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

Abstract

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 aerophilum and 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. acidophilum cell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ϳ15 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 singlenucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.In all cells, deamination of cytosine to uracil is only surpassed by depurination as the most common hydrolytic DNAdamaging event (38). While the apurinic/apyrimidinic (AP) site is primarily a cytotoxic lesion inhibiting replication or transcription, deaminated cytosines result in G ⅐ C to A ⅐ T transition mutations if they are not repaired before DNA synthesis. In addition, some dUTP escapes hydrolysis by dUTPase causing a certain amount of uracil introduced into DNA opposite adenine during replication (5, 32).Uracil is excised from DNA by a specific monofunctional uracil-DNA glycosylase (UDG) enzyme in virtually all organisms, including those living at the highest temperatures (22,33,47). After such enzyme-catalyzed, as well as spontaneous, base removal, a single nucleotide is reinserted by the sequential action of a 5Ј-acting AP endonuclease, 5Ј-deoxyribose phosphate (5Ј-dRP) lyase, DNA polymerase, and DNA ligase (22, 32), which constitute the short-patch mode of the base excision repair (BER) pathway. In eukaryotes, like mammals, the 5Ј-dRP lyase activity (35, 41) is a function of DNA polymerase ␤ (41, 62). BER is quantitatively the most important repair mechanism for the removal of spontaneously generated base modifications in DNA and is thus necessary to secure genomic integrity (38). The 5Ј-AP site remnant following strand incision blocks ligation, and when it is chemically modified in such a way that efficient removal by the 5Ј-dRP lyase is obstructed, polymerization will continue past one nucleotide and the downstream DNA strand is displaced. This long-patch mode of BER ...

The mesophilic archaeon Methanosarcina acetivorans counteracts uracil in DNA with multiple enzymes: EndoQ, ExoIII, and UDG

“…This high conservation implies a significant contribution of this protein in maintenance of the archaeal genome; however, this issue has been poorly investigated to date. Importantly, considering their high conservation, most of the archaeal organisms seem to conduct BER using EndoIV and family-4 UDGs, which is supported by previous studies 56 – 59 . We speculate that the archaeal BER pathway is regulated by the interplay of DNA glycosylases (UDG, MIG, AlkA, OGG1), AP lyase (EndoIII), and AP endonucleases (EndoIV, ExoIII).…”

Molecular Basis of Substrate Recognition of Endonuclease Q from the Euryarchaeon Pyrococcus furiosus

“…Except for some orders in Euryarchaeota, almost all archaea have family 4 UDGs (18, 22). Thus far, several family 4 UDGs from bacteria and archaea have been investigated (22,(26)(27)(28)(29)(30)(31); however, their substrate specificity range has not been fully examined. We examined the substrate specificity of family 4 UDG from the closely related T. kodakarensis.…”

Transfer RNA Methyltransferases from Thermoplasma acidophilum, a Thermoacidophilic Archaeon

“…Furthermore, lipoylation of proteins [ 5 ], biosynthesis of lipids [ 6 ], cell surface glycoproteins [ 7 ] and a channel protein [ 8 ] have been also studied in T. acidophilum . Given that a prokaryotic histone-like DNA binding protein was discovered first from T. acidophilum [ 9 , 10 ], the bacterium has been used as a model system to investigate DNA replication, DNA repair, and transcriptional initiation in archaea [ 11 , 12 , 13 , 14 , 15 , 16 ]. Furthermore, the energy metabolism of T. acidophilum has been studied in detail because it can grow under an extreme microaerophilic environment [ 17 , 18 ].…”