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Stable expression of MutLĪ³ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.
Roesner, Lennart M Mielke, Christian FƤhnrich, Silke Merkhoffer, Yvonne Dittmar, Kurt E J Drexler, Hans G Dirks, Wilhelm G
Roesner, Lennart M
Mielke, Christian
FƤhnrich, Silke
Merkhoffer, Yvonne
Dittmar, Kurt E J
Drexler, Hans G
Dirks, Wilhelm G
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2013-10
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Abstract
The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLĪ± (MLH1-PMS2), MutLĪ² (MLH1-PMS1), and MutLĪ³ (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 MutLĪ± 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 MutLĪ±-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. MutLĪ³ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLĪ±. Surprisingly, splicing variant MLH3Ī7 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 MutLĪ± led to full recovery of DNA damage response in MMR deficient cells, expression of MutLĪ³ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLĪ³-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLĪ±-deficient background no DNA repair-specific function carried out by MutLĪ³ can be detected in living cells.
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Stable expression of MutLĪ³ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites. 2013, 114 (10):2405-14 J. Cell. Biochem.
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en
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1097-4644
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Archived with thanks to Journal of cellular biochemistry