A bifunctional DNA repair protein from Ferroplasma acidarmanus exhibits O ⁶ -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 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. deamination ͉ genotoxicity ͉ alkylationA GT (O 6 -alkylguanine-DNA alkyltransferase) is a DNA repair protein that removes alkyl adducts from the O 6 -position of guanine and O 4 -position of thymine in DNA (1, 2). If they are not removed, these lesions are highly mutagenic and carcinogenic (1,3,4). AGTs repair these DNA adducts by transferring the alkyl group onto a cysteine amino acid residue at the active site of the protein, thus restoring the DNA to its unmodified form in a single step. This alkyl transfer reaction permanently inactivates the AGT, and synthesis of new protein is required for further repair of the DNA damage. The presence of a characterized AGT gene product or a putative gene coding for the protein has been reported so far in Ͼ100 different species from the three domains of life, Archaea, Bacteria, and Eukarya. However, it is not ubiquitous: it is apparently absent from plants, Schizosaccharomyces pombe, and Deinococcus radiodurans. The AGT proteins share a highly conserved -PCHRV-amino acid sequence in their active site and contain a number of other conserved amino acids in the active site and DNA-binding domain of the protein. The widespread presence of AGT indicates the occurrence of alkylation damage to DNA under a variety of living conditions and the need to repair such damage in the cell. Alkylating agents including N-nitroso compounds are present in the environment and can be generated endogenously.Crystal structures for AGT from three different species (Escherichia coli, human, and Pyrococcus kodakaraensis) have been deter...

Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon “Ferroplasma acidarmanus” Fer1

“…Similar observations were made for an unusual type of DNA repair protein from F. acidarmanus Fer1 (Kanugula et al 2005). These results suggest that at least some of the enzymes involved in DNA metabolism are optimized to function in the intracellular environment of this organism, which has been measured to be at pH 5.6 (Macalady et al 2004).…”

“…Perhaps more likely is that the differences are due to the studies of different types of enzymes and that this may indicate that different compartments within the organisms experience different pH (Golyshina et al 2006). In this case, the pH optima of enzymes acting on DNA are closest to neutral (Kanugula et al 2005), perhaps suggesting that the organisms have evolved mechanisms to protect their genome from harsh, acidic environments. A further explanation for the observations of different pH optima for proteins is that some of them may not be functioning at their optimal level within the cell.…”

“…Thus, the cytoplasmic conditions of these organisms are distinct from their environment, which is aided by the fact that their single cytoplasmic membrane has low permeability to protons (Golyshina et al 2006;Golyshina and Timmis 2005). It would, therefore, be expected that the proteins of these organisms have optimal activity close to the mildly-acidic cytoplasmic pH, as supported by characterization of a novel DNA repair protein encoded by F. acidarmanus Fer1 (Kanugula et al 2005). By contrast, a recent study identified that several intracellular or membrane-bound proteins of F. acidiphilum had optimal activity in the pH range 2-4 (Golyshina et al 2006).…”

Differential inactivation of polymorphic variants of human O6-alkylguanine-DNA alkyltransferase

“…2a and 2e, and summarized in Table 1, there was a small increase in the ED 50 for BG with W65C and I143V/K178R but no change from wild type and L84F. This was seen in assays conducted in the absence (Fig.…”

“…This alteration occurs in the S. cerevisiae and B. subtilis AGTs that are known to be active. However, it is well established that there are striking species differences in the ability of AGTs to repair more bulky adducts [2,49,50]. The I143V/ K178R hAGT was active in the repair of O 6 -n-butylguanine, O 6 -[4-oxo-4-(3-pyridyl)butyl] guanine or BG when these were contained in oligodeoxynucleotides [39].…”