The hyperthermophilic crenarchaeon Sulfolobus solfataricus P2 encodes three B-family DNA polymerase genes, B1 (Dpo1), B2 (Dpo2), and B3 (Dpo3), and one Y-family DNA polymerase gene, Dpo4, which are related to eukaryotic counterparts. Both mRNAs and proteins of all four DNA polymerases were constitutively expressed in all growth phases. Dpo2 and Dpo3 possessed very low DNA polymerase and 3 to 5 exonuclease activities in vitro. Steady-state kinetic efficiencies (k cat /K m ) for correct nucleotide insertion by Dpo2 and Dpo3 were several orders of magnitude less than Dpo1 and Dpo4. Both the accessory proteins proliferating cell nuclear antigen and the clamp loader replication factor C facilitated DNA synthesis with Dpo3, as with Dpo1 and Dpo4, but very weakly with Dpo2. DNA synthesis by Dpo2 and Dpo3 was remarkably decreased by singlestranded binding protein, in contrast to Dpo1 and Dpo4. DNA synthesis in the presence of proliferating cell nuclear antigen, replication factor C, and single-stranded binding protein was most processive with Dpo1, whereas DNA lesion bypass was most effective with Dpo4. Both Dpo2 and Dpo3, but not Dpo1, bypassed hypoxanthine and 8-oxoguanine. Dpo2 and Dpo3 bypassed uracil and cis-syn cyclobutane thymine dimer, respectively. High concentrations of Dpo2 or Dpo3 did not attenuate DNA synthesis by Dpo1 or Dpo4. We conclude that Dpo2 and Dpo3 are much less functional and more thermolabile than Dpo1 and Dpo4 in vitro but have bypass activities across hypoxanthine, 8-oxoguanine, and either uracil or cis-syn cyclobutane thymine dimer, suggesting their catalytically limited roles in translesion DNA synthesis past deaminated, oxidized base lesions and/or UV-induced damage.The timely and faithful replication of genomic information by DNA polymerases is crucial for survival in all living organisms (1). An important issue in understanding chromosomal DNA replication is why so many different kinds of DNA polymerases exist and how they function differently and can be regulated in cells, with the discovery of at least 10 new human DNA polymerases during the past 12 years (2). Numerous DNA polymerases from the bacterial, eukaryal, and archaeal domains of life are divided into six families (A, B, C, D, X, and Y) (3), some of which have been suggested to be utilized for replicative and/or non-replicative tasks, such as DNA repair, translesion DNA synthesis, and hypermutation (4), but their precise roles are still in question. The information-processing systems of eukarya are considered to be more similar to those of archaea rather than to bacteria (5, 6), offering a practical advantage of studying archaeal systems as a model for DNA replication. Indeed, many of the B-family DNA polymerases in eukaryotes and archaea, but not in bacteria, are believed to be mainly involved in chromosomal DNA replication (7). Crenarchaeota has only B-family (but at least two) replicative DNA polymerases, whereas Eryarchaeota has one B-family and one D-family replicative DNA polymerase (7).Sulfolobus solfataricus, a thermoaci...

adducts are formed in DNA by 1,2,3,4-diepoxybutane (metabolite of human carcinogen 1,3-butadiene). Results: hpols and carry out translesion synthesis, incorporating T, G, or A opposite the 1,N

