Genetic evidence for the involvement of mismatch repair proteins,PMS2 and MLH3, in a late step of homologous recombination |
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Authors: | Md Maminur Rahman Mohiuddin Mohiuddin Islam Shamima Keka Kousei Yamada Masataka Tsuda Hiroyuki Sasanuma Jessica Andreani Raphael Guerois Valerie Borde Jean-Baptiste Charbonnier Shunichi Takeda |
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Affiliation: | 1.Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan;2.Institute for Integrative Biology of the Cell (I2BC), Commissariat à l''Energie Atomique (CEA), CNRS, Université Paris-Saclay, Gif-sur-Yvette, France;3.Institut Curie, CNRS, UMR3244, PSL Research University, Paris, France |
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Abstract: | Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3−/− and PMS2−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3−/− and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2−/− and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells. |
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Keywords: | homologous recombination PMS2 MLH3 MLH1 MUS81 GEN1 resolvase joint molecules mutL homolog 1 (MLH1) mutL homolog 3 (MLH3) Holliday junction endonuclease |
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