Biomedical subjects

S H Wilson

Publications and source records attributed to S H Wilson.

At least 73 records · Page 4Linked to original sources

PubMed1999

The fidelity of DNA polymerase beta during distributive and processive DNA synthesis.

During base excision repair, DNA polymerase beta fills 1-6-nucleotide gaps processively, reflecting a contribution of both its 8- and 31-kDa domains to DNA binding. Here we report the fidelity of pol beta during synthesis to fill gaps of 1, 5, 6, or >300 nucleotides. Error rates during distributive synthesis by recombinant rat and human polymerase (pol) beta with a 390-base gap are similar to each other and to previous values with pol beta purified from tissues. The base substitution fidelity of human pol beta when processively filling a 5-nucleotide gap is similar to that with a 361-nucleotide gap, but "closely-spaced" substitutions are produced at a rate at least 60-fold higher than for distributive synthesis. Base substitution fidelity when filling a 1-nucleotide gap is higher than when filling a 5-nucleotide gap, suggesting a contribution of the 8-kDa domain to the dNTP binding pocket and/or a difference in base stacking or DNA structure imposed by pol beta. Nonetheless, 1-nucleotide gap filling is inaccurate, even generating complex substitution-addition errors. Finally, the single-base deletion error rate during processive synthesis to fill a 6-nucleotide gap is indistinguishable from that of distributive synthesis to fill a 390-nucleotide gap. Thus the mechanism of processivity by pol beta does not allow the enzyme to suppress template misalignments.

Animals↗

PubMed1999

Enhanced coronary vasoconstriction to oxidative stress product, 8-epi-prostaglandinF2 alpha, in experimental hypercholesterolemia.

OBJECTIVES: The F2-isoprostanes are a family of novel prostaglandin isomers and a stable product of in vivo oxidative stress. 8-epi-prostaglandinF2 alpha, a member of this isoprostane family, is a vasoconstrictor and its local release may contribute to the abnormal vasomotor tone associated with hypercholesterolemia. We therefore aimed to outline the role of 8-epi-prostaglandinF2 alpha as a coronary vasoconstrictor in experimental hypercholesterolemia. METHODS AND RESULTS: Pigs were randomized to two experimental groups (each n = 9): normal (N) and high cholesterol (HC) diet. To determine the vasoconstrictive effects of 8-epi-prostaglandinF2 alpha in vitro, doses from 10(-9) to 10(-5) M were used to constrict coronary epicardial rings. Plasma total and LDL cholesterol levels were significantly higher in the HC group compared with the N group (P < 0.005) as were plasma 8-epi-prostaglandinF2 alpha levels (P < 0.001). 8-epi-prostaglandinF2 alpha immunoreactivity was present in the vessel wall in both groups. Normal vessels with intact endothelium (n = 8 rings) contracted to 8-epi-prostaglandinF2 alpha (maximal contraction 15.5 +/- 8.74%). In the HC group, rings with intact endothelium had a greater contractile response to 8-epi-prostaglandinF2 alpha compared to normals (72.3 +/- 7.9%; n = 8; P < 0.0001). This was reversed by preincubation with NOR-3, a NO donor (maximal contraction 6.7 +/- 1.56%; n = 5; P < 0.0001). Enhanced contraction in normal vessels occurred with endothelial denudation (98.4 +/- 3.56%; n = 6; P < 0.0001) and with preincubation of the endothelium-intact rings with L-NMMA (N-monomethyl-L-arginine), an NO synthase inhibitor (85.5 +/- 10.3%, n = 6, P < 0.001). The enhanced contraction seen with hypercholesterolemia did not occur with other prostanoid vasoconstrictors. CONCLUSION: Experimental hypercholesterolemia leads to a significant increase in 8-epi-prostaglandinF2 alpha levels in addition to enhanced 8-epi-prostaglandinF2 alpha-induced coronary vasoconstriction, in vitro. These findings support a role for the F2-isoprostanes in the regulation of coronary vasomotor tone in pathophysiologic states.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1999

HIV-1 reverse transcriptase is phosphorylated in vitro and in a cellular system.

