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DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair
Mark C Hall1, Polina V Shcherbakova, John M Fortune
1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Abstract:
The yeast Mlh1-Pms1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1. We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1-Pms1 heterodimer. Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.
Insights
The N-terminal domains of Mlh1 and Pms1 independently bind DNA, crucial for mismatch repair (MMR). Mutations affecting Mlh1 DNA binding impair MMR, highlighting Mlh1
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The Mlh1-Pms1 heterodimer is essential for DNA mismatch repair (MMR) in yeast.
- Understanding the DNA binding mechanisms of MMR proteins is key to deciphering repair pathways.
Purpose of the Study:
- To map the DNA binding regions within the Mlh1-Pms1 heterodimer.
- To investigate the role of specific amino acid residues in Mlh1 and Pms1 DNA binding and MMR function.
Main Methods:
- Site-directed mutagenesis to alter conserved amino acids in Mlh1 and Pms1.
- DNA binding assays using N-terminal fragments and full-length proteins.
- Assessing mutator phenotypes in yeast strains with modified Mlh1/Pms1 genes.
Main Results:
- Mlh1 and Pms1 N-terminal domains (NTDs) independently bind single- and double-stranded DNA with distinct affinities.
- Mutations in conserved residues of the Mlh1 NTD significantly reduce DNA binding and MMR efficiency, causing mutator phenotypes.
- Homologous mutations in Pms1 had minimal impact on DNA binding and mutation rates.
- Full-length Mlh1 and the Mlh1-Pms1 heterodimer also exhibit DNA binding, potentially involving C-terminal regions of Mlh1.
Conclusions:
- Yeast Mlh1 and Pms1 possess independent DNA binding sites within their N-terminal domains.
- Mlh1's DNA binding activity is critical for MMR, whereas Pms1's role in DNA interaction appears less significant.
- These findings support the hypothesis that Mlh1 DNA binding is a vital step in the MMR process.