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

2.50
Hdl Handle:
http://hdl.handle.net/10541/74819
Title:
Inhibition of O6-methylguanine-DNA methyltransferase by an alkyltransferase-like protein from Escherichia coli.
Authors:
Pearson, Steven J; Ferguson, Jennifer; Santibanez-Koref, Mauro F; Margison, Geoffrey P
Abstract:
The alkyltransferase-like (ATL) proteins contain primary sequence motifs resembling those found in DNA repair O(6)-alkylguanine-DNA alkyltransferase proteins. However, in the putative active site of ATL proteins, a tryptophan (W(83)) 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 C(83) or A(83) mutants transferred [(3)H] from [(3)H]-methylated DNA to themselves, and the levels of O(6)-methyl guanine (O(6)-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 O(6)-meG in the substrate is not changed by ATL. Gel-shift assays show that ATL binds to short single- or double-stranded oligonucleotides containing O(6)-meG, but not to oligonucleotides containing 8-oxoguanine, ethenoadenine, 5-hydroxycytosine or O(4)-methylthymine. There was no evidence of demethylation of O(6)-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 O(6)-meG and possibly other O(6)-alkylation lesions for processing by other repair pathways.
Affiliation:
Cancer Research-UK Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust Wilmslow Road, Manchester, UK.
Citation:
Inhibition of O6-methylguanine-DNA methyltransferase by an alkyltransferase-like protein from Escherichia coli. 2005, 33 (12):3837-44 Nucleic Acids Res.
Journal:
Nucleic Acids Research
Issue Date:
2005
URI:
http://hdl.handle.net/10541/74819
DOI:
10.1093/nar/gki696
PubMed ID:
16027108
Type:
Article
Language:
en
ISSN:
1362-4962
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorPearson, Steven J-
dc.contributor.authorFerguson, Jennifer-
dc.contributor.authorSantibanez-Koref, Mauro F-
dc.contributor.authorMargison, Geoffrey P-
dc.date.accessioned2009-07-21T16:41:11Z-
dc.date.available2009-07-21T16:41:11Z-
dc.date.issued2005-
dc.identifier.citationInhibition of O6-methylguanine-DNA methyltransferase by an alkyltransferase-like protein from Escherichia coli. 2005, 33 (12):3837-44 Nucleic Acids Res.en
dc.identifier.issn1362-4962-
dc.identifier.pmid16027108-
dc.identifier.doi10.1093/nar/gki696-
dc.identifier.urihttp://hdl.handle.net/10541/74819-
dc.description.abstractThe alkyltransferase-like (ATL) proteins contain primary sequence motifs resembling those found in DNA repair O(6)-alkylguanine-DNA alkyltransferase proteins. However, in the putative active site of ATL proteins, a tryptophan (W(83)) 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 C(83) or A(83) mutants transferred [(3)H] from [(3)H]-methylated DNA to themselves, and the levels of O(6)-methyl guanine (O(6)-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 O(6)-meG in the substrate is not changed by ATL. Gel-shift assays show that ATL binds to short single- or double-stranded oligonucleotides containing O(6)-meG, but not to oligonucleotides containing 8-oxoguanine, ethenoadenine, 5-hydroxycytosine or O(4)-methylthymine. There was no evidence of demethylation of O(6)-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 O(6)-meG and possibly other O(6)-alkylation lesions for processing by other repair pathways.en
dc.language.isoenen
dc.subject.meshAlkyl and Aryl Transferases-
dc.subject.meshDNA-
dc.subject.meshDNA Glycosylases-
dc.subject.meshEndonucleases-
dc.subject.meshEscherichia coli-
dc.subject.meshEscherichia coli Proteins-
dc.subject.meshGuanine-
dc.subject.meshMethylnitronitrosoguanidine-
dc.subject.meshMutation-
dc.subject.meshO(6)-Methylguanine-DNA Methyltransferase-
dc.titleInhibition of O6-methylguanine-DNA methyltransferase by an alkyltransferase-like protein from Escherichia coli.en
dc.typeArticleen
dc.contributor.departmentCancer Research-UK Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust Wilmslow Road, Manchester, UK.en
dc.identifier.journalNucleic Acids Researchen

Related articles on PubMed

All Items in Christie are protected by copyright, with all rights reserved, unless otherwise indicated.