Phosphorylation modulates the activity of many proteins that interact with nucleic acids including DNA and RNA polymerases. The HIV-1 reverse transcriptase (RT) is essential during the replicative cycle of the HIV-1 virus. HIV-1 RT has several potential sites for phosphorylation that could regulate its activities. In this work, the phosphorylation of HIV-1 RT is examined in vitro and in vivo, to evaluate any role for this modification in regulating RT metabolism. Recombinant unphosphorylated HIV-1 RT heterodimer expressed in bacteria can be phosphorylated in vitro by several purified mammalian protein kinases. Seven kinases were tested, and five of these enzymes phosphorylated HIV-1 RT. Using an insect baculovirus expression system, the 66 kDa HIV-1 RT was also phosphorylated in vivo. However, HIV-1 RT immunoprecipitated from H9-lymphoma cells infected with HIV-1 showed negligible phosphorylation. Our results indicate that purified HIV-1 RT can be phosphorylated by several mammalian protein kinases in vitro and during expression in baculovirus infected insect cells.

Amino Acid Sequence↗

PubMed1999

DNA polymerase beta expression differences in selected human tumors and cell lines.

A long-standing question in cancer biology has been the extent to which DNA repair may be altered during the process of carcinogenesis. We have shown recently that DNA polymerase beta (beta-pol) provides a rate-determining function during in vitro repair of abasic sites by one of the mammalian DNA base excision repair pathways. Therefore, altered expression of beta-pol during carcinogenesis could alter base excision repair and, consequently, be critical to the integrity of the mammalian genome. We examined the expression of beta-pol in several cell lines and human adenocarcinomas using a quantitative immunoblotting method. In cell lines from normal breast or colon, the level of beta-pol was approximately 1 ng/mg cell extract, whereas in all of the breast and colon adenocarcinoma cell lines tested, a higher level of beta-pol was observed. In tissue samples, colon adenocarcinomas had a higher level of beta-pol than adjacent normal mucosa. Breast adenocarcinomas exhibited a wide range of beta-pol expression: one tumor had a much higher level of beta-pol (286 ng/mg cell extract) than adjacent normal breast tissue, whereas another tumor had the same level of beta-pol as adjacent normal tissue. Differences in beta-pol expression level, from normal to elevated, were also observed with prostate adenocarcinomas. All kidney adenocarcinomas tested had a slightly lower beta-pol level than adjacent normal tissue. This study reveals that the base excision repair enzyme DNA polymerase beta is up-regulated in some types of adenocarcinomas and cell lines, but not in others.

Adenocarcinoma↗

PubMed1999

Vascular risk factors correlate to the extent as well as the severity of coronary atherosclerosis.

BACKGROUND: Because most acute coronary events result from thrombosis at sites of minor plaque, the extent of non-obstructive coronary artery disease (CAD), rather than simply the number of severe stenoses, might be clinically relevant. OBJECTIVE: To examine the relationship between vascular risk factors and a novel extent score for CAD that measures the percentage of the coronary tree involved with atheromatous plaque, as judged by coronary angiography. METHODS: We assessed the extent and severity of CAD and the presence of vascular risk factors of 429 consecutive eligible patients (296 men, aged 61 +/- 11 years) who presented for elective coronary angiography. Detailed analyses of lipid levels were performed for 126 subjects. RESULTS: The mean extent score was 54 (range 0-100). The presence of diabetes (P < 0.001), current or former smoking (P < 0.005) and a history of hypertension (P < 0.001) were all strongly associated with the CAD extent score, as was severity of disease. For the 283 patients with one or no severe stenosis, diabetes was associated with a greater extent score (57 versus 41%, P < 0.005), as was smoking (49 versus 34%, P < 0.005). For the 126 patients with detailed data on lipid levels, extent of coronary artery disease was independently correlated to age (P < 0.005), male sex (P < 0.05), presence of diabetes (P < 0.05), hypertension (P < 0.05), level of lipoprotein (a) (P < 0.005) and low-density:high-density lipoprotein ratio (P < 0.01) in multivariable analysis. CONCLUSIONS: Extent of CAD, as well as its severity, is significantly associated with traditional vascular risk factors. Because most acute coronary events occur at sites of minor plaque, this might explain the mechanism whereby risk factors confer adverse prognostic significance.

Coronary Artery Disease↗

PubMed1999

In vivo renal vascular and tubular function in experimental hypercholesterolemia.

