Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis

Abstract

Proliferating cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSβ and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function.is an essential genome stability pathway that removes mismatches introduced by nucleotide misincorporation during DNA replication (1-4). MMR in eukaryotic cells is nick-directed and targeted specifically to the newly synthesized DNA strand (5, 6). MMR is carried out in three phases: initiation, excision, and resynthesis. The initiation phase involves mismatch recognition by MutSα or MutSβ; assembly of the initiation complex containing MutSα (or MutSβ), MutLα, and proliferating cell nuclear antigen (PCNA); and localization of the strand discrimination signal (a single-strand nick) by this complex (7-11) in a process that is incompletely understood. During the excision phase, exonuclease 1 (Exo1) removes nascent DNA exonucleolytically from a distal nick to the mismatch in a reaction that requires MutSα (or MutSβ), MutLα, and PCNA. Replication protein A (RPA) stimulates DNA excision by Exo1 and protects single-stranded DNA from cleavage by nucleases (12, 13). During the resynthesis phase of MMR, DNA polymerase δ fills in the single-strand DNA gap left by DNA excision in a concerted reaction that requires replication factor C (RFC), PCNA, and RPA, followed by DNA ligase I-catalyzed nick ligation.Mutations or promoter hypermethylation in coding or regulatory regions of MMR genes MSH2, MSH6, MLH1, and PMS2 are linked to susceptibility to colorectal and other cancers in humans and other organisms (2, 14-16). The cellular phenotype associated with defects in MMR includes an elevated genomewide mutation rate as well as frequent changes in DNA microsatellite repeats, a phenomenon called microsatellite instability (MSI). Thus, MSI is frequently used as a biomarker for (or hallmark of) MMR deficiency (1-4). However, a significant number of MSIpositive tumors do not carry mutations in known MMR genes (17). Recent studies have shown that mutations affecting the proofreading nuclease activity of DNA polymerases δ and e are associated with some colorectal and/or endometrial cancers (18), and that defects in the gene encoding histone H3 Lys36 (H3K36) trimethyltran...

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“…These results were consistent with previous reports that EGFR can drive cell cycle progression by affecting the expression of proliferationrelated proteins (Ortega et al, 2015).…”

EGFR inhibits DNA mismatch repair

“…In PNAS, Ortega et al reveal that phosphorylation of PCNA by EGFR inhibits MMR in cells and switches DNA synthesis in vitro from highfidelity to error-prone ( Fig. 2) (10). This intersection between MMR and EGFR underscores the importance of understanding the nuclear roles of EGFR and other RTKs, and highlights the relationship between the control of cell proliferation and the maintenance of genome stability, two central aspects of tumorigenesis.…”

“…Ortega et al use in vitro assays to dissect the effects of PCNA-pY211 on MMR in several MMR-deficient colorectal cancer cell lines with elevated levels of EGFR, and observe a correlation between levels of EGFR, PCNA-pY211, and MMR loss (10). Restoration of MMR in these colorectal cancer cells requires the addition of MMR proteins and unphosphorylated PCNA.…”

“…However, there are broader implications as well. Tyr211 is positioned in close proximity to a critical loop and hydrophobic pocket that mediate binding to PCNA-interacting protein box-containing proteins, and modeling suggests that phosphorylation might disrupt local geometry, threatening many PCNA interactions (10). Is DNA synthesis affected in replication, translesion synthesis repair, or recombination?…”

“…Understanding the intersection between cell proliferation and genome stability will reveal much about what diverts EGFR from normal paths to more reckless ones. (10). ER, endoplasmic reticulum; INM, inner nuclear membrane; NPC, nuclear pore complex; ONM, outer nuclear membrane; RFC, replication factor C.…”