Biochemical characterization and mutational studies of a novel 3-methlyadenine DNA glycosylase II from the hyperthermophilic Thermococcus gammatolerans

Abstract

The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes a putative 3-methlyadenine DNA glycosylase II (Tg-AlkA).Herein, we report biochemical characterization and catalytic mechanism of Tg-AlkA.The recombinant Tg-AlkA can excise hypoxanthine (Hx) and 1-methlyadenine (1-meA) from dsDNA with varied efficiencies at high temperature. Notably, Tg-AlkA is a bi-functional glycosylase, which is sharply distinct from all the reported AlkAs.Biochemical data show that the optimal temperature and pH of Tg-AlkA for removing Hx from dsDNA are ca.70°C and ca.7.0-8.0, respectively. Furthermore, the Tg-AlkA activity is independent of a divalent metal ion, and Mg 2+ stimulates the Tg-AlkA activity whereas other divalent ions inhibit the enzyme activity with varied degrees.Mutational studies show that the Tg-AlkA W204A and D223A mutants abolish completely the excision activity, thereby suggesting that residues W204 and D223 are involved in catalysis. Surprisingly, the mutations of W204, D223, Y139 and W256 to alanine in Tg-AlkA lead to the increased affinity for binding DNA substrate with varied degrees, suggesting that these residues are flexible for conformational change of the enzyme. Therefore, Tg-AlkA is a novel AlkA that can remove Hx and 1-meA from dsDNA, thus providing insights into repair of deaminated and alkylated bases in DNA from hyperthermophilic Thermococcus.

Repair of Hypoxanthine in DNA Revealed by DNA Glycosylases and Endonucleases From Hyperthermophilic Archaea

“…A number of structural and biochemical studies have revealed damaged base recognition and removal mechanisms of Escherichia coli AlkA ( Nakabeppu et al, 1984 ; Bjelland and Seeberg, 1996 ; Labahn et al, 1996 ; Yamagata et al, 1996 ; O’Brien and Ellenberger, 2004 ), thus enabling the enzyme to be a model alkylated DNA glycosylase. Currently, only two archaeal AlkA homologs have been reported from the hyperthermophilic euryarchaeon Archaeoglobus fulgidus and Thermococcus gammatolerans ( Figure 1 ; Birkeland et al, 2002 ; Mansfield et al, 2003 ; Leiros et al, 2007 ; Jiang et al, 2021 ). Thus, our understanding on biochemical function and catalytic mechanism of archaeal AlkA remains incomplete.…”

“…Biochemical data have demonstrated that A. fulgidus AlkA and T. gammatolerans AlkA are able to remove Hx from dsDNA ( Mansfield et al, 2003 ; Jiang et al, 2021 ). Although, the two protein hosts are hyperthermophiles, T. gammatolerans AlkA displays biochemical characteristics distinct from A. fulgidus AlkA.…”

“…Although, the two protein hosts are hyperthermophiles, T. gammatolerans AlkA displays biochemical characteristics distinct from A. fulgidus AlkA. Firstly, T. gammatolerans AlkA is able to excise Hx from dsDNA rather than from ssDNA ( Jiang et al, 2021 ), suggesting that the enzyme might be critical for repair of Hx in genomic DNA. By comparison, A. fulgidus AlkA can remove Hx from ssDNA ( Mansfield et al, 2003 ).…”

“…By comparison, A. fulgidus AlkA can remove Hx from ssDNA ( Mansfield et al, 2003 ). Additionally, T. gammatolerans AlkA is a bi-functional DNA glycosylase, harboring the activity for not only excising Hx from dsDNA but further cleaving the generated apurinic/apyrimidinic (AP) site ( Jiang et al, 2021 ), while A. fulgidus AlkA is monofunctional DNA glycosylase, only removing Hx from DNA ( Mansfield et al, 2003 ). Overall, effective excision of Hx from DNA by the two archaeal AlkA proteins might provide an alternative pathway for repair of Hx in DNA.…”

“…Analysis of amino acid sequences of AlkA homologs suggests that these AlkA proteins harbor a conserved helix-hairpin-helix (HhH)-GPD motif that is composed of an HhH motif for binding DNA and a glycine/proline-rich loop followed by an invariable Asp (GPD; Kuznetsov and Fedorova, 2020 ). Mutational analyses have demonstrated that the conserved Asp residue in the HhH-GPD motif serves as a catalytic residue in T. gammatolerans AlkA and A. fulgidus AlkA ( Mansfield et al, 2003 ; Jiang et al, 2021 ), which is also observed in Helicobacter pylori AlkA ( Eichman et al, 2003 ). Additionally, residue W204 in T. gammatolerans AlkA is another essential residue for catalysis since the replacement of residue W204 with alanine abolishes the enzyme activity ( Jiang et al, 2021 ).…”