Hypercholesterolemia (HC) is often associated with impaired peripheral and coronary vascular responses to endothelium-dependent vasodilators, which are probably due to low bioavailability of nitric oxide. To examine the effect of HC on renal vascular and tubular function, 22 domestic pigs were studied after being fed a 12-week normal (n=11) or HC (n=11) diet. Renal regional perfusion and intratubular contrast media concentration in each nephron segment (representing fluid reabsorption) were quantified in vivo with electron-beam computed tomography before and after a suprarenal infusion of either acetylcholine (6 pigs of each diet) or sodium nitroprusside (SNP; 5 pigs of each diet). An increase in cortical perfusion, observed in normal pigs with acetylcholine (+35+/-6%, P=0. 002) and SNP (+12+/-4%, P=0.005), was blunted in the HC group (+8. 8+/-4.0, P=0.01, and -4.6+/-4.0%, P=0.1, respectively, P=0.003 and P=0.005 compared with normal) as was an increase in medullary perfusion (+58+/-21 in normal versus +24+/-11% in HC, P=0.04). A decrease in the intratubular contrast media concentration in the distal tubule and collecting duct of normal pigs was observed in all tubular segments (and was significantly enhanced in the proximal tubule and Henle's loop) in the HC group, which was associated with increased sodium excretion. The tubular and renal excretory responses to SNP were similar between the groups. In conclusion, early experimental HC in the pig attenuates renal perfusion response to both endothelium-dependent and -independent vasodilators possibly because of decreased bioavailability or decreased vascular responsiveness to nitric oxide. This vascular impairment may play a role in maladjusted renovascular responses and contribute to renal damage in later stages of atherosclerosis.

Acetylcholine↗

PubMed1998

Vertical-scanning mutagenesis of a critical tryptophan in the minor groove binding track of HIV-1 reverse transcriptase. Molecular nature of polymerase-nucleic acid interactions.

While sequence-specific DNA-binding proteins interact predominantly in the DNA major groove, DNA polymerases bind DNA through interactions in the minor groove that are sequence nonspecific. Through functional analyses of alanine-substituted mutant enzymes that were guided by molecular dynamics modeling of the human immunodeficiency virus type 1-reverse transcriptase and DNA complex, we previously identified a structural element in reverse transcriptase, the minor groove binding track (MGBT). The MGBT is comprised of five residues (Ile94, Gln258, Gly262, Trp266, and Gln269) which interact 2-6 base pairs upstream from the polymerase active site in the DNA minor groove and are important in DNA binding, processivity, and frameshift fidelity. These residues do not contribute equally; functional analysis of alanine mutants suggests that Trp266 contributes the most to binding. To define the molecular interactions between Trp266 and the DNA minor groove, we have analyzed the properties of eight mutants, each with an alternate side chain at this position. A refined molecular dynamics model was used to calculate relative binding free energies based on apolar surface area buried upon complex formation. In general, there was a strong correlation between the relative calculated binding free energies for the alternate residue 266 side chains and the magnitude of the change in the properties which reflect template-primer interactions (template-primer dissociation rate constant, Ki,AZTTP, processivity, and frameshift fidelity). This correlation suggests that hydrophobic interactions make a major contribution to the stability of the polymerase-DNA complex. Additionally, tyrosine and arginine substitutions resulted in mutant enzymes with DNA binding properties better than predicted by buried surface area alone, suggesting that hydrogen bonding could also play a role in DNA binding at this position.

Antiviral Agents↗

PubMed1998

Identification of 5'-deoxyribose phosphate lyase activity in human DNA polymerase gamma and its role in mitochondrial base excision repair in vitro.

Mitochondria have been proposed to possess base excision repair processes to correct oxidative damage to the mitochondrial genome. As the only DNA polymerase (pol) present in mitochondria, pol gamma is necessarily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol gamma to participate in uracil-provoked base excision repair reconstituted in vitro with purified components. Subsequent to actions of uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, human pol gamma was able to fill a single nucleotide gap in the presence of a 5' terminal deoxyribose phosphate (dRP) flap. We report here that the catalytic subunit of human pol gamma catalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensional protein structure. The dRP lyase activity does not require divalent metal ions, and the ability to trap covalent enzyme-DNA complexes with NaBH4 strongly implicates a Schiff base intermediate in a beta-elimination reaction mechanism.

Base Sequence↗

PubMed1998

Mammalian base excision repair by DNA polymerases delta and epsilon.

Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA. We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the repair reaction is delayed compared to single nucleotide replacement by Pol beta. Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.

Animals↗

PubMed1998

Mammalian abasic site base excision repair. Identification of the reaction sequence and rate-determining steps.

