A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities (original) (raw)
Related papers
Journal of Biological Chemistry, 2010
Alkyltransferase-like proteins (ATLs) are a novel class of DNA repair proteins related to O 6 -alkylguanine-DNA alkyltransferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair pathway. Here, we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus (vpAtl). We demonstrate that vpAtl adopts an AGT-like fold and that the protein is capable of tightly binding to O 6 -methylguanine-containing DNA and disrupting its repair by human AGT, a hallmark of ATLs. Mutation of highly conserved residues Tyr 23 and Arg 37 demonstrate their critical roles in a conserved mechanism of ATL binding to alkylated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair. . 3 The abbreviations used are: AGT, O 6 -alkylguanine-DNA alkyltransferase;
Repair of O4-Alkylthymine by O6-Alkylguanine-DNA Alkyltransferases
Journal of Biological Chemistry, 2010
O 6 -Alkylguanine-DNA alkyltransferase (AGT) plays a major role in repair of the cytotoxic and mutagenic lesion O 6 -methylguanine (m 6 G) in DNA. Unlike the Escherichia coli alkyltransferase Ogt that also repairs O 4 -methylthymine (m 4 T) efficiently, the human AGT (hAGT) acts poorly on m 4 T. Here we made several hAGT mutants in which residues near the cysteine acceptor site were replaced by corresponding residues from Ogt to investigate the basis for the inefficiency of hAGT in repair of m 4 T. Construct hAGT-03 (where hAGT sequence -V 149 CSSGAVGN 157 -was replaced with the corresponding Ogt -I 143 GRNGTMTG 151 -) exhibited enhanced m 4 T repair activity in vitro compared with hAGT. Three AGT proteins (hAGT, hAGT-03, and Ogt) exhibited similar protection from killing by
The EMBO Journal, 1988
Communicated by J.Cairns The in vivo removal of three different 0-alkylated bases from DNA was measured in Escherichia coli. Using monoclonal antibodies specific for 06-methylguanine, 06-ethylguanine and 04-ethylthymine we have monitored the removal of these lesions from six different strains to assess the relative contributions of the adaptive response and of nucleotide excision repair. During the first hour after DNA alkylation, Ob-methylguanine, 06-ethylguanine and 04-ethylthymine lesions were repaired almost exclusively by nucleotide excision, except when the adaptive response was being constitutively expressed. In wild-type E.coli the adaptive response began to contribute to 06-methylguanine repair about one hour after alkylation, the time required for the full induction of the ada DNA methyltransferase. In contrast, the adaptive response did not play such a large role in the repair of 06-ethylguanine and 04-ethylthymine in wild-type E.coli, presumably because DNA ethylation damage is a poor inducer of the adaptive response; possible reasons for this poor induction are discussed. The repair of all three 0-alkylated lesions was virtually absent in adauvr-bacteria suggesting that no alternative pathway is available for their repair, at least during the first two hours after alkylation. When the repair of 0-alkylated bases was compromised by an adaor by a uvr-mutation, the bacteria became more sensitive to alkylation induced killing and mutation.
Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes
Proceedings of the National Academy of Sciences, 1986
The ada-alkB operon protects Escherichia coli against the effects of many alkylating agents. We have subcloned it into the pSV2 mammalian expression vector to yield pSV2ada-alkB, and this plasmid has been introduced into Mer-HeLa S3 cells, which are extremely sensitive to killing and induction of sister chromatid exchange by alkylating agents. One transformant (the S3-9 cell line) has several integrated copies of pSV2ada-alkB and was found to express a very high level of the ada gene product, the 39-kDa 06. methylguanine-DNA methyltransferase. S3-9 cells were found to have become resistant to killing and induction of sister chromatid exchange by two alkylating agents, N-methyl-N'nitro-N-nitrosoguanidine and N,N'-bis(2-chloroethyl)-N-nitrosourea. This shows that bacterial DNA alkylation-repair genes are able to suppress the alkylation-repair defects in human Mer-cells.
Ada response - a strategy for repair of alkylated DNA in bacteria
FEMS Microbiology Letters, 2014
Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N 3 -methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N 1 -methyladenine (1meA) and N 3 -methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O 6 -methylguanine (O 6 meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA.
