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. 2005 Mar 8;102(10):3617-22.
doi: 10.1073/pnas.0408719102. Epub 2005 Feb 24.

"V体育安卓版" A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities

Sreenivas Kanugula  1 , Gary T Pauly, Robert C Moschel, Anthony E Pegg
Affiliations

"V体育安卓版" A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O6-alkylguanine-DNA alkyltransferase and endonuclease V activities

Sreenivas Kanugula et al. Proc Natl Acad Sci U S A. .

Abstract

A recently discovered DNA repair protein of 303 aa from the archaeal organism Ferroplasma acidarmanus was studied VSports手机版. This protein (AGTendoV) consists of a fusion of the C-terminal active site domain of O(6)-alkylguanine-DNA alkyltransferase (AGT) with an endonuclease V domain. The AGTendoV recombinant protein expressed in Escherichia coli and purified to homogeneity repaired O(6)-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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Figures

Fig. 1.
Fig. 1.
Amino acid sequence and alignment of F. acidarmanus AGTendoV. The F. acidarmanus AGTendoV sequence (Fa AGTendoV) is aligned with portions of the AGT sequences from E. coli Ada (Ec Ada-C) and human (Hs AGT) and with endonuclease V sequences from E. coli (Ec EndoV) and mouse (mouse EndoV). Key residues that are identical (or highly conservative changes) are shown in red for the AGT domain and blue for the endonuclease V domain. The active site Cys in AGT and the amino acid residues in endonuclease V that are involved in incision activity are indicated by asterisks above the amino acids.
Fig. 2.
Fig. 2.
AGT assay of purified AGTendoV. (A) The effect of temperature. (B) The effect of pH. (C) The inactivation by BG.
Fig. 3.
Fig. 3.
Protection of cells from killing and mutagenic effects of MNNG by AGTendoV. GWR 109 cells with no AGT (empty vector; circles) or expressing either AGTendoV (triangles) or human AGT (squares) were treated with MNNG, and survival was determined (A). (A Inset) The level of expression of the human AGT and AGTendoV determined by Western blotting using Penta-His antibody, which is specific for a (His)5 epitope. Purified AGTendoV (50 ng) was loaded in lane 1 and purified hAGT (50 ng) was loaded in lane 2 as controls. Lanes 3–5 show extracts from GWR109/pQE-30 cells, GWR109/pQE-Fa AGTendoV cells and GWR109/pQE-hAGT cells, respectively. (B) The occurrence of Rifr mutants. The values are shown above each bar. (Bars for cells expressing AGTendoV are too small to show up well above the x axis.) In the absence of MNNG, there were fewer than five mutants per 108 cells. Results are means ± SD for three separate experiments.
Fig. 4.
Fig. 4.
Endonuclease V activity of AGTendoV. (A) Substrate oligonucleotide sequence (asterisk indicates uracil, hypoxanthine, or xanthine) and predicted reaction products. (B) Activity was assessed on double-stranded oligonucleotide substrates containing uracil (U), hypoxanthine (H), xanthine (X), or O6-methylguanine (m6G) at pH 7.6. The purified AGTendoV was incubated with 5′ end 32P-labeled double stranded-oligonucleotide at 46°C for 15 min in 20 mM Tris·HCl (pH 7.6) 1 mM DTT, and 5 mM MgCl2 buffer. The reaction products were separated on a denaturing polyacrylamide gel as described in Materials and Methods. Single-stranded oligonucleotides of the predicted reaction product (14-mers) containing U or H were also loaded on the gel as markers.
Fig. 5.
Fig. 5.
Suppression of spontaneous mutations in endonuclease V-deficient E. coli strain by FaAGTendoV protein. The E. coli MG1655 wild-type (nfi+alkA+), DNA repair-deficient (nfi-alkA-), and nfi-alkA- deficient cells transformed with pQE-FaAGTendoV plasmid were plated in the presence and absence of 100 μg/ml rifampicin. These results were obtained from four independent experiments and are shown as the mean ± SD.
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