Base excision repair (BER) is one of the cellular defense mechanisms repairing damage to nucleoside 5'-monophosphate residues in genomic DNA. This repair pathway is initiated by spontaneous or enzymatic N-glycosidic bond cleavage creating an abasic or apurinic-apyrimidinic (AP) site in double-stranded DNA. Class II AP endonuclease, deoxyribonucleotide phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities complete repair of the AP site. In mammalian cell nuclear extract, BER can be mediated by a macromolecular complex containing DNA polymerase beta (beta-pol) and DNA ligase I. These two enzymes are capable of contributing the latter three of the four BER enzymatic activities. In the present study, we found that AP site BER can be reconstituted in vitro using the following purified human proteins: AP endonuclease, beta-pol, and DNA ligase I. Examination of the individual enzymatic steps in BER allowed us to identify an ordered reaction pathway: subsequent to 5' "nicking" of the AP site-containing DNA strand by AP endonuclease, beta-pol performs DNA synthesis prior to removal of the 5'-dRP moiety in the gap. Removal of the dRP flap is strictly required for DNA ligase I to seal the resulting nick. Additionally, the catalytic rate of the reconstituted BER system and the individual enzymatic activities was measured. The reconstituted BER system performs repair of AP site DNA at a rate that is slower than the respective rates of AP endonuclease, DNA synthesis, and ligation, suggesting that these steps are not rate-determining in the overall reconstituted BER system. Instead, the rate-limiting step in the reconstituted system was found to be removal of dRP (i.e. dRP lyase), catalyzed by the amino-terminal domain of beta-pol. This work is the first to measure the rate of BER in an in vitro reaction. The potential significance of the dRP-containing intermediate in the regulation of BER is discussed.

Base Sequence↗

PubMed1998

Thermodynamics of human DNA ligase I trimerization and association with DNA polymerase beta.

The interaction between human DNA polymerase beta (pol beta) and DNA ligase I, which appear to be responsible for the gap filling and nick ligation steps in short patch or simple base excision repair, has been examined by affinity chromatography and analytical ultracentrifugation. Domain mapping studies revealed that complex formation is mediated through the non-catalytic N-terminal domain of DNA ligase I and the N-terminal 8-kDa domain of pol beta that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol beta, a 39-kDa bi-domain enzyme, undergoes indefinite self-association, forming oligomers of many sizes. The binding sites for self-association reside within the C-terminal 31-kDa domain. DNA ligase I undergoes self-association to form a homotrimer. At temperatures over 18 degreesC, three pol beta monomers attached to the DNA ligase I trimer, forming a stable heterohexamer. In contrast, at lower temperatures (<18 degreesC), pol beta and DNA ligase I formed a stable 1:1 binary complex only. In agreement with the domain mapping studies, the 8-kDa domain of pol beta interacted with DNA ligase I, forming a stable 3:3 complex with DNA ligase I at all temperatures, whereas the 31-kDa domain of pol beta did not. Our results indicate that the association between pol beta and DNA ligase I involves both electrostatic binding and an entropy-driven process. Electrostatic binding dominates the interaction mediated by the 8-kDa domain of pol beta, whereas the entropy-driven aspect of interprotein binding appears to be contributed by the 31-kDa domain.

Chromatography, Affinity↗

PubMed1998

Human DNA polymerase beta deoxyribose phosphate lyase. Substrate specificity and catalytic mechanism.

DNA polymerase beta (beta-pol) cleaves the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5'-incised by AP endonuclease, resulting in a 5' 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of beta-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of beta-pol are similar. However, kcat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5' dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of beta-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5'-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.

Carbon-Oxygen Lyases↗

PubMed1998

Functional analysis of the amino-terminal 8-kDa domain of DNA polymerase beta as revealed by site-directed mutagenesis. DNA binding and 5'-deoxyribose phosphate lyase activities.

The amino-terminal 8-kDa domain of DNA polymerase beta functions in binding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in gapped DNA structures, and as a 5'-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)16. Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5'-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5' terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys35 is involved in 5'-phosphate recognition of DNA polymerase beta. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA. Alanine mutants of Lys35 and Lys60 are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. More importantly, alanine substitution for Lys72 resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Alanine mutants of Lys68 and Lys84 had wild-type dRP lyase activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase activity, suggesting that the effects of the alanine substitution at Lys72 and Lys35 were additive. The results suggest that Lys72 is directly involved in formation of a covalent imino intermediate and are consistent with Lys72 as the predominant Schiff base nucleophile in the dRP lyase beta-elimination catalytic reaction.

Catalysis↗

PubMed1998

Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.

DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.

Animals↗

PubMed1998

Different DNA polymerases are involved in the short- and long-patch base excision repair in mammalian cells.

