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Nucleic Acids Research, 2000, Vol. 28, No. 13 2551-2556
© 2000 Oxford University Press

Identification of proteins of Escherichia coli and Saccharomyces cerevisiae that specifically bind to C/C mismatches in DNA

Takehisa Nakahara, Qiu-Mei Zhang, Kazunari Hashiguchi and Shuji Yonei*

Department of Biological Sciences, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan

The pathways leading to G:C->C:G transversions and their repair mechanisms remain uncertain. C/C and G/G mismatches arising during DNA replication are a potential source of G:C->C:G transversions. The Escherichia coli mutHLS mismatch repair pathway efficiently corrects G/G mismatches, whereas C/C mismatches are a poor substrate. Escherichia coli must have a more specific repair pathway to correct C/C mismatches. In this study, we performed gel-shift assays to identify C/C mismatch-binding proteins in cell extracts of E.coli. By testing heteroduplex DNA (34mers) containing C/C mismatches, two specific band shifts were generated in the gels. The band shifts were due to mismatch-specific binding of proteins present in the extracts. Cell extracts of a mutant strain defective in MutM protein did not produce a low-mobility complex. Purified MutM protein bound efficiently to the C/C mismatch-containing heteroduplex to produce the low-mobility complex. The second protein, which produced a high-mobility complex with the C/C mismatches, was purified to homogeneity, and the amino acid sequence revealed that this protein was the FabA protein of E.coli. The high-mobility complex was not formed in cell extracts of a fabA mutant. From these results it is possible that MutM and FabA proteins are components of repair pathways for C/C mismatches in E.coli. Furthermore, we found that Saccharomyces cerevisiae OGG1 protein, a functional homolog of E.coli MutM protein, could specifically bind to the C/C mismatches in DNA.

* To whom correspondence should be addressed. Tel: +81 75 753 4097; Fax: +81 75 753 4087; Email: yonei@kingyo.zool.kyoto-u.ac.jp


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