A novel DNA damage recognition protein in Schizosaccharomyces pombe

Abstract

Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N′nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6-methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.

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

“…1A), all of which are reported to be repaired less efficiently by AGT proteins (6-10). This result, along with the observation that the AGT proteins Ogt and Ada from Escherichia coli are even less capable than the human protein of repair of large O 6 -alkylguanine lesions (38-40) is consistent with ATLs having a much broader substrate specificity than AGT proteins (16,17), as would be required to highlight subsequent processing by NER.…”

“…The MBP-Atl1 fusion protein was cleaved with Factor Xa (NEB) and purified over a Superdex 200 column (HiLoad 16/60; GE Healthcare). To introduce Arg69 to Phe and Ala point mutations into the atl1 gene of pMAL-2c-atl1 vector (17), the Phusion site-directed mutagenesis kit was used (Finnzymes, NEB) and the constructs verified by sequencing. E. coli clones harboring the pMAL-2c-atl1-R69F and pMAL-2c-atl1-R69A constructs were grown to OD 260 of ∼0.6, induced for 3 h by the addition of isopropyl-β-thiogalactoside and the protein purified as above.…”

“…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).…”

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).…”

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

“…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). Similarly, the Atl1 gene in Schizosaccharomyces pombe stimulates alkylating agent repair by the NER pathway (21,22).…”