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Inactivation of DNA Mismatch Repair by Increased Expression of Yeast MLH1

机译：通过酵母MLH1表达的增加使DNA错配修复失活

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Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177–3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)+ mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3′→5′ exonuclease activity of replicative DNA polymerases δ and ? but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, orPMS1. This suggests that overexpression ofMLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K d of 3.14 μM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressingMLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.
机译：通过突变或 MLH1 基因的转录沉默使DNA错配修复失活会导致基因组不稳定和易患癌症。我们最近发现（P. V. Shcherbakova和T. A. Kunkel，Mol。Cell。Biol。19：3177-3183，1999），自发突变率的升高也可能是由于酵母 MLH1 的表达增加所致。在这里，我们研究这种突变效应的机制。 poly（A） + mRNA与含有96.4％酵母开放阅读框的DNA微阵列的杂交显示， MLH1 的过表达不会诱导其他参与DNA复制的基因表达的变化或维修。 MLH1 的过表达强烈增强了酵母菌株中的自发诱变，但复制DNA聚合酶δ和δ的3'→5'核酸外切酶活性存在缺陷但并没有提高缺失 MSH2 ， MLH1，或 PMS1 的菌株的突变率。这表明 MLH1 的过表达会激活复制错误的错配修复。仅 PMS1 基因的过表达引起突变率的适度增加，并强烈抑制了 MLH1 过表达引起的突变效应。突变体的作用还被 MLH1 基因的错义突变所破坏，该突变破坏了Mlh1p-Pms1p​​的相互作用。超离心分析实验表明，纯化的Mlh1p在溶液中形成同型二聚体，尽管 K d 为3.14μM，比其高36倍。 Mlh1p-Pms1p​​异二聚化。这些观察结果表明，过表达 MLH1 的细胞中的错配修复缺陷是由于Mlh1p和Pms1p​​的水平失衡造成的，并且这种失衡可能导致含有Mlh1p同型二聚体的非功能错配修复复合物的形成。

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