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)

DNA polymerase beta (polbeta) is one of mammalian DNA polymerases and is known to be involved in a G:T/G:U mismatch repair. In order to investigate an involvement of this enzyme in a base excision repair, we searched a mutation of human polbeta in human gastric cancer and studied a function of the mutation. We observed cancer-specific missense mutations in 6 of 20 samples. All of these mutations were, however, heterozygous. We further analyzed the base excision repair activity of these mutants to know whether these mutants cause an error of mismatch repair. One of these mutants, which resulted in an amino acid substitution of Glu for Lys at codon 295, showed an inhibitory effect by in vitro base excision repair assay, suggesting that this mutation might play some role in carcinogenesis of the gastric mucosa.
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PMID:Functional mutation of DNA polymerase beta found in human gastric cancer--inability of the base excision repair in vitro. 1055 92

Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.
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PMID:The motif D loop of human immunodeficiency virus type 1 reverse transcriptase is critical for nucleoside 5'-triphosphate selectivity. 1058 59

The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.
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PMID:The E249K mutator mutant of DNA polymerase beta extends mispaired termini. 1058 71

Amino acid Lys(65) is part of the highly flexible beta3-beta4 loop in the fingers domain of the 66 kDa subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Recent crystal data show that the epsilon-amino group of Lys(65) interacts with the gamma-phosphate of the bound deoxynucleoside triphosphate ('dNTP') substrate [Huang, Chopra, Verdine and Harrison (1998) Science 282, 1669-1675]. In order to biochemically define the function of RT Lys(65), we have used site-specific mutagenesis to generate RT with a variety of substitutions at this position, including K65E, K65Q, K65A and K65R. Kinetic analyses demonstrate that if Lys(65) in RT is substituted with an amino acid other than arginine the enzyme exhibits dramatic decreases in the binding affinity (K(m)) for all dNTP substrates, in RT catalytic efficiency (k(cat)/K(m)) and in the mutant enzyme's ability to carry out pyrophosphorolysis, the reverse reaction of DNA synthesis. The pH optimum for the DNA polymerase activity of K65E RT was 6.5, compared to 7.5 for the wild-type enzyme, and 8.0 for the K65R, K65A and K65Q mutants. Molecular modelling studies show that mutations of Lys(65) do not affect the geometry of the loop's alpha-carbon backbone, but rather lead to changes in positioning of the side chains of residues Lys(70) and Arg(72). In particular, Glu in K65E can form a salt bridge with Arg(72), leading to the diminution of the latter residue's interaction with the alpha-phosphate of the dNTP residue. This alteration in dNTP-binding may explain the large pH-dependent changes in both dNTP-binding and catalytic efficiency noted with the enzyme. Furthermore, the K65A, K65Q and K65E mutant enzymes are 100-fold less sensitive to all dideoxynucleoside triphosphate ('ddNTP') inhibitors, whereas the K65R mutation results in a selective 10-fold decrease in binding of ddCTP and ddATP only. This implies that mutations at position 65 in HIV-1 RT influence the nucleotide-binding specificity of the enzyme.
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PMID:Mutational analysis of Lys65 of HIV-1 reverse transcriptase. 1079 16

DNA replication and repair require a specific mechanism to join the 3'- and 5'-ends of two strands to maintain DNA continuity. In order to understand the details of this process, we studied the activity of the 5' nucleases with substrates containing an RNA template strand. By comparing the eubacterial and archaeal 5' nucleases, we show that the polymerase domain of the eubacterial enzymes is critical for the activity of the 5' nuclease domain on RNA containing substrates. Analysis of the activity of chimeric enzymes between the DNA polymerases from Thermus aquaticus (TaqPol) and Thermus thermophilus (TthPol) reveals two regions, in the "thumb" and in the "palm" subdomains, critical for RNA-dependent 5' nuclease activity. There are two critical amino acids in those regions that are responsible for the high activity of TthPol on RNA containing substrates. Mutating glycine 418 and glutamic acid 507 of TaqPol to lysine and glutamine, respectively, increases its RNA-dependent 5' nuclease activity 4-10-fold. Furthermore, the RNA-dependent DNA polymerase activity is controlled by a completely different region of TaqPol and TthPol, and mutations in this region do not affect the 5' nuclease activity. The results presented here suggest a novel substrate binding mode of the eubacterial DNA polymerase enzymes, called a 5' nuclease mode, that is distinct from the polymerizing and editing modes described previously. The application of the enzymes with improved RNA-dependent 5' nuclease activity for RNA detection using the invasive signal amplification assay is discussed.
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PMID:RNA template-dependent 5' nuclease activity of Thermus aquaticus and Thermus thermophilus DNA polymerases. 1082 84

The structures of open and closed conformations of DNA polymerase beta (pol beta) suggests that the rate of single-nucleotide deletions during synthesis may be modulated by interactions in the DNA minor groove that align the templating base with the incoming dNTP. To test this hypothesis, we measured the single-base deletion error rates of wild-type pol beta and lysine and alanine mutants of Arg(283), whose side chain interacts with the minor groove edge of the templating nucleotide at the active site. The error rates of both mutant enzymes are increased >100-fold relative to wild-type pol beta. Template engineering experiments performed to distinguish among three possible models for deletion formation suggest that most deletions in repetitive sequences by pol beta initiate by strand slippage. However, pol beta also generates deletions by a different mechanism that is strongly enhanced by the substitutions at Arg(283). Analysis of error specificity suggests that this mechanism involves nucleotide misinsertion followed by primer relocation, creating a misaligned intermediate. The structure of pol beta bound to non-gapped DNA also indicates that the templating nucleotide and its downstream neighbor are out of register in the open conformation and this could facilitate misalignment (dNTP or primer terminus) with the next template base.
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PMID:Minor groove interactions at the DNA polymerase beta active site modulate single-base deletion error rates. 1085 Dec 38

The gene 32 protein (gp32) of bacteriophage T4 is the essential single-stranded DNA (ssDNA)-binding protein required for phage DNA replication and recombination. gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by DNA polymerase or recombination enzymes. The precise roles of gp32 affinity and cooperativity in promoting replication and recombination have yet to be defined, however. Previous work established that the N-terminal "B-domain" of gp32 is essential for cooperativity and that point mutations at Arg(4) and Lys(3) positions have varying and dramatic effects on gp32-ssDNA interactions. Therefore, we examined the effects of six different gp32 B-domain mutants on T4 in vitro systems for DNA synthesis and homologous pairing. We find that the B-domain is essential for gp32's stimulation of these reactions. The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e. wild-type gp32 approximately R4K > K3A approximately R4Q > R4T > R4G gp32-B. However, the functional defect of a particular mutant is often greater than can be explained simply by its ability to saturate the ssDNA at equilibrium, suggesting additional defects in the proper assembly and activity of DNA polymerase and recombinase complexes on ssDNA, which may derive from a decreased lifetime of gp32-ssDNA clusters.
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PMID:Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems. 1090 24

The nucleotide (nt) sequence of the Treponema denticola (Td) DNA gyrase beta-subunit gene (gyrB) has been determined. Southern blot analysis of Td chromosomal DNA indicated that gyrB is present as a single copy. Approximately 3.2kb of the nt sequence 5' and 0.7kb of nucleotide sequence 3' of gyrB were obtained. Analysis of the deduced amino acid (aa) sequence revealed two complete open reading frames (ORFs) (ORF1 and ORF3) and a truncated ORF (ORF4'). ORF1 has no homology to sequences in the databases, whereas ORF3 and ORF4' have significant homology to several bacterial DnaA (replication initiator) and DnaE (DNA polymerase III) proteins respectively. RT-PCR data showed that orf1-gyrB are co-transcribed, while dnaA-dnaE are co-transcribed but in the opposite direction. These data indicated that the gene organization of the Td gyrB region is unique compared with that of other bacteria. Eighteen putative DnaA boxes with several AT-rich regions were identified in the dnaA-dnaE intergenic region, and three putative DnaA boxes were identified in the gyrB-dnaA intergenic region. Spontaneous coumermycin A(1)-resistant Td mutants were isolated and characterized. The mutants have a >20-fold higher resistance to coumermycin A(1) than wild-type Td. A single point mutation in gyrB that changed GyrB Lys(136) to Glu or Thr appears to be responsible for the coumermycin A(1) resistance.
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PMID:Molecular characterization of the gyrB region of the oral spirochete, Treponema denticola. 1094 May 64

DNA polymerase slippage occurs frequently in tracts of a tandemly repeated nucleotide, and such slippage events can be genetically detected as frameshift mutations. In long mononucleotide runs, most frameshift intermediates are repaired by the postreplicative mismatch repair (MMR) machinery, rather than by the exonucleolytic proofreading activity of DNA polymerase. Although mononucleotide runs are hotspots for polymerase slippage events, it is not known whether the composition of a run and the surrounding context affect the frequency of slippage or the efficiency of MMR. To address these issues, 10-nucleotide (10N) runs were inserted into the yeast LYS2 gene to create +1 frameshift alleles. Slippage events within these runs were detected as Lys(+) revertants. 10G or 10C runs were found to be more unstable than 10A or 10T runs, but neither the frequency of polymerase slippage nor the overall efficiency of MMR was greatly influenced by sequence context. Although complete elimination of MMR activity (msh2 mutants) affected all runs similarly, analyses of reversion rates in msh3 and msh6 mutants revealed distinct specificities of the yeast Msh2p-Msh3p and Msh2p-Msh6p mismatch binding complexes in the repair of frameshift intermediates in different sequence contexts.
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PMID:Sequence composition and context effects on the generation and repair of frameshift intermediates in mononucleotide runs in Saccharomyces cerevisiae. 1101 7

Most translesion DNA synthesis (TLS) in Escherichia coli is dependent upon the products of the umuDC genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA-single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD'. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD' with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD' homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD' proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD(2) and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD(2) for self-cleavage.
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PMID:Genetic and biochemical characterization of a novel umuD mutation: insights into a mechanism for UmuD self-cleavage. 1111 35


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