Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha.

A series of 6-anilinouracils, dGTP analogues which selectively inhibit specific bacterial DNA polymerases, were examined for their capacity to inhibit purified DNA polymerases from HeLa cells. The p-n-butyl derivative (BuAU) was found to inhibit DNA polymerase alpha with a Ki of approximately 60 microM. The inhibitory effect of BuAU was reversed specifically by dGTP and was observed only for DNA polymerase alpha; polymerases beta and lambda were not inhibited by drug at concentrations as high as 1 mM. BuAU also was inhibitory in vivo in HeLa cell culture; at 100 microM it reversibly inhibited cell division and selectively depressed DNA synthesis. The results of these studies indicate that BuAU is an inhibitor with considerable potential as a specific probe with which to dissect the structure of mammalian polymerase alpha and its putative role in cellular DNA replication.     

Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.     

Inhibitory effects of 5-alkyl- and 5-alkenyl-1-beta-D-arabinofuranosyluracil 5'-triphosphates on herpes virus-induced DNA polymerases

Various 5-substituted 1-beta-D-arabinofuranosyluracil 5'-triphosphates (H, methyl, ethyl, n-propyl, n-butyl, (E)-bromovinyl, styryl, and beta-phenylethyl derivatives) were prepared and their inhibitory effects on two different herpes virus-induced DNA polymerases (OMV and HCMV) were studied. These dTTP analogues inhibited the incorporation of [3H]dTMP into DNA in vitro. Among them, analogues having a vinyl group at the 5-position were strongly active against DNA polymerases induced on herpes virus infection. Kinetic analysis showed that the inhibition by the analogues was essentially competitive with respect to the substrate, dTTP. The K1 values (microM) for AraUTP (2.4), AraTTP (1.0), BVAUTP (0.8), and StUAUTP (0.8) were smaller than the Km value (microM) for dTTP (3.4), but those for AraEtUTP, AraPrUTP, and AraBuUTP (5-14) were larger than the Km for dTTP in the case of HCMV-induced DNA polymerase. In contrast to these results, OMV-induced DNA polymerase seemed to be more resistant to these inhibitors than HCMV-induced DNA polymerase. However, the mode of the structure of substituent groups at the 5-position of base moieties is almost the same for the two DNA polymerases, except for in the case of AraUTP itself.     

Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases lambda and beta

In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) damage, many DNA polymerases exhibit a dual coding potential which facilitates efficient incorporation of matched dCTP or mismatched dATP. This also holds true for the insertion of 8-oxodGTP opposite template bases dC and dA. Employing single-turnover kinetic methods, we examined human DNA polymerase beta and its novel X-family homolog, human DNA polymerase lambda, to determine which nucleotide and template base was preferred when encountering 8-oxodG and 8-oxodGTP, respectively. While DNA polymerase beta preferentially incorporated dCTP over dATP, DNA polymerase lambda did not modulate a preference for either dCTP or dATP when opposite 8-oxodG in single-nucleotide gapped DNA, as incorporation proceeded with essentially equal efficiency and probability. Moreover, DNA polymerase lambda is more efficient than DNA polymerase beta to fill this oxidized single-nucleotide gap. Insertion of 8-oxodGTP by both DNA polymerases lambda and beta occurred predominantly against template dA, thereby reiterating how the asymmetrical design of the polymerase active site differentially accommodated the anti and syn conformations of 8-oxodG and 8-oxodGTP. Although the electronegative oxygen at the C8 position of 8-oxodG may induce DNA structural perturbations, human DNA ligase I was found to effectively ligate the incorporated 8-oxodGMP to a downstream strand, which sealed the nicked DNA. Consequently, the erroneous nucleotide incorporations catalyzed by DNA polymerases lambda and beta as well as the subsequent ligation catalyzed by a DNA ligase during base excision repair are a threat to genomic integrity.     