DNA Repair, 2009
a b s t r a c t O 6 -methylguanine adducts are potent pre-mutagenic lesions owing to their high capacity to direct misinsertion of thymine when bypassed by replicative DNA polymerases. The strong mutagenic potential of these adducts is prevented by alkyltransferases such as Ada and Ogt in Escherichia coli that transfer the methyl group to one of their cysteine residues. Alkyl residues larger than methyl are generally weak substrates for reversion by alkyltransferases. In this paper we have investigated the genotoxic potential of the O 6 -alkylguanine adducts formed by ethylene and propylene oxide using single-adducted plasmid probes. Our work shows that the ybaZ gene product, a member of the alkyltransferase-like protein family, strongly enhances the repair by nucleotide excision repair of the larger O 6 -alkylguanine adducts that are otherwise poor substrates for alkyltransferases. The YbaZ protein is shown to interact with UvrA. This factor may thus enhance the efficiency of nucleotide excision repair in a way similar to the Transcription-Repair Coupling factor Mfd, by recruiting the UvrA 2 ·UvrB complex to the adduct site via its interaction with UvrA.
Dna Repair, 2009
O 6-methylguanine adducts are potent pre-mutagenic lesions owing to their high capacity to direct misinsertion of thymine when bypassed by replicative DNA polymerases. The strong mutagenic potential of these adducts is prevented by alkyltransferases such as Ada and Ogt in Escherichia coli that transfer the methyl group to one of their cysteine residues. Alkyl residues larger than methyl are generally weak substrates for reversion by alkyltransferases. In this paper we have investigated the genotoxic potential of the O 6-alkylguanine adducts formed by ethylene and propylene oxide using single-adducted plasmid probes. Our work shows that the ybaZ gene product, a member of the alkyltransferase-like protein family, strongly enhances the repair by nucleotide excision repair of the larger O 6-alkylguanine adducts that are otherwise poor substrates for alkyltransferases. The YbaZ protein is shown to interact with UvrA. This factor may thus enhance the efficiency of nucleotide excision repair in a way similar to the Transcription-Repair Coupling factor Mfd, by recruiting the UvrA 2 •UvrB complex to the adduct site via its interaction with UvrA.
Mutagenesis, 1998
In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increase in mutation induction by iV-butyl-iVnitrosourea (BNU). Irrespective of the presence or absence of the Ogt ATase, little mutagenic response was detected in Uvr + bacteria in the concentration range 0-8 mM BNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system. Increased susceptibility to mutagenesis by BNU was detected in Uvr~ Ogt + bacteria, but the Uvr~ Ogt" double mutant exhibited much higher sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM BNU within the initial part of the lacl gene of E.coli were recovered from Uvr + Ogt~, Uvr" Ogt+ and Uvr~ Ogtb acteria. A total of 454 independent mutations were characterized by DNA sequence analysis. The BNU-induced spectra were dominated by G:C-»A:T transitions, consistent with the major role of the 0*-alkylguanine miscoding lesion in mutagenesis by alkylating agents. Specific sites for G:C-»A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10"" 6 ). We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base associated with the BNU-induced G:C->A:T transitions; preferences different from those previously reported for other alkylnitrosoureas were detected. We discuss how these differences might be caused by BNU producing branched chain derivatives, in addition to the expected linear chain adducts, and by possible preferences with respect to both the initial distribution of O'-butylguanine lesions and their repairability.
Mutation research, 2000
O(6)-alkylguanine-DNA alkyltransferase (AGT) directly repairs alkylation damage at the O(6)-position of guanine in a unique, stoichiometric reaction. Crystal structures of AGT homologs from the three kingdoms of life reveal that despite their extremely low primary sequence homology, the topology and overall structure of AGT has been remarkably conserved. The C-terminal domain of the two-domain, alpha/beta fold bears a helix-turn-helix (HTH) motif that has been implicated in DNA-binding by structural and mutagenic studies. In the second helix of the HTH, the recognition helix, lies a conserved RAV[A/G] motif, whose "arginine finger" promotes flipping of the target nucleotide from the base stack. Recognition of the extrahelical guanine is likely predominantly through interactions with the protein backbone, while hydrophobic sidechains line the alkyl-binding pocket, as defined by product complexes of human AGT. The irreversible dealkylation reaction is accomplished by an acti...