Stable Expression of MutLγ in Human Cells Reveals No Specific Response to Mismatched DNA, But Distinct Recruitment to Damage Sites

The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutL (MLH1-PMS2), MutL (MLH1-PMS1), and MutL (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutL is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutL-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutL dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutL. Surprisingly, splicing variant MLH37 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutL led to full recovery of DNA damage response in MMR deficient cells, expression of MutL or single MLH3 failed to do so. These experiments show recruitment and persistence of MutL-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutL-deficient background no DNA repair-specific function carried out by MutL can be detected in living cells. J. Cell. Biochem. 114: 2405-2414, 2013. (c) 2013 Wiley Periodicals, Inc.