Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequence specificity of interstrand cross-links induced in DNA by mononuclear and dinuclear platinum complexes in a 49-base-pair DNA duplex has been determined directly. This new assay takes advantage of the fact that 3'-->5' exonuclease digestion of randomly platinated DNA produces a pool of fragments of different lengths. This treatment allows identification of the spectrum of adducts impeding the exonuclease scission. Interstrand cross-linked adducts produce fragments that may remain complementary in the proximity of the binding site. As a result, these fragments may act as primer templates for extension upon subsequent treatment with a DNA polymerase. This extension increases the size of the oligonucleotide fragments, which may be evidenced by a more slowly migrating band on a sequencing gel. Concomitantly, the original band corresponding to the digested cross-link decreases in intensity. Therefore, comparison of a sequencing gel after digestion only and after the "digestion-extension" treatment should show the disappearance, or diminished band intensity, of only those fragments with interstrand cross-links. This approach was applied to the analysis of DNA interstrand cross-links formed by cis-[PtCl2(NH3)2] (cis-DDP) and [(trans-PtCl(NH3)2)2H2N(CH2)4NH2]Cl2. Cis-DDP was confirmed to form interstrand cross-links at d(GC) sequences but, interestingly, interstrand cross-links predominated in a sequence GCGG, with possible 1,3-intrastrand but no 1,2-intrastrand cross-links forming. The dinuclear compound formed 1,2, 1,3, and 1,4 DNA interstrand cross-links between guanines on opposite strands. In 1,3 and 1,4 cross-links, the guanines are separated by one and two base pairs, respectively, whereas a 1,2 cross-link is formed from guanines on neighboring base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sequence specificity of DNA-DNA interstrand cross-link formation by cisplatin and dinuclear platinum complexes. 818 Jan 63

3'-Amino-2',3'-dideoxycytidine (3'-NH2-ddCyd) is a 3'-modified deoxycytidine analog that specifically inhibits DNA synthesis. Inhibition of chain elongation at the replication fork was examined utilizing a batch hydroxylapatite chromatography method. Exponentially growing cells were exposed to 3'-NH2-ddCyd and the diterpene aphidicolin for 9.5 hr at concentrations that inhibited DNA synthesis by approximately 60 and 90%, as determined by precursor uptake. Both agents demonstrated a concentration-dependent inhibition of pulse labeling of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) generated by a limited alkaline lysis procedure. Upon removal of drug, the rate of elongation of pulse-labeled DNA was similar to that of untreated cells at both concentrations of aphidicolin and at the low concentration of the amino analog. Under these conditions, no reduction in cell survival was observed using the clonogenic assay technique. However, at the high concentration of 3'-NH2-ddCyd, the rate of elongation following drug removal was one-third that of untreated cultures, and a 50% loss in cell viability was observed. Furthermore, upon incubation of purified dsDNA with the Klenow fragment of Escherichia coli DNA polymerase I or purified ssDNA with calf thymus terminal deoxynucleotidyl transferase, only DNA from cells treated with the high concentration of 3'-NH2-ddCyd served as a poor template for further synthesis. The results indicate that 3'-NH2-ddCyd, in a concentration-dependent manner, inhibits DNA synthesis by reducing the rate of chain elongation at the replication fork, which subsequently leads to a functional blocking of 3'-ends in DNA. The data suggest that there may be a relationship between loss of cell viability and reduction in the number of 3'-ends available for DNA replication.
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PMID:Effect of 3'-amino-2',3'-dideoxycytidine on DNA replicative intermediates. 818 37

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.
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PMID:Identification of residues in the single-stranded DNA-binding site of the 8-kDa domain of rat DNA polymerase beta by UV cross-linking. 822 85

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.
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PMID:Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme. 826 41

The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.
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PMID:The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication. 828 32

We report the crystal structure of an NH2-terminal 388-residue fragment of T4 DNA polymerase (protein N388) refined at 2.2 A resolution. This fragment contains both the 3'-5' exonuclease active site and part of the autologous mRNA binding site (J. D. Karam, personal communication). The structure of a complex between the apoprotein N388 and a substrate, p(dT)3, has been refined at 2.5 A resolution to a crystallographic R-factor of 18.7%. Two divalent metal ion cofactors, Zn(II) and Mn(II), have been located in crystals of protein N388 which had been soaked in solutions containing Zn(II), Mn(II), or both. The structure of the 3'-5' exonuclease domain of protein N388 closely resembles the corresponding region in the Klenow fragment despite minimal sequence identity. The side chains of four carboxylate residues that serve as ligands for the two metal ions required for catalysis are located in geometrically equivalent positions in both proteins with a rms deviation of 0.87 A. There are two main differences between the 3'-5' exonuclease active site regions of the two proteins: (I) the OH of Tyr-497 in the Klenow fragment interacts with the scissile phosphate in the active site whereas the OH of the equivalent tyrosine (Tyr-320) in protein N388 points away from the active center; (II) different residues form of the binding pocket for the 3'-terminal bases of the substrate. In the protein N388 complex the 3'-terminal base of p(dT)3 is rotated approximately 60 degrees relative to the position that the corresponding base occupies in the p(dT)3 complex with the Klenow fragment. Finally, a separate domain (residues 1-96) of protein N388 may be involved in mRNA binding that results in translational regulation of T4 DNA polymerase (Pavlov & Karam, 1994).
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PMID:Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions. 867 62

The tau subunit dimerizes DNA polymerase III via interaction with the alpha subunit, allowing DNA polymerase III holoenzyme to synthesize both leading and lagging strands simultaneously at the DNA replication fork. Here, we report a general method to map the limits of domains required for heterologous protein-protein interactions using surface plasmon resonance. The method employs fusion of a short biotinylation sequence at either the NH2 or COOH terminus of the protein to be immobilized on streptavidin-derivatized biosensor chips. Inclusion of a hexahistidine sequence permits rapid purification and separation of the fusion protein from the endogenous Escherichia coli biotin carboxyl carrier protein. Ten deletions of the alpha subunit were constructed and purified by Ni2+-nitrilotriacetic acid chromatography and, when required, monomeric avidin chromatography. Each alpha deletion protein was captured by streptavidin immobilized on a Pharmacia Biosensor BIAcore chip, and the tau binding activity of each alpha deletion was analyzed using surface plasmon resonance. The tau subunit bound very tightly to a full-length amino-terminal fusion of the biotinylation sequence with alpha (KD approximately 70 pm). Four additional NH2-terminal alpha deletion proteins (60, 240, 360, and 542 residues deleted) retained strong binding activity to the tau subunit (KD = 0.19-0.39 nM), whereas deletion of 705 residues or more from the NH2 terminus of the alpha subunit abolished tau binding activity. Full-length alpha that contained a carboxyl-terminal fusion with the biotinylation sequence bound tau strongly (KD = 0.37 nM). However, deletion of 48 amino acids from the COOH terminus totally eliminated tau binding. These results indicate that the COOH-terminal half of the alpha subunit is involved in tau interaction.
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PMID:Biotin tagging deletion analysis of domain limits involved in protein-macromolecular interactions. Mapping the tau binding domain of the DNA polymerase III alpha subunit. 870 19

Rapid and processive DNA synthesis by Escherichia coli DNA polymerase III holoenzyme is achieved by the direct interaction between the alpha subunit of DNA polymerase III core and the beta sliding clamp (LaDuca, R. J., Crute, J. J., McHenry, C. S., and Bambara, R. A. (1986) J. Biol. Chem. 261, 7550-7557; Stukenberg, T. P., Studwell-Vaughan, P. S., and O'Donnell, M. (1991) J. Biol. Chem. 266, 11328-11334). In this study, we localized the beta-binding domain of alpha to a carboxyl-terminal region by quantifying the interaction of beta with a series of alpha deletion proteins. Purification and binding analysis was facilitated by insertion of hexahistidine and short biotinylation sequences on the deletion terminus of alpha. Interaction of beta with alpha deletion proteins was studied by gel filtration and surface plasmon resonance. alpha lacking 169 COOH-terminal residues still possessed beta-binding activity; whereas deletion of 342 amino acids from the COOH terminus abolished beta binding. Deletion of 542 amino acids from the NH2 terminus of the 1160 residue alpha subunit resulted in a protein that bound beta 10-20-fold more strongly than native alpha. Hence, portions of alpha between residues 542 and 991 are involved in beta binding. DNA binding to alpha apparently triggers an increased affinity for beta (Naktinis, V., Turner, J., and O'Donnell, M. (1996) Cell 84, 137-145). Our findings extend this observation by implicating the amino-terminal polymerase domain in inducing a low affinity taut conformation in the carboxyl-terminal beta-binding domain. Deletion of the polymerase domain (or, presumably, its occupancy by DNA) relaxes the COOH-terminal domain, permitting it to assume a conformation with high affinity for beta.
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PMID:Identification of the beta-binding domain of the alpha subunit of Escherichia coli polymerase III holoenzyme. 870 20

We report the identification of the PPS1 gene of Saccharomyces cerevisiae. The deduced amino acid sequence of PPS1p shows similarity with protein-tyrosine phosphatases (PTPases) and is most closely related to a subfamily of PTPases that are capable of dephosphorylating phosphoseryl and phosphothreonyl residues as well as phosphotyrosyl residues. Analysis of the predicted amino acid sequence suggests that the protein consists of an active phosphatase domain, an inactive phosphatase-like domain, and an NH2-terminal extension. Mutation of the catalytic cysteinyl residue in the active phosphatase domain reduced the in vitro activity of the mutant protein to less than 0.5% of wild type activity, while mutation of the corresponding cysteinyl residue of the inactive phosphatase-like domain had no effect on in vitro activity. The PPS1 protein was expressed in Escherichia coli, and the protein was shown to catalyze the hydrolysis of p-nitrophenyl phosphate, dephosphorylate phosphotyrosyl, and phosphothreonyl residues in synthetic diphosphorylated peptides and to inactivate the human ERK1 protein. PPS1 transcript abundance is coregulated with that of the divergently transcribed DPB3 gene, which codes for a subunit of DNA polymerase II, with both transcripts showing peak abundance in S phase. pps1Delta mutant strains did not differ from PPS1 strains under any of the conditions tested, but overexpression of the PPS1 protein in S. cerevisiae led to synchronous growth arrest and to aberrant DNA synthesis. A screen for suppressors of this growth arrest identified the RAS2 gene as a multicopy suppressor of the PPS1p overexpression arrest. The arrest was not suppressed by the presence of multicopy RAS1, TPK2, or TPK3 genes or by the presence of 5 mM cAMP in the growth medium, suggesting that PPS1 functions in a pathway involving RAS2, but not TPK kinases or adenylate cyclase.
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PMID:The PPS1 gene of Saccharomyces cerevisiae codes for a dual specificity protein phosphatase with a role in the DNA synthesis phase of the cell cycle. 908 70

The catalytic subunit of human DNA polymerase (pol) delta was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells. The recombinant enzyme was separated from endogenous DNA polymerases by phosphocellulose, Mono Q-Sepharose, and single-stranded DNA-cellulose chromatography. Recombinant DNA pol delta was also purified by immunoaffinity chromatography. The enzymatic properties of the purified catalytic subunit were characterized. The enzyme was active and possessed both DNA polymerase and associated 3' to 5' exonuclease activities. NH2-terminal deletion mutants retained polymerase activity, whereas the core and COOH-terminal deletion mutants were devoid of any measurable activities. Coinfection of Sf9 cells with recombinant baculovirus vectors for pol delta and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with 32Pi showed that the recombinant catalytic subunit of pol delta could be hyperphosphorylated by G1 phase-specific cdk-cyclins. When cdk2 was coexpressed with pol delta in Sf9 cells, pol delta was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol delta showed that the NH2-terminal region was essential for this interaction. Coimmunoprecipitation and Western blot experiments in Molt 4 cells confirmed the interaction in vivo. Preliminary experiments showed that phosphorylation of the catalytic subunit of pol delta by cdk2-cyclins had little or no effect on the specific activity of the enzyme.
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PMID:Characterization of the p125 subunit of human DNA polymerase delta and its deletion mutants. Interaction with cyclin-dependent kinase-cyclins. 954 86


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