DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.

The DNA mismatch repair (MMR) pathway and its regulation are critical for genomic stability. Mismatch repair (MMR) follows replication and repairs misincorporated bases and small insertions or deletions that are not recognized and removed by the proofreading polymerase. Cells deficient in MMR exhibit an increased overall mutation rate and increased expansion and contraction of short repeat sequences in the genome termed microsatellite instability (MSI). MSI is often a clinical measure of genome stability in tumors and is used to determine the course of treatment. MMR is also critical for inducing apoptosis after alkylation damage from environmental agents or DNA-damaging chemotherapy. MLH1 is essential for MMR, and loss or mutation of MLH1 leads to defective MMR, increased mutation frequency, and MSI. In this study, we report that tyrosine kinase inhibitors, imatinib and nilotinib, lead to decreased MLH1 protein expression but not decreased MLH1 mRNA levels. Of the seven cellular targets of Imatinib and nilotinib, we show that silencing of ABL1 also reduces MLH1 protein expression. Treatment with tyrosine kinase inhibitors or silencing of ABL1 results in decreased apoptosis after treatment with alkylating agents, suggesting the level of MLH1 reduction is sufficient to disrupt MMR function. We also report MLH1 is tyrosine phosphorylated by ABL1. We demonstrate that MLH1 downregulation by ABL1 knockdown or inhibition requires chaperone protein Hsp70 and that MLH1 degradation can be abolished with the lysosomal inhibitor bafilomycin. Taken together, we propose that ABL1 prevents MLH1 from being targeted for degradation by the chaperone Hsp70 and that in the absence of ABL1 activity at least a portion of MLH1 is degraded through the lysosome. This study represents an advance in understanding MMR pathway regulation and has important clinical implications as MMR status is used in the clinic to inform patient treatment, including the use of immunotherapy.