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

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.

DNA Repair by Reversal of DNA Damage

“…ATLs tightly bind ss/ dsDNA that contain O 6 -alkyl guanine, but they display no alkyltransferase, demethylase, glycosylase, or endonuclease activity (Pearson et al 2005(Pearson et al , 2006Chen et al 2008;Morita et al 2008). In fact, preincubation of alkylated oligonucleotides inhibits the repair activity of hAGT (Pearson et al 2005(Pearson et al , 2006).…”

“…In fact, preincubation of alkylated oligonucleotides inhibits the repair activity of hAGT (Pearson et al 2005(Pearson et al , 2006). Yet it was clear that ATL must be playing a role in alkylation damage protection in vivo, because S. pombe and Thermus thermophilus were rendered more sensitive to alkylating reagents on inactivation of their ATL genes (Pearson et al 2006;Morita et al 2008). Through a series of genetic and biochemical experiments, ATL was discovered to be linked to the nucleotide excision repair (NER) pathway (Mazon et al 2009;Tubbs et al 2009).…”

Activity and Regulation of Archaeal DNA Alkyltransferase

“…the AGTlike protein of Thermus thermophilus (12)) or poorly characterized because they were not soluble when heterologously expressed (e.g. the AGTs from the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus (13)).…”

Alkyltransferase-like protein (Atl1) distinguishes alkylated guanines for DNA repair using cation–π interactions

“…Comparable values were obtained using fluorescence anisotropy. Because native TTHA1564 is unstable and prone to aggregation in vitro, its MBP fusion protein (19) was used. In a control experiment MBP had no discernable binding interaction with a 5′-SIMA(HEX)-labeled O 6 -methylguanine (O 6 -MeG)-containing ODN.…”

“…3, 11, and 12) are highly homologous to AGTs but have a different amino acid (typically Trp or Ala) in place of the nucleophilic active site Cys. ATL proteins retain the ability to bind single-stranded or duplex DNA containing O 6 -alkylguanine, and although unable to undertake the de-alkylative repair reaction, they protect against the adverse effects of DNA alkylation damage by downstream recruitment of nucleotide excision repair (NER) (13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”