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S H Wilson

Publications and source records attributed to S H Wilson.

At least 127 records · Page 7Linked to original sources
PubMed1995

DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract.

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.

Animals↗
PubMed1995

DNA damage-induced transcriptional activation of a human DNA polymerase beta chimeric promoter: recruitment of preinitiation complex in vitro by ATF/CREB.

Treatment of hamster cells in culture with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces DNA polymerase beta (beta-pol) gene expression and cellular levels of the enzyme. Transcriptional activity of a cloned beta-pol promoter in transient expression assays is also stimulated. Among the requirements for these responses are methylation damage to genomic DNA, cellular cAMP-dependent protein kinase, and the ATF/CREB site of the cloned beta-pol promoter. In the present study, HeLa cell nuclear extract from MNNG-treated cells was much more active in an in vitro transcription assay than nuclear extract from normal cells. By using an oligonucleotide affinity column to deplete the nuclear extract of ATF/CREB, we showed that the difference was due to ATF/CREB activator. Purified ATF/CREB activator from MNNG-treated cells was approximately 10-fold more active than ATF/CREB purified from normal cells as a transcriptional activator for the depleted nuclear extract. ATF/CREB in the extract from normal cells is known to activate in vitro transcription by increasing the rate of promoter clearance [Narayan, S., Widen, S. G., Beard, W. A., & Wilson, S. H. (1994) J. Biol. Chem. 269, 12755-12763]. With ATF/CREB from MNNG-treated cells, the amount of preinitiation complex formed was much greater than with ATF/CREB from normal cells, and the kinetics of both the closed to open preinitiation complex isomerization and promoter clearance were altered. These results indicate that the mechanism of transcriptional activation secondary to DNA alkylation damage is recruitment of more preinitiation complex and alteration of the kinetic scheme of transcription initiation.

Base Sequence↗
PubMed1995

Evaluation of lymphocyte stimulation tests for diagnosis of bovine tuberculosis in elk (Cervus elaphus).

Lymphocyte stimulation tests (LST), performed using 6 antigen preparations, were compared individually and in pairs. The tests were performed on 433 blood samples collected from elk in Mycobacterium bovis-infected herds. These elk were killed as part of Agriculture and Agri-Food Canada's bovine tuberculosis eradication policy, and mycobacterial culture results were obtained from tissues of each animal. The LST, which had the highest total sum of sensitivity and specificity, was a comparative test that used M bovis purified protein derivative (PPD) and M paratuberculosis (johnin) PPD. This test had a sensitivity of 76%, with confidence limits (CL) of 63 to 85% for this estimate, and specificity of 77% (CL, 72 to 81%). The LST, using only M bovis PPD antigen, had a sensitivity of 70% (CL, 57 to 80%) and specificity of 74% (CL, 69 to 79%); when it was compared with culture results, using the kappa statistic, agreement was only 32%. This indicated that the LST identified different elk than did M bovis isolation tests.

Animals↗
PubMed1994

Characterization of the tryptophan fluorescence and hydrodynamic properties of rat DNA polymerase beta.

We have examined the biophysical properties of DNA polymerase beta (beta-pol) in solution. Time-resolved and steady-state fluorescence were used to investigate the microenvironment of the lone tryptophanyl residue (Trp324), and a combination of sedimentation equilibrium, sedimentation velocity and fluorescence anisotropy decay measurements were used to study the hydrodynamic properties of the enzyme. Trp324 appears to be exposed to water as judged by the tryptophan emission and steady-state and lifetime quenching experiments. The fluorescence is easily quenched by a neutral quencher acrylamide (kq = 1.59 x 10(9)M-1S-1), and by a negatively charged ionic quencher, I- (kq = 1.60 x 10(9) M-1S-1), but not by a positively charged ionic quencher, Cs+ (kq = 0.2 x 10(9) M-1S-1). The fluorescence lifetime of beta-pol is best described by the sum of two exponentials with a longer lifetime component of 8.4 ns and a shorter lifetime component of 1.3 ns. Decay associated spectra (DAS) show emission maxima at 340 nm and at 345 nm for the shorter lifetime and longer lifetime components, respectively, with corresponding centers of gravity at 347 nm and 348 nm. Sedimentation equilibrium experiments show that the enzyme exists as a monomer at the KCl concentrations (> 0.05 M) studied in the absence of divalent metals. Zn2+ causes higher order aggregation, but no such aggregates are seen with Mg2+ and Mn2+. In the presence of 1 mM manganese, the average lifetime decreased approximately 10%, from 8.14 ns to 7.38 ns, with a concomitant increase of average rotational correlational time (phi) from 24 ns to 28 ns. The accessibility of the positively charged quencher (Cs+) to tryptophan also decreases approximately 50%, indicating alteration of the tryptophan microenvironment. By contrast, Mg2+ causes minor changes in fluorescence properties. The hydrodynamic shape of the intact enzyme and its single-stranded (8 kDa) and double-stranded (31 kDa) DNA binding domains were further investigated by sedimentation velocity measurements. The value of S0(20),W for the intact enzyme is 2.97 S, and the calculated axial ratio is 5.0. In contrast to the 8 kDa domain, which has a less asymmetric shape with an axial ratio of 2.3, the 31 kDa domain shows an elongated structure with an axial ratio of 5.5. These data suggest that the axial ratio of the intact enzyme may be the result of marked bending of the molecule at the flexible hinge region between the two domains.

