The Hyperthermophilic Euryarchaeon Archaeoglobus fulgidus Repairs Uracil by Single-Nucleotide Replacement

Abstract

Hydrolytic deamination of cytosine to uracil in cellular DNA is a major source of C-to-T transition mutations if uracil is not repaired by the DNA base excision repair (BER) pathway. Since deamination increases rapidly with temperature, hyperthermophiles, in particular, are expected to succumb to such damage. There has been only one report of crenarchaeotic BER showing strong similarities to that in most eukaryotes and bacteria for hyperthermophilic Archaea. Here we report a different type of BER performed by extract prepared from cells of the euryarchaeon Archaeoglobus fulgidus. Although immunodepletion showed that the monofunctional family 4 type of uracil-DNA glycosylase (UDG) is the principal and probably only UDG in this organism, a ␤-elimination mechanism rather than a hydrolytic mechanism is employed for incision of the abasic site following uracil removal. The resulting 3 remnant is removed by efficient 3-phosphodiesterase activity followed by single-nucleotide insertion and ligation. The finding that repair product formation is stimulated similarly by ATP and ADP in vitro raises the question of whether ADP is more important in vivo because of its higher heat stability.After depurination, hydrolytic deamination of cytosine to uracil is the most frequent event that damages DNA (36), and it results in G ⅐ C-to-A ⅐ T transition mutations if the damage is not repaired. In addition, some dUTP molecules escape hydrolysis by dUTPase, which results in a certain amount of dUMP introduced into DNA opposite adenine during replication (32). Irrespective of the mode of appearance, all cells contain uracil-DNA glycosylase (UDG) (EC 3.2.2.3) enzymes to remove uracil from DNA (17). The resulting abasic or apurinic/apyrimidinic (AP) site can subsequently be removed, and the integrity of the DNA can be restored by the so-called base excision repair (BER) pathway, which consists in its simplest form of the sequential actions of 5Ј-acting AP endonuclease, 5Ј-deoxyribose phosphate (dRP) lyase, DNA polymerase, and DNA ligase. The BER pathway can be initiated by one of several DNA glycosylases with different substrate specificities (17,36,57), and quantitatively it is the most important repair mechanism for the removal of spontaneously generated base modifications. Genes encoding bacterial and eukaryotic UDGs exhibiting significant selectivity for uracil have been cloned and sequenced in the last 2 decades, and the results have demonstrated that there is a high degree of conservation between distantly related species. Family 1 UDGs (for a review of UDG families 1 to 3, see reference 44), typified by the Escherichia coli Ung enzyme (37), recognize uracil in an extrahelical or flipped-out conformation in double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). Several family 1 enzymes have been extensively characterized, both structurally and at the cell and organism levels. Family 2 UDGs, which includes E. coli Mug and mammalian thymine-DNA glycosylase, are mismatch specific and recognize guanine on the complementary strand...

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

“…Only single-nucleotide insertion or short-patch BER has been reported for P. aerophilum (54) and A. fulgidus (30). The results presented here suggest that this subpathway is the most important mode of BER in T. acidophilum as well (Fig.…”

“…For instance, P. aerophilum contains one family 4, one family 5, and one MIG enzyme (53,55,76), while A. fulgidus contains only one UDG of family 4, Afung (30). Similar to the latter organism, only one gene coding for a putative UDG, of family 5, is present in the T. acidophilum genome (51).…”

“…4, step 2a), followed by 3Ј-phosphodiesterase activity (Fig. 4, step 3a) (30), implying that the latter is more important than the 5Ј-dRP lyase activity (Fig. 4, step 3b) to prepare for the reconstruction steps of BER.…”

“…Reasonably, BER of uracil in DNA has been studied on a variety of organisms representing the three domains of life. However, BER has thus far only been studied in two archaeons; the aerobic crenarchaeon P. aerophilum (54) and the anaerobic euryarchaeon A. fulgidus (30). To pursue the investigation on archaeal BER, T. acidophilum became our organism of choice.…”

“…Thus far, only two hyperthermophilic archaeons, the aerobic crenarchaeon Pyrobaculum aerophilum (54) and the anaerobic euryarchaeon Archaeoglobus fulgidus (30), have been investigated. Whereas P. aerophilum accomplishes uracil replacement by the common mode as outlined above, A. fulgidus prefers to use a 3Ј-acting AP lyase, followed by a 3Ј-phosphodiesterase, to excise the AP site intermediate, although both organisms use the short-patch repair subpathway.…”