A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities

Sreenivas Kanugula, Gary T. Pauly, Robert C. Moschel, Anthony E. Pegg

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

A recently discovered DNA repair protein of 303 aa from the archaeal organism Ferroplasma acidarmanus was studied. This protein (AGTendoV) consists of a fusion of the C-terminal active site domain of O6-alkylguanine- DNA alkyltransferase (AGT) with an endonuclease V domain. The AGTendoV recombinant protein expressed in Escherichia coli and purified to homogeneity repaired O6-methylguanine lesions in DNA via alkyl transfer action despite the complete absence of the N-terminal domain and some differences in key active site residues present in known AGTs. The AGTendoV recombinant protein also cleaved DNA substrates that contained the deaminated bases uracil, hypoxanthine, or xanthine in a similar manner to E. coli endonuclease V. Expression of AGTendoV in E. coli GWR109, a strain that lacks endogenous AGT activity, protected against both the killing and mutagenic activity of N-methyl-N′-nitro-N-nitrosoguanidine and was more effective in preventing mutations than human alkyltransferase, suggesting that the endonuclease V activity may also repair a promutagenic lesion produced by this alkylating agent. Expression of AGTendoV in a DNA repair-deficient E. coli nfi -alkA- strain protected from spontaneous mutations arising in saturated cultures and restored the mutation frequency to that found in the nfi+ alkA+ strain. These results demonstrate the physiological occurrence of two completely different but functional DNA repair activities in a single polypeptide chain.

Original languageEnglish (US)
Pages (from-to)3617-3622
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number10
DOIs
StatePublished - Mar 8 2005

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Deoxyribonuclease (Pyrimidine Dimer)
Deoxyribonuclease I
DNA Repair
Escherichia coli
Recombinant Proteins
Catalytic Domain
Proteins
Alkyl and Aryl Transferases
DNA Repair-Deficiency Disorders
Methylnitronitrosoguanidine
Mutation
Hypoxanthine
Xanthine
Uracil
Alkylating Agents
DNA
Mutation Rate
Peptides
DNA alkyltransferase

All Science Journal Classification (ASJC) codes

  • General

Cite this

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title = "A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities",
abstract = "A recently discovered DNA repair protein of 303 aa from the archaeal organism Ferroplasma acidarmanus was studied. This protein (AGTendoV) consists of a fusion of the C-terminal active site domain of O6-alkylguanine- DNA alkyltransferase (AGT) with an endonuclease V domain. The AGTendoV recombinant protein expressed in Escherichia coli and purified to homogeneity repaired O6-methylguanine lesions in DNA via alkyl transfer action despite the complete absence of the N-terminal domain and some differences in key active site residues present in known AGTs. The AGTendoV recombinant protein also cleaved DNA substrates that contained the deaminated bases uracil, hypoxanthine, or xanthine in a similar manner to E. coli endonuclease V. Expression of AGTendoV in E. coli GWR109, a strain that lacks endogenous AGT activity, protected against both the killing and mutagenic activity of N-methyl-N′-nitro-N-nitrosoguanidine and was more effective in preventing mutations than human alkyltransferase, suggesting that the endonuclease V activity may also repair a promutagenic lesion produced by this alkylating agent. Expression of AGTendoV in a DNA repair-deficient E. coli nfi -alkA- strain protected from spontaneous mutations arising in saturated cultures and restored the mutation frequency to that found in the nfi+ alkA+ strain. These results demonstrate the physiological occurrence of two completely different but functional DNA repair activities in a single polypeptide chain.",
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A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities. / Kanugula, Sreenivas; Pauly, Gary T.; Moschel, Robert C.; Pegg, Anthony E.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 10, 08.03.2005, p. 3617-3622.

Research output: Contribution to journalArticle

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