defects also occur in ATAD5-depleted cells via a similar mechanism. Interestingly, the same report showed that the mutation rate also increases through MMRindependent but accumulated PCNA-dependent processes in elg1Δ mutants 84 . Based on the results in human cells, one such possible candidate process has been identified as monoubiquitinated PCNA-directed errorprone translesion DNA synthesis 77 .…”

“…PCNA participates in several steps during the DNA MMR process, including mismatch recognition by MSH2–MSH6 or MSH2–MSH3, strand discrimination, strand excision, and repair DNA synthesis. Other negative effects of PCNA accumulation on DNA metabolism have recently been reported in the MMR pathway 84 . The mutation rate is increased in elg1∆ mutants, but this is not observed in a disassembly-prone PCNA or msh2∆ / msh6∆ -mutant background.…”

PCNA antagonizes cohesin-dependent roles in genomic stability

“…This intersection between PCNA and cohesin is consistent with prior studies that document that 1) highly elevated levels of PCNA result in genomic instability [ 63 ], 2) cohesin are both recruited to stalled replication forks and promote fork restart [ 77 – 79 , 89 ], and 3) DNA replication fork protection complexes and cohesin pathways are intimately linked [ 24 , 63 , 77 , 80 , 108 ]. During the final stages of this study, independent analyses revealed that elevated levels of chromatin-bound PCNA results in hyper-recruitment of mismatch repair factors [ 109 ]. Cells that exhibit elevated levels of mismatch repair intermediates normally activate cohesin recruitment pathways to promote efficient DNA repair through homologous recombination [ 110 , 111 ].…”

“…Cells that exhibit elevated levels of mismatch repair intermediates normally activate cohesin recruitment pathways to promote efficient DNA repair through homologous recombination [ 110 , 111 ]. Cells utilize Msh2-Msh3-dependent mismatch repair (MMR) to correct mismatches (including short insertions and deletions) that accumulate during DNA replication [ 109 , 112 – 114 ], which increase upon high levels of PCNA expression [ 109 ]. However, numerous mcd1-1 msh3Δ elg1Δ triple mutant cells were obtained from crossing msh3Δ single mutant cells to mcd1-1 elg1Δ double mutant cells ( S4 Table ).…”