Mammalian cells possess two distinct pathways for completion of base excision repair (BER): the DNA polymerase beta (Pol beta)-dependent short-patch pathway (replacement of one nucleotide), which is the main route, and the long-patch pathway (resynthesis of 2-6 nucleotides), which is PCNA-dependent. To address the issue of how these two pathways share their role in BER the ability of Pol beta-defective mammalian cell extracts to repair a single abasic site constructed in a circular duplex plasmid molecule was tested in a standard in vitro repair reaction. Pol beta-deficient extracts were able to perform both BER pathways. However, in the case of the short-patch BER, the repair kinetics was significantly slower than with Pol beta-proficient extracts, while the efficiency of the long-patch synthesis was unaffected by the loss of Pol beta. The repair synthesis was fully dependent on PCNA for the replacement of long patches. These data give the first evidence that in cell extracts DNA polymerases other than Pol beta are specifically involved in the long-patch BER. These DNA polymerases are also able to perform short-patch BER in the absence of PCNA, although less efficiently than Pol beta. These findings lead to a novel model whereby the two BER pathways are characterized by different protein requirements, and a functional redundancy at the level of DNA polymerases provides cells with backup systems.

Animals↗

PubMed1998

Interactions between DNA polymerase beta and the major covalent adduct of the carcinogen (+)-anti-benzo[a]pyrene diol epoxide with DNA at a primer-template junction.

A molecular dynamics simulation has been carried out with DNA polymerase beta (beta pol) complexed with a DNA primer-template. The templating guanine at the polymerase active site was covalently modified by the carcinogenic metabolite of benzo[a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, to form the major (+)-trans-anti-benzo[a]pyrene diol epoxide covalent adduct. Thus, the benzo[a]pyrenyl moiety (BP) is situated in the single-stranded template at the junction between double- and single-stranded DNA. The starting structure was based on the X-ray crystal structure of the rat beta pol primer-template and ddCTP complex [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903]. During the simulation, the BP and its attached templating guanine rearrange to form a structure in which the BP is closer to parallel with the adjacent base pair. In addition, the templating attached guanine is displaced toward the major groove side and access to its Watson-Crick edge is partly obstructed. This structure is stabilized, in part, by new hydrogen bonds between the BP and beta pol Asn279 and Arg283. These residues are within hydrogen bonding distance to the incoming ddCTP and templating guanine, respectively, in the crystal structure of the beta pol ternary complex. Site-directed mutagenesis has confirmed their role in dNTP binding, discrimination, and catalytic efficiency [Beard, W. A., Osheroff, W. P., Prasad, R., Sawaya, M. R., Jaju, M., Wood, T. G., Kraut, J., Kunkel, T. A., and Wilson, S. H. (1996) J. Biol. Chem. 271, 12141-12144]. The predominant biological effect of the BP is DNA polymerase blockage. Consistent with this biological effect, the computed structure suggests the possibility that the BP's main deleterious impact on DNA synthesis might result at least in part from its specific interactions with key polymerase side chains. Moreover, relatively modest movement of BP and its attached guanine, with some concomitant enzyme motion, is necessary to relieve the obstruction and permit the observed rare incorporation of a dATP opposite the guanine lesion.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1998

Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases.

Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonucleotide was synthesized in situ by DNA polymerases. 5-[N-(2-nitro-5-azidobenzoyl)-trans -3-aminopropenyl-1]deoxyuridine-5'-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer logical ortemplate either by human DNA polymerase alpha primase (polalpha), human DNA polymerase beta (polbeta) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The polymerase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA. The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE. The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA. The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3'-end of the primer, while the p14 subunit locates relatively far from the 3'-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.

Azides↗

PubMed1998

Impairment of proliferating cell nuclear antigen-dependent apurinic/apyrimidinic site repair on linear DNA.

Repair of apurinic/apyrimidinic (AP) sites by mammalian cell extracts was compared using circular and linear DNA substrates. Extracts prepared from DNA polymerase beta (polbeta)-proficient mouse fibroblasts repaired AP sites on both circular and linear DNA. However, extracts from the isogenic polbeta-knockout cells repaired AP sites on circular DNA but not efficiently on linear DNA. The circularity-dependent repair by the polbeta-knockout cell extract was completely inhibited by anti-proliferating cell nuclear antigen (PCNA) antibody but fully restored by addition of purified PCNA. Pretreatment of the linear DNA with AP endonuclease did not improve repair, indicating that impairment of AP site repair on linear DNA by polbeta-knockout cell extracts is not due to inefficiency of damage incision but rather to deficiency at the subsequent steps. These results indicate that AP sites can be repaired on circular DNA by the PCNA-dependent pathway in addition to the polbeta-dependent pathway and that the PCNA-dependent repair mechanism is poorly functional on linear DNA in vitro.

Animals↗