Inhibition of O6-methylguanine-DNA methyltransferase by an alkyltransferase-like protein from Escherichia coli

Abstract

The alkyltransferase-like (ATL) proteins contain primary sequence motifs resembling those found in DNA repair O6-alkylguanine-DNA alkyltransferase proteins. However, in the putative active site of ATL proteins, a tryptophan (W83) residue replaces the cysteine at the known active site of alkyltransferases. The Escherichia coli atl gene was expressed as a fusion protein and purified. Neither ATL nor C83 or A83 mutants transferred [3H] from [3H]-methylated DNA to themselves, and the levels of O6-methyl guanine (O6-meG) in substrate DNA were not affected by ATL. However, ATL inhibited the transfer of methyl groups to human alkyltransferase (MGMT). Inhibition was reduced by prolonged incubation in the presence of MGMT, again suggesting that O6-meG in the substrate is not changed by ATL. Gel-shift assays show that ATL binds to short single- or double-stranded oligonucleotides containing O6-meG, but not to oligonucleotides containing 8-oxoguanine, ethenoadenine, 5-hydroxycytosine or O4-methylthymine. There was no evidence of demethylation of O6-meG or of glycosylase or endonuclease activity. Overexpression of ATL in E.coli increased, or did not affect, the toxicity of N-methyl-N′-nitro-N-nitrosoguanidine in an alklyltransferase-proficient and -deficient strain, respectively. These results suggest that ATL may act as a damage sensor that flags O6-meG and possibly other O6-alkylation lesions for processing by other 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).…”

“…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). 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).…”

Structural Basis of O6-Alkylguanine Recognition by a Bacterial Alkyltransferase-like DNA Repair Protein

“…ATLs tightly bind a wide range of O 6 -alkylguanine adducts and block the repair of O 6 -mG by human AGT but exhibit no alkyltransferase, glycosylase, or endonuclease activities (7)(8)(9). The recent first structural study of an ATL (10), from the fission yeast Schizosaccharomyces pombe (spAtl1) which lacks an AGT, provides strong evidence for a novel mechanism of DNA repair in which an ATL binds alkylated DNA in a manner analogous to AGTs, and the resulting nonenzymatic ATL⅐DNA complex triggers the NER pathway (10,11).…”

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O 6 -alkylguanine adducts in Escherichia coli

“…17), stimulates the repair by NER of O 6 -alkylguanine adducts (16). Whereas eATL binds to O 6 -alkylguanine adducts, it is devoid of any alkyltransferase activity (18,19) and thus, unlike Ada or Ogt, does not act as a repair factor per se. eATL merely promotes repair via an "enhanced NER" pathway by facilitating the recruitment of the NER factors to the O 6 -alkylguanine lesion sites that are otherwise poor substrates (16), akin to the stimulation of photoproduct excision by the binding of photolyase in the absence of light (20).…”