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

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 β-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·C-to-A·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 5′-acting AP endonuclease, 5′-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 rather than the lesion itself and thus are inactive with ssDNA. Family 3 UDGs, typified by the SMUG1 enzyme of human cells, have similar substrate requirements but exhibit a stronger preference for uracil in ssDNA than family 1 enzymes (17, 27, 57, 67).UDG activity in hyperthermophilic microorganisms was first reported in 1996 (33). Three years later, Sandigursky and Franklin (47) cloned and overexpressed an open reading frame (ORF) of the hyperthermophilic bacterium Thermotoga maritima that typifies the family 4 UDGs that are able to remove uracil from U·G and U·A base pairs, as well as from ssDNA. By means of homology searches, these workers found ORFs homologous to the T. maritima UDG gene in several prokaryotic genomes, including that of the hyperthermophilic archaeon Archaeoglobus fulgidus, a strict anaerobe that grows optimally at 83°C (60; for a review of DNA repair in hyperthermophilic archaea, see reference 20). Subsequently, they cloned and overexpressed the A. fulgidus ORF in E. coli by producing a His-tagged fusion protein. As expected, the purified A. fulgidus recombinant Afung (rAfung) protein exhibited UDG activity (48). However, whether Afung is the major UDG of A. fulgidus or is just a minor glycosylase with uracil-releasing ability remained to be determined.As a continuation of previous biochemical and physicochemical studies (31) of non-His-tagged rAfung protein, here we characterized a family 4 UDG in archaeon cell extract. The abundance of Afung in vivo was determined, and evidence indicates that this enzyme is the principal UDG of A. fulgidus. Here we also describe the mechanism of dUMP repair employed by this euryarchaeon, which differs in important ways from the mechanism reported for the crenarchaeon Pyrobaculum aerophilum (50).