Nucleotide incorporation and extension opposite N 2 -ethylGua by DNA polymerase was measured and structures of the DNA polymerase -N 2 -ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase opposite N 2 -ethyl-Gua was determined by steady state kinetic analysis with Mg 2؉ or Mn 2؉ as the activating metal. DNA polymerase incorporates dCMP opposite N 2 -ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg 2؉ and with greater efficiencies in the presence of Mn 2؉ . However, the fidelity of nucleotide incorporation by DNA polymerase opposite N 2 -ethyl-Gua and Gua using Mn 2؉ is lower relative to that using Mg 2؉ indicating a metal-dependent effect. DNA polymerase extends from the N 2 -ethyl-Gua:Cyt 3 terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase catalyzed reaction is well suited for N 2 -ethyl-Gua bypass. The structure of DNA polymerase with N 2 -ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase complexed with N 2 -ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.2 is an acetaldehyde-derived DNA adduct generated from the reduction of acetaldehyde with 2Ј-deoxyguanosine-3Ј-monophosphate (1). Humans are exposed to acetaldehyde from the environment and through the formation of acetaldehyde by the oxidation of ethanol (2). N 2 -Ethyl-Gua has been detected in the DNA of both alcoholic and nonalcohol drinkers (2, 3). Ethanol is classified as a human carcinogen, and acetaldehyde is known to contribute to the formation of malignant tumors (4). The formation of N 2 -ethylGua during the reduction of acetaldehyde could cause ethanolrelated cancers (5).The ethyl moiety of N 2 -ethyl-Gua is predicted to project into the minor groove of duplex DNA. The N 2 -ethyl-Gua adduct is a strong block to DNA replication by replicative DNA polymerases in vitro and in cells (6, 7). Structures of bacteriophage DNA polymerase (pol) RB69, a homolog of human DNA pol ␣, indicate a possible mechanism of N 2 -ethyl-Gua blocked DNA replication. The structures reveal a DNA-binding motif that contacts the DNA minor groove and functions as an important safeguard to replication fidelity (8). The blocking of replicative DNA pols by N 2 -ethyl-Gua could arise when the ethyl group, protruding into the minor groove, disrupts protein:DNA contacts involved in the proposed "checking mechanism" (8). N 2 -Ethyl-Gua also has a high mis-coding potential during DNA replication with the Klenow fragment of Escherichia coli DNA po...

DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases and . DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50 -100-fold lower than for native G. Both human polymerase (hPol) and hPol inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol . Molecular dynamics simulation of an hPol ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.

O6 -Methylguanine (O 6 -methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase core enzyme was determined for nucleoside triphosphate incorporation opposite O 6 -methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol , which showed that dTTP incorporation occurs with high efficiency opposite O Alkylating agents damage DNA by reacting with the nitrogen and oxygen atoms in DNA bases. Human exposure to alkylating agents arises from endogenous (e.g. food-derived nitrosamines) and exogenous (e.g. tobacco-specific nitrosoamines and chemotherapeutic agents including temozolomide and streptozotocin) sources (1). The endogenously produced compound S-adenosylmethionine also methylates DNA (2). Many alkylating agents that react with DNA form the mutagenic and cytotoxic DNA lesion O 6 -alkylguanine (3), along with other methylated bases. The mutagenicity of O 6 -alkylated DNA is a factor in human diseases such as cancer, teratogenic defects, and premature aging (1). Work focused on the adduct O 6 -methylG 2 has shown that it causes G:C3 A:T transition mutations (4).The mutagenic potential of O -methylG:T base pair fits in the active site of the DNA polymerase without distorting the DNA, thereby contributing to the incorporation of dTTP opposite the lesion (17). Also, the sequence context of O 6 -methylG has been shown to influence the extent of dTTP incorporation by DNA polymerases opposite the lesion (18).Replicative DNA polymerases catalyze the misincorporation of dTTP opposite O 6 -alkylG with similar or even higher efficiency than incorporation of the correct dCTP (4,7,14,18). Similarly, the Y-family DNA polymerases , , and all show poor nucleotide discrimination when bypassing O 6 -alkylG (5, 8). Pols and have similar efficiencies for dTTP * This work was supported, in whole or in part, by National Institutes of Health Grants R01 ES010375 (to F. P. G.), P01 ES005355 (to M. E.), P30 ES000267 (to F. P. G. and M. E.), and T32 ES007028 (to F. P. G. and M. G. P.). Vanderbilt University is a member institution of LS-CAT at the Advanced Photon Source (APS), Argonne, Illinois. Use of the APS was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-06CH11357. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. The atomic coordinates and structure factors (codes 3NGD and 3OSN)