The overexpression of specialized DNA polymerases in cancer

Specialized DNA polymerases are required to bypass DNA damage lesions that would otherwise cause replication arrest and cell death. When operating on non-canonical templates, such as undamaged DNA or on non-cognate lesions, these polymerases exhibit considerably reduced fidelity, resulting in the generation of mutations. Ectopic overexpression of these polymerases can also lead to an increased mutation rate and an enhanced capability of DNA repair, suggesting that they could potentially act as oncogenes if they were overexpressed in cancers. Here, we examine expression patterns of DNA polymerases in matched normal and tumor samples from a diverse range of tissues. As well as investigating the specialized polymerases beta, lambda, iota and kappa, we also investigate the expression of the replicative polymerases alpha, delta and epsilon. The data presented provide evidence for the overexpression of specialized polymerases in tumors, with more than 45% of the 68 tumor samples studied demonstrating greater than two-fold enhanced expression of at least one specialized polymerase. Of particular note, DNA polymerase beta (pol beta) was found to be overexpressed at both the mRNA and protein level in approximately one third of all tumor types studied, with overexpression being particularly frequent in uterus, ovary, prostate and stomach samples. Pols lambda, and iota were also found to be overexpressed to a significant extent in a range of tumor types, albeit less frequently than pol beta. In contrast, pol kappa was rarely found to be overexpressed in tumors but was found to be commonly underexpressed in many samples. Downregulation of pol beta expression by siRNA resulted in an increased sensitivity to the chemotherapeutic agent cisplatin, suggesting a role for this polymerase in providing tolerance to cisplatin-induced damage. These observations suggest that specialised DNA polymerases, and particularly pol beta, could be considered both as caretaker genes altered during tumorigenesis, and as potential drug targets to sensitise tumors to chemotherapy.     

Interplay between DNA polymerases beta and lambda in repair of oxidation DNA damage in chicken DT40 cells

DNA polymerase lambda (Pol lambda) is a DNA polymerase beta (Pol beta)-like enzyme with both DNA synthetic and 5'-deoxyribose-5'-phosphate lyase domains. Recent biochemical studies implicated Pol lambda as a backup enzyme to Pol beta in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol lambda and Pol beta in BER of DNA damage in living cells, we disrupted the genes for both enzymes either singly or in combination in the chicken DT40 cell line and then characterized BER phenotypes. Disruption of the genes for both polymerases caused hypersensitivity to H(2)O(2)-induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in cells after H(2)O(2) exposure was lower in Pol beta(-/-)/Pol lambda(-/-) cells than in Pol beta(-/-), wild-type, and Pol lambda(-/-) cells, which were equivalent. These results suggest that these polymerases can complement for one another in counteracting oxidative DNA damage. Similar results were obtained in assays for in vitro BER capacity using cell extracts. With MMS-induced cytotoxicity, there was no significant effect on either survival or BER capacity from Pol lambda gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol beta(-/-)/Pol lambda(-/-) and Pol beta(-/-) cells, suggesting that Pol beta had a dominant role in counteracting alkylation DNA damage in this cell system.     

Role of the catalytic metal during polymerization by DNA polymerase lambda

The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.     

Mechanism and dynamics of translesion DNA synthesis catalyzed by the Escherichia coli Klenow fragment

Translesion DNA synthesis represents the ability of a DNA polymerase to incorporate and extend beyond damaged DNA. In this report, the mechanism and dynamics by which the Escherichia coli Klenow fragment performs translesion DNA synthesis during the misreplication of an abasic site were investigated using a series of natural and non-natural nucleotides. Like most other high-fidelity DNA polymerases, the Klenow fragment follows the "A-rule" of translesion DNA synthesis by preferentially incorporating dATP opposite the noninstructional lesion. However, several 5-substituted indolyl nucleotides lacking classical hydrogen-bonding groups are incorporated approximately 100-fold more efficiently than the natural nucleotide. In general, analogues that contain large substituent groups in conjunction with significant pi-electron density display the highest catalytic efficiencies ( k cat/ K m) for incorporation. While the measured K m values depend upon the size and pi-electron density of the incoming nucleotide, k cat values are surprisingly independent of both biophysical features. As expected, the efficiency by which these non-natural nucleotides are incorporated opposite templating nucleobases is significantly reduced. This reduction reflects minimal increases in K m values coupled with large decreases in k cat values. The kinetic data obtained with the Klenow fragment are compared to that of the high-fidelity bacteriophage T4 DNA polymerase and reveal distinct differences in the dynamics by which these non-natural nucleotides are incorporated opposite an abasic site. These biophysical differences argue against a unified mechanism of translesion DNA synthesis and suggest that polymerases employ different catalytic strategies during the misreplication of damaged DNA.     

