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

摘要

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.