Molecular Cloning and Functional Analysis of the MutY Homolog of Deinococcus radiodurans

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American Society for Microbiology

RESUMO

The mutY homolog gene (mutYDr) from Deinococcus radiodurans encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the Escherichia coli MutY (MutYEc) protein. Expressed MutYDr is able to complement E. coli mutY mutants but not mutM mutants to reduce the mutation frequency. The glycosylase and binding activities of MutYDr with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutYEc protein, purified recombinant MutYDr expressed in E. coli has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutYDr exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutYDr at the position corresponding to the position of MutYEc Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutYDr are similar to those of MutYEc. Although MutYDr has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutYEc does, the binding affinities for both mismatches are slightly lower for MutYDr than for MutYEc. Thus, MutYDr can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.

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