Thermostable DNA polymerases can perform translesion synthesis using 8-oxoguanine and tetrahydrofuran-containing DNA templates

The translesion synthesis (TLS) capacity of the thermostable DNA polymerases Taq, Tte and Tte-seq utilizing a synthetic abasic site, tetrahydrofuran (THF), and an 8-oxoguanine-containing DNA template was investigated. Measurements with human DNA polymerase beta were used as a "positive control". Thermostable DNA polymerases were observed to perform TLS with different specificities on both substrates. With a THF-containing template, dGMP was preferentially inserted by all the DNA polymerases. In the presence of Mn(II) as a cofactor, all the polymerases incorporated dCMP opposite 8-oxoguanine whereas, in the presence of Mg(II) ions, dAMP was incorporated. It was found that none of the thermophilic DNA polymerases utilized dTTP with either an 8-oxoguanine or a THF-containing template. In all cases, DNA duplex containing THF as damage was processed to full length less effectively than DNA duplex containing 8-oxoguanine.     

Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.     

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases

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-OH-dATP 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.     

A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase beta and lambda.

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for pol lambda. Because pol beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to analyze the biochemical relationship with solanapyrone A. On pol beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BIAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of pol beta. This domain is known to bind single-stranded DNA, provide 5'-phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity) including 5'-deoxyribose phosphate lyase activity. Solanapyrone A inhibited the single-stranded DNA-binding activity but did not influence the activities of the 5'-phosphate recognition in gapped DNA structures and the AP lyase. Based on these results, the inhibitory mechanism of solanapyrone A is discussed.     

Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases.

Multiple DNA polymerases have been described in all organisms studied to date. Their specific functions are not easy to determine, except when powerful genetic and/or biochemical tools are available. However, the processivity of a DNA polymerase could reflect the physiological role of the enzyme. In this study, analogies between plant and animal DNA polymerases have been investigated by analyzing the size of the products synthesized by wheat DNA polymerases A, B, CI, and CII as a measure of their processivity. Thus, incubations have been carried out with poly(dA)-oligo(dT) as a template-primer under varying assay conditions. In the presence of MgCl2, DNA polymerase A was highly processive, whereas DNA polymerases B, CI, and CII synthesized much shorter products. With MnCl2 instead of MgCl2, DNA polymerase A was highly processive, DNA polymerases B and CII were moderately processive, and DNA polymerase CI remained strictly distributive. The effect of calf thymus proliferating cell nuclear antigen (PCNA) on wheat polymerases was studied as described for animal DNA polymerases. The high processivity of DNA polymerase A was PCNA independent, whereas both enzyme activity and processivity of wheat DNA polymerases B and CII were significantly stimulated by PCNA. On the other hand, DNA polymerase CI was not stimulated by PCNA and, like animal DNA polymerase beta, was distributive in all cases. From these results, we propose that wheat DNA polymerase A could correspond to a DNA polymerase alpha, DNA polymerases B and CII could correspond to the delta-like enzyme, and DNA polymerase CI could correspond to DNA polymerase beta.     

Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III

Bacterial replicative DNA polymerases such as Polymerase III (Pol III) share no sequence similarity with other polymerases. The crystal structure, determined at 2.3 A resolution, of a large fragment of Pol III (residues 1-917), reveals a unique chain fold with localized similarity in the catalytic domain to DNA polymerase beta and related nucleotidyltransferases. The structure of Pol III is strikingly different from those of members of the canonical DNA polymerase families, which include eukaryotic replicative polymerases, suggesting that the DNA replication machinery in bacteria arose independently. A structural element near the active site in Pol III that is not present in nucleotidyltransferases but which resembles an element at the active sites of some canonical DNA polymerases suggests that, at a more distant level, all DNA polymerases may share a common ancestor. The structure also suggests a model for interaction of Pol III with the sliding clamp and DNA.