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Journal of Biochemistry Advance Access originally published online on May 15, 2008
Journal of Biochemistry 2008 144(2):267-277; doi:10.1093/jb/mvn065
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© 2008 The Japanese Biochemical Society

An O6-methylguanine-DNA Methyltransferase-like Protein from Thermus thermophilus Interacts with a Nucleotide Excision Repair Protein

Rihito Morita1, Noriko Nakagawa1,2, Seiki Kuramitsu1,2 and Ryoji Masui1,2,*

1Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043; and 2RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan

*To whom correspondence should be addressed. Tel: +81-6-6850-5434, Fax: +81-6-6850-5442, E-mail: rmasui{at}bio.sci.osaka-u.ac.jp

Received March 6, 2008; Accepted May 2, 2008


  Abstract

The major damage to DNA caused by alkylating agents involves the formation of O6-methylguanine (O6-meG). Almost all species possess O6-methylguanine-DNA-methyltransferase (Ogt) to repair such damage. Ogt repairs O6-meG lesions in DNA by stoichiometric transfer of the methyl group to a cysteine residue in its active site (PCHR). Thermus thermophilus HB8 has an Ogt homologue, TTHA1564, but in this case an alanine residue replaces cysteine in the putative active site. To reveal the possible function of TTHA1564 in processing O6-meG-containing DNA, we characterized the biochemical properties of TTHA1564. No methyltransferase activity for synthetic O6-meG-containing DNA could be detected, indicating TTHA1564 is an alkyltransferase-like protein. Nevertheless, gel shift assays showed that TTHA1564 can bind to DNA containing O6-meG with higher affinity (9-fold) than normal (unmethylated) DNA. Experiments using a fluorescent oligonucleotide suggested that TTHA1564 recognizes O6-meG in DNA using the same mechanism as other Ogts. We then investigated whether TTHA1564 functions as a damage sensor. Pull-down assays identified 20 proteins, including a nucleotide excision repair protein UvrA, which interacts with TTHA1564. Interaction of TTHA1564 with UvrA was confirmed using a surface plasmon resonance assay. These results suggest the possible involvement of TTHA1564 in DNA repair pathways.

Key Words: DNA repair, methyltransferase, nucleotide excision repair, O6-methylguanine, Ogt

Abbreviations: 2AP, 2-aminopurine; AGT, O6-alkylguanine-DNA alkyltransferase; ATL protein, alkyltransferase-like protein; dsDNA, double-stranded DNA; DTT, dithiothreitol; hMGMT, MGMT from Homo sapiens; MBP, maltose-binding protein; MGMT, O6-methylguanine-DNA methyltransferase; MJOgt, Ogt from Methanocaldococcus jannaschii; NER, nucleotide excision repair; O6-meG, O6-methylguanine; Ogt, O6-methylguanine-DNA methyltransferase; ssDNA, single-stranded DNA; TRCF, transcription-repair coupling factor; TTHB8, Thermus thermophilus HB8; WT, wild-type


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