Animals↗
PubMed1994

Structure/function studies of human immunodeficiency virus type 1 reverse transcriptase. Alanine scanning mutagenesis of an alpha-helix in the thumb subdomain.

Human immunodeficiency virus type 1 reverse transcriptase has subunits of 66 and 51 kDa (p66 and p51, respectively). Structural studies indicate that each subunit consists of common subdomains. The polymerase domain of p66 forms a nucleic acid binding cleft, and, by analogy with a right hand, the subdomains are referred to as fingers, palm, and thumb (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Residues 257-266 correspond to a highly conserved region of primary structure among retroviral pol genes. Crystallographic evidence indicates that these residues are in the thumb subdomain and form part of an alpha-helix (alpha H), which interacts with DNA (Jacobo-Molina, A., Ding, J., Nanni, R. G., Clark, A. D., Jr., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H., and Arnold, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6320-6324). To define the role of this region during catalytic cycling, we performed systematic site-directed mutagenesis from position 253 through position 271 by changing each residue, one by one, to alanine. Each mutant protein was expressed and purified, and their substrate-specific activities were surveyed. The results are consistent with alpha H (residues 255-268) of p66 interacting with the template and/or primer strand. The core of alpha H appears to play an important role in template-primer binding (residues Gln-258, Gly-262, and Trp-266), and in protein-protein interactions (residues Val-261 and Leu-264). The periodicity of the effects observed suggest that a segment of one face of alpha H interacts with the template-primer. The lower fidelity observed with alanine mutants of Gly-262 and Trp-266 correlated with an over 200-fold increase in the dissociation rate constant for template-primer relative to wild type enzyme and suggests that enzyme-DNA interactions in the template-primer stem are important fidelity determinants.

Alanine↗
PubMed1994

Regulation of in vitro nucleic acid strand annealing activity of heterogeneous nuclear ribonucleoprotein protein A1 by reversible phosphorylation.

Phosphorylation in vivo of several proteins in the mammalian heterogeneous nuclear ribonucleoprotein complex (hnRNP), including A1, has been observed and proposed as a regulatory step in pre-mRNA splicing [Maryland, S. H., Dwen, P., & Pederson, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7764-7768]. We examined the ability of recombinant hnRNP protein A1 to act as a substrate for a number of purified Ser/Thr protein kinases in vitro. A survey of seven protein kinases showed that A1 was heavily phosphorylated by protein kinase C (PKC) and also was phosphorylated by casein kinase II, protamine kinase, and protein kinase A. In contrast, autophosphorylation-activated protein kinase and two forms of myelin basic protein kinase failed to phosphorylate A1. Proteolysis with trypsin and V8 protease revealed that PKC phosphorylates A1 at three main sites, two in the N-terminal domain (spanning residues 2-196) and one in the C-terminal domain (spanning residues 197-320). Amino acid sequencing revealed that these sites were Ser95, Ser192, and Ser199; phosphorylation at Ser192 was more abundant than at Ser95 and Ser199. Phosphorylation by PKC inhibited the strand annealing activity of A1. Protein phosphatase 2A, but not protein phosphatase 1, dephosphorylated A1 and reversed the inhibitory effect of PKC phosphorylation on the strand annealing activity. A conformational change in the C-terminal domain of A1 was observed upon PKC phosphorylation, and this was associated with a decrease in A1's affinity for single-stranded polynucleotides. The results are consistent with a role of phosphorylation of A1 in regulating its strand annealing activity in vivo.

Amino Acid Sequence↗
PubMed1994

Assignments of 1H, 15N, and 13C resonances for the backbone and side chains of the N-terminal domain of DNA polymerase beta. Determination of the secondary structure and tertiary contacts.

DNA polymerase beta consists of an N-terminal single-stranded DNA binding domain and a C-terminal catalytic domain separable by mild proteolysis [Kumar et al. (1990) J. Biol. Chem. 265, 2124-2131]. The N-terminal domain participates in template and gapped DNA recognition and contributes significantly to catalysis. The secondary structure and tertiary contacts within the cloned N-terminal domain (residues 2-87) of mammalian DNA polymerase beta have been determined using multidimensional NMR. Assignments of backbone 1H, 15N, and 13C resonances and side chain 1H and 13C resonances have been obtained from double- and triple-resonance 3D NMR experiments. The 13C-edited TOCSY experiment has allowed nearly complete assignments of 1H and 13C resonances within side chains. The 13C-edited NOESY experiment has been used for determination of medium- and long-range NOEs and a determination of tertiary contacts. The N-terminal domain is found to consist of four helices, helix-1 (15-26), helix-2 (36-47), helix-3 (56-61), and helix-4 (69-78), which on the basis of long-range NOEs are tightly packed of form a hydrophobic core. The remainder of the domain consists of two turns (48-51 and 62-65), an omega-type loop (27-35), and extended structure. The aromatic side chains of Y36, Y39, Y49, and F76 display tertiary contacts indicative of at least partial hydrophobic packing. The S30 and H34 residues which cross-link to single-stranded DNA [Prasad et al. (1993) J. Biol. Chem. 268, 15906-15911] are contained within the K27-K35 loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The human DNA polymerase beta gene structure. Evidence of alternative splicing in gene expression.

DNA polymerase beta (beta-pol) is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. Using two human genomic libraries we have cloned the complete human beta-pol gene and determined the organization of the beta-pol coding sequence within the gene. The human beta-pol gene spans 33 kb and contains 14 exons that range from 50 to 233 bp. The 13 introns vary from 96 bp to 6.5 kb. Information derived from this study was used in defining the location of a deletion/insertion type restriction fragment length polymorphism (RFLP) 5' to exon I of the human beta-pol gene. This RFLP was utilized in linkage analysis of DNAs from CEPH families and the results confirm the previous assignment of the human beta-pol gene to chromosome 8 (p12-p11). Analysis of mRNA from six human cell lines using the polymerase chain reaction showed the expression of two beta-pol transcripts. Sequence analysis revealed that the size difference in these transcripts was due to deletion of the 58 bp sequence encoded by exon II, suggesting that the smaller transcript results from an alternative splicing of the exon II sequence during processing of the beta-pol precursor RNA.

Animals↗
PubMed1994

Studies of gapped DNA substrate binding by mammalian DNA polymerase beta. Dependence on 5'-phosphate group.

Purified mammalian DNA polymerase beta (beta-pol) fills short gaps of up to 6 nucleotides by a processive mechanism, and this gap-filling activity requires a PO4 group on the 5'-side of the gap (Singhal, R. K., and Wilson, S. H. (1993) J. Biol. Chem. 268, 15906-15911). To assess details of bimolecular binding between beta-pol and a 5-nucleotide (nt) gapped radiolabeled heteropolymeric DNA substrate, beta-pol.DNA complexes were formed, photochemically cross-linked using UV light, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A 39-nt template was annealed with two 17-mer oligonucleotides, generating a 5-nt gap. The results indicate that beta-pol binds to both the template and primer strands, and binding is strongly enhanced by a 5'-PO4 on the downstream oligonucleotide, even though little cross-linking is observed to this oligonucleotide. The results suggest that beta-pol recognizes the 5'-side of a long single-stranded gap in DNA, provided it contains a 5'-PO4. Additional beta-pol.DNA binding measurements were performed using a competition assay to assess the ability of heteropolymeric DNA to inhibit synthesis on a homopolymeric template-primer system. The results indicate that in addition to the 5'-PO4, the length of the single-stranded template nucleic acid adjacent to the 5'-PO4 is also important for tight binding. Proteolysis of the cross-linked beta-pol.DNA complex with trypsin resulted in a single radiolabeled tryptic product corresponding to nucleic acid cross-linked to the 8-kDa domain. The results demonstrate that the role of the 8-kDa domain is to direct beta-pol binding to the phosphorylated 5'-position in gapped DNA substrates.

Base Sequence↗
PubMed1994

Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism.

Structures of the 31-kilodalton catalytic domain of rat DNA polymerase beta (pol beta) and the whole 39-kilodalton enzyme were determined at 2.3 and 3.6 angstrom resolution, respectively. The 31-kilodalton domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel reminiscent of the polymerase domains of the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, and bacteriophage T7 RNA polymerase. The amino-terminal 8-kilodalton domain is attached to the fingers subdomain by a flexible hinge. The two invariant aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly re-evolved, a common nucleotidyl transfer mechanism. The location of Mn2+ and deoxyadenosine triphosphate in pol beta confirms the role of the invariant aspartates in metal ion and deoxynucleoside triphosphate binding.

Animals↗
PubMed1994

Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP.

Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.

Animals↗
PubMed1994

RNA polymerase II transcription. Rate of promoter clearance is enhanced by a purified activating transcription factor/cAMP response element-binding protein.

The core promoter of the human DNA beta-polymerase (beta-pol) gene is regulated by proteins binding at 3 GC boxes and the single activating transcription factor/cAMP response element (ATF/CRE) centered at -45; the central 8 residues of this ATF/CRE match the ATF/CRE consensus sequence, TGACGTCA. Previously, we purified a beta-pol promoter ATF/CRE-binding protein (named palindrome-binding protein or PBP) from bovine testes and found that this protein is a beta-pol promoter transcriptional activator in vitro using a HeLa nuclear extract transcription system (Widen, S. G., and Wilson, S. H. (1991) Biochemistry 30, 6296-6305). In this study, we determined the mechanism of in vitro transcriptional activation by this purified PBP. We used a PBP-depleted HeLa nuclear extract transcription system with an artificial promoter containing a solitary activator element corresponding to the entire 22-nucleotide beta-pol promoter ATF/CRE-binding site. Kinetic analyses of the 180-nucleotide run-off product formation indicated that stimulation of transcriptional activity by PBP was due entirely to an increase in the rate constant for promoter clearance. Thus, under our conditions, the purified PBP had no effect on the rate of closed preinitiation complex formation or for the closed complex to open complex transition. Instead, the rate of productive initiation leading to the 180-nucleotide transcript was stimulated by PBP. We found that the rate of closed preinitiation complex formation was not in rapid equilibrium with promoter and RNA polymerase II, in contrast to the model with prokaryotic RNA polymerase transcription. The results also indicated that PBP binding to the ATF/CRE is required for the stimulation of promoter clearance. These studies define the kinetic mechanism of a purified ATF/CRE-binding protein in stimulation of the in vitro transcription of a designed mammalian promoter.

Activating Transcription Factor 2↗
PubMed1994

Studies on primer binding of HIV-1 reverse transcriptase using a fluorescent probe.

The fluorescent nucleotide analog, 2',3'-trinitrophenyladenosine-5'-triphosphate (TNP-ATP), was utilized to quantify the affinities of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) for its substrates. Interaction of this probe with the enzyme brings about a twofold increase in the magnitude of fluorescence emission from the probe, and a blue-shift in wavelength maximum, from 561 to 553 nm. TNP-ATP binds HIV-1 RT with a dissociation constant of 21 microM. The presence of millimolar levels of deoxynucleoside triphosphates or micromolar levels of an oligonucleotide primer analogue, p(dT)12-18, suppressed this enhancement of fluorescence. The fact that inhibition was achieved with much lower levels of primer than of dNTPs suggests that TNP-ATP is a probe for the binding site of primer on the enzyme, rather than that of deoxynucleoside triphosphate. In support of this, the effect of TNP-ATP on the kinetics of DNA synthesis catalyzed by the enzyme indicated that the probe is a competitive inhibitor with respect to template-primer. The ability of primers and primer analogs to reverse the fluorescence enhancement was determined, and the corresponding affinities of these compounds for reverse transcriptase were calculated. The affinity increased with primer length, increasing more than 50-fold from a span of 5 to 15 nucleotide residues. The interaction of polydeoxynucleotides was consistent with a model in which the enzyme bound at adjacent internal sites of about 15 residues in length. Several mammalian and bacterial transfer RNA primers were tested, including the natural primer, tRNA(3Lys). The affinities were found to be between 0.55 and 1.2 microM, with no obvious selectivity for the natural primer, which had a Kd of 0.79 microM. These results are discussed within the context of data for HIV-1 RT obtained by other methodologies.

Adenosine Triphosphate↗
PubMed1994

Site-directed mutagenesis of HIV reverse transcriptase to probe enzyme processivity and drug binding.

Site-directed mutagenesis has demonstrated that changes within the human immunodeficiency virus reverse transcriptase coding sequence alone can account for viral resistance to inhibitors. Inhibitor sensitivity of mutant enzymes in vitro correlates with the sensitivity of the virus to non-nucleoside inhibitors observed in vivo, but this is not the case with nucleoside analogs. Recent structural, kinetic, and site-directed mutagenesis studies demonstrate the importance of enzyme-nucleic acid contacts in determining enzyme sensitivity to inhibitors in vitro, as well as how accurately the reverse transcriptase synthesizes DNA.

Antiviral Agents↗
PubMed1993

Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme.

We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.

Amino Acid Sequence↗
PubMed1993

HIV-1 reverse transcriptase: inhibition by 2',5'-oligoadenylates.

2',5'-Oligoadenylates (2-5A) and derivatives are noncompetitive inhibitors of primer/HIV-1 reverse transcriptase complex formation. The mechanism and specificity of this inhibitory action of 2-5A and 2-5A derivatives have been evaluated with 2-5A molecules modified in ribosyl moiety, chain length, extent of 5'-phosphorylation, and 2',5'-phosphodiester linkage. UV covalent cross-linking of preformed complexes of p66/p66 homodimer or p66/p51 heterodimer recombinant HIV-1 reverse transcriptase and the primer analog pd(T)16 allowed analysis of the initial step in HIV-1 reverse transcriptase-catalyzed DNA synthesis. Utilizing this primer binding assay, it is demonstrated that 2-5A and 2-5A derivatives inhibit the binding of pd(T)16 to HIV-1 reverse transcriptase. This inhibition is specific for the 2',5'-internucleotide linkage in that the corresponding 3',5'-adenylate derivatives do not exhibit inhibitory activity. Enhanced inhibitory properties were observed following modifications of the 2-5A molecule which result in an increase in hydrophobicity. Replacement of the D-ribosyl moiety of 2-5A with the 3'-deoxyribosyl moiety increased the inhibition of primer/HIV-1 reverse transcriptase complex formation 15-20%. 2',5'-Phosphorothioate substitution yielded the most effective inhibitors, with Ki's of 7-13 microM. In all cases, inhibition of primer/HIV-1 reverse transcriptase complex formation showed a preference for the 5'-triphosphate moiety. Nonphosphorylated derivatives were not inhibitory; 5'-monophosphate derivatives exhibited little or no inhibition. The inhibition of primer binding to HIV-1 reverse transcriptase correlated well with the inhibition of DNA-directed DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1993

Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase beta by UV cross-linking.

Rat DNA polymerase beta (beta-pol) is a 39-kDa monomeric protein, organized in two structurally and functionally distinct domains. The 8-kDa NH2-terminal domain binds single-stranded (ss) DNA, whereas the 31-kDa COOH-terminal domain does not. To facilitate studies on ssDNA binding structure-function relationships of beta-pol, we overexpressed the 8-kDa domain in Escherichia coli, and purified the recombinant protein to homogeneity. Single-stranded nucleic acid binding of the recombinant 8-kDa domain was found to be similar to that previously reported for the 8-kDa fragment prepared by proteolysis of intact beta-pol (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P. Karpel, R. L., and Wilson, S. H. (1990b) J. Biol. Chem. 265, 2124-2131; Casas-Finet, J. R., Kumar, A., Morris, G., Wilson, S. H., and Karpel, R. L. (1991) J. Biol. Chem. 266, 19618-19625). Residues in or near the DNA-binding pocket of the recombinant 8-kDa domain were examined by photochemical cross-linking to [32P] p(dT)16. Cross-linking was localized to a tryptic fragment spanning residues 28 through 35 and a V8 protease fragment spanning residues 27 through 58. Sequence analysis of the various [32P]p(dT)16-labeled proteins indicated that Ser30 and His34 were modified by cross-linking to p(dT)16. Therefore, these residues of the ssDNA-binding domain of beta-pol appear to be in close contact with this nucleic acid probe.

Amino Acid Sequence↗