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Stable expression of MutLγ in human cells reveals no specific response to mismatched DNA, but distinct recruitment to damage sites.

机译:MutLγ在人细胞中的稳定表达没有显示出对错配DNA的特异性反应,但对损伤部位的募集却不同。

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摘要

The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLβ (MLH1-PMS1), and MutLγ (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 MutLα 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 MutLα-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. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 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 MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.
机译:人类DNA错配修复(MMR)基因家族包含四个MutL旁系同源物,能够形成异二聚体MutLα(MLH1-PMS2),MutLβ(MLH1-PMS1)和MutLγ(MLH1-MLH3)蛋白复合物。人MutL亚基PMS2和MLH3包含进化上保守的氨基酸基序DQHA(X)2E(X)4E,被鉴定为能够切出有缺陷的DNA链的内切核酸域。普遍认为MutLα的PMS2是内切核酸酶活性的唯一执行者,但是由于MLH3被证明能够在体外进行低水平的DNA修复,因此我们的目的是研究MLH3是否在MutLα-下被激活作为备用。条件不足。在这里,我们报告等离子细胞系293和293T的GFP标记的MLH3的稳定表达,它们分别对MLH1表达具有功能或缺陷。如预期的那样,MLH3与内源和重组MLH1形成了二聚体复合物。如MutLα所示,将MutLγ二聚体募集到UVA微辐照诱导的DNA损伤位点。令人惊讶地,缺少内切核酸基序的剪接变体MLH3Δ7显示出一致的病灶形成,这暗示募集不一定代表主动DNA修复。作为修复酶活性的替代测试,我们将烷基化导向的DNA损伤与彗星形成分析相结合。虽然重组MutLα导致MMR缺陷细胞中DNA损伤反应的完全恢复,但MutLγ或单个MLH3的表达未能做到。这些实验表明MutLγ-异二聚体在UVA诱导的DNA损伤中的募集和持久性。但是,我们证明在MutLα缺乏的背景下,在活细胞中无法检测到MutLγ进行的DNA修复特异性功能。

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