Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OHdATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase η, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.

Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80 degrees C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3-4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP.

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/ RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the ␤ subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.The recently identified novel family of DNA polymerases (pol) 1 (DinB/UmuC/Rev1/Rad30 superfamily or family Y) comprises proteins from different species including Bacteria, Eukarya, and Archaea (1, 2). Members of this superfamily can bypass lesions on template DNA, such as ultraviolet light photoproducts, and mismatched primer/template DNA (3-7). Thus, the new DNA polymerases seem to function by assisting the conventional DNA replicases to cope with faulty DNA templates by taking over the DNA synthesis at aberrant sites (for recent reviews, see Refs. 1 and 8 -12). Common features of family Y DNA polymerases include the lack of 3Ј-exonuclease proofreading activity and synthesis of DNA with low fidelity in a distributive fashion. The human DNA pol has been shown recently to possess 5Ј-deoxyribose phosphate lyase activity, suggesting a role in base excision repair (13). It has been hypothesized that the very low processivity of these polymerases prevents excessive introduction of mutations because of extended error-prone DNA synthesis. This has been, however, questioned recently when DNA polymerase IV of Escherichia coli (pol IV) (7) was found to synthesize tracks of more than 300 nucleotides upon interaction with the ␤ subunit of pol III (14). The ␤ subunit and its eukaryotic counterpart, i.e. PCNA, play essential roles in processive chromosomal replication by forming a sliding platform that mediates the interaction of DNA polymerases with DNA (15). In addition, the sliding clamps also interact with a variety of proteins other than DNA polymerases involved in DNA processing and even in cell cycle control (16). It is therefore of general interest to elucidate the relationships between various members of Y family DNA polymerases and the corresponding sliding clamps.Archaea, the third domain of life, is t...

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.