Structural Basis for Enzymatic Excision of N1 Methyladenine and N3 Methylcytosine from DNA

N1 methyladenine m1A and N3 methylcytosine m3C are major toxic and mutagenic lesions induced by alkylation in single stranded DNA. In bacteria and mammals, m1A and m3C were recently shown to be repaired by AlkB mediated oxidative demethylation, a direct DNA damage reversal mechanism. No AlkB gene homologues have been identified in Archaea. We report that m1A and m3C are repaired by the AfAlkA base excision repair glycosylase of Archaeoglobus fulgidus, suggesting a different repair mechanism for these lesions in the third domain of life. In addition, AfAlkA was found to effect a robust excision of 1,N6 ethenoadenine. We present a high resolution crystal structure of AfAlkA, which, together with the characterization of several site directed mutants, forms a molecular rationalization for the newly discovered base excision activit

Uracil-DNA Glycosylase of Thermoplasma acidophilumDirects Long-Patch Base Excision Repair, Which Is Promoted by Deoxynucleoside Triphosphates and ATP/ADP, into Short-Patch Repair ▿

Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilumand the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5′-acting AP endonuclease and a 5′-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3′-acting AP lyase, followed by a 3′-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilumcell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ∼15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid single-nucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER