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 (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.
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PMID:The human DNA polymerase beta gene structure. Evidence of alternative splicing in gene expression. 791 64

Replication protein A (RPA), also known as human single-stranded DNA-binding protein, is a three-subunit protein complex with multiple functions. Here, we investigated the role of the 70-kDa RPA subunit (p70) in DNA replication, by generating a series of deletion mutants. Mutant p70, which lacked 50 amino acids at the C-terminus, failed to interact with the 11-kDa RPA subunit (p11) and, when deleted further at the C terminus, was unable to interact with either the 34-kDa subunit (p34) or with p11, suggesting that p70 directly interacts with both p34 and p11. Studies with purified RPA mutants indicated that deletions at the N-terminal domain of p70 had very little effect on RPA's single-stranded DNA (ssDNA) binding activity, whereas deletion of amino acids 169-246 significantly weakened the DNA binding ability of RPA. By deleting amino acids 296-373 or 374-458, we totally abolished p70's ssDNA binding activity, suggesting that multiple p70 domains are involved in DNA binding. Two p70 domains, the N-terminal domain and the DNA binding domain, were required to stimulate DNA polymerase (pol) alpha, yet the DNA binding domain alone supported pol delta activity. Interestingly, RPA containing p70 with a zinc-finger domain deletion retained its DNA binding activity, but inhibited pol alpha and delta activity. RPA that lacked ssDNA binding activity failed to support simian virus 40 (SV40) DNA replication in vitro, whereas mutant RPA that lacked pol alpha stimulatory activity (including the zinc-finger p70 mutant) functioned normally. We conclude that RPA's DNA binding activity, but not its pol alpha stimulatory activity, is required for DNA replication.
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PMID:Role of the 70-kDa subunit of human replication protein A (I). Single-stranded dna binding activity, but not polymerase stimulatory activity, is required for DNA replication. 866 11

The DNA polymerase beta gene (POLB), which encodes a DNA polymerase believed to be involved in short gap-filling DNA synthesis, has been mapped to the proximal region of 8p (8p12-p11), a region commonly deleted in bladder carcinoma and a wide variety of other neoplasms. Also mapped to this region (8p12-p11.2) is the gene encoding the beta isoform of the catalytic subunit of protein phosphatase 2A (PPP2CB), a major serine/threonine phosphatase thought to play a regulatory role in many cellular pathways. The known functions of these proteins make them good candidates for 8p tumor suppressor genes. To test this hypothesis, we assessed a series of bladder tumors and bladder tumor cell lines for sequence variation in POLB and PPP2CB. Single strand conformation polymorphism (SSCP) analysis and direct sequencing of POLB cDNA derived from cell lines and tumors, many with known deletions of proximal 8p, revealed one sequence variant that was shown to represent a normal sequence polymorphism. No tumor-specific sequence variants were identified. The promotor sequence in genomic DNA from tumors with 8p LOH was also screened by SSCP. Four polymorphisms were identified but no tumor-specific mutations were found. PPP2CB was analyzed by SSCP analysis of all 7 coding exons in genomic DNA of bladder tumors and cell lines. Polymorphisms were detected in exons 4 and 5 but no tumor-specific mutations were found. We conclude that these genes are unlikely to be the suppressor genes for bladder cancer targeted by deletions of chromosome arm 8p.
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PMID:Mutation analysis of 8p genes POLB and PPP2CB in bladder cancer. 907 3

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase alpha-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys-->Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase delta activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2-3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.
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PMID:In vitro analysis of the zinc-finger motif in human replication protein A. 988 30

Primed in situ labeling (PRINS) can be used to localize single-copy genes and unique sequences. Using a modified PRINS method that incorporates multiple primers for the same sequence, single-step annealing and extension, anti-Taq DNA polymerase antibody, and stringent washing, we localized the human SRY gene to Yp11.31-p11.32 in chromosome preparations in situ. Locus-specific oligonucleotide probes (i.e., PRINS primers) were annealed to chromosomal DNA fixed on glass slides and extended in the presence of the four trinucleotide precursors, biotin-16-2'-deoxyuridine 5'-triphosphate, Tris-HCl, KCl, MgCl2, bovine serum albumin, and Taq DNA polymerase. After reaction with avidin-conjugated fluorophores, the resulting signals could be visualized by fluorescence microscopy in metaphase spreads and in interphase nuclei. This method could prove useful for unique sequences in general.
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PMID:PRINS for the detection of unique sequences. 1686 50

Primed in situ labeling (PRINS) is a sensitive and specific method that can be used for the localization of single copy genes and sequences too small for detection by conventional fluorescence in situ hybridization. By the use of PRINS, the human SRY gene was localized to Yp11.31-p11.32 and the SOX3 gene to Xq26-q27. In other studies, we localized specific deletions of RBM and DAZ, candidate genes for AZF (azoospermia factor) to proximal Yq11.2, the AZF region, in an infertile male. Locus-specific oligonucleotide probes (PRINS primers) were annealed to chromosomal DNA in situ and extended on preparations fixed on glass slides in the presence of dATP, dCTP, dGTP, dTTP, biotin-16-dUTP, and Taq DNA polymerase. After addition of avidin-conjugated fluorophore, signals were visualized by fluorescence microscopy in metaphase spreads from patients and controls. With further development, the PRINS method may prove useful for localization of single-copy genes, in general, and for the detection of gene deletions.
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PMID:PRINS for mapping single-copy genes. 1688 50

Bipolar disorder (BPD) is characterized by vulnerability to episodic depression and mania and spontaneous cycling. Because of marked advances in candidate-gene and genome-wide association studies, the list of risk genes for BPD is growing rapidly, creating an unprecedented opportunity to understand the pathophysiology of BPD and to develop novel therapeutics for its treatment. However, genetic findings are associated with major unresolved issues, including whether and how risk variance leads to behavioral abnormalities. Although animal studies are key to resolving these issues, consensus is needed regarding how to define and monitor phenotypes related to mania, depression and mood swing vulnerability in genetically manipulated rodents. In this study we discuss multiple facets of this challenging area, including theoretical considerations, available tests, limitations associated with rodent behavioral modeling and promising molecular-behavioral findings. These include CLOCK, glycogen synthase kinase 3beta (GSK-3beta), glutamate receptor 6 (GluR6), extracellular signal-regulated kinase-1 (ERK1), p11 (or S100A10), vesicular monoamine transporter 2 (VMAT2 or SLC18A2), glucocorticoid receptors (GRs), Bcl-2-associated athanogene-1 (BAG1) and mitochondrial DNA polymerase-gamma (POLG). Some mutant rodent strains show behavioral clusters or activity patterns that cross-species phenocopy objective/observable facets of mood syndromes, and changes in these clustered behaviors can be used as outcome measures in genetic-behavioral research in BPD.
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PMID:Translational research in bipolar disorder: emerging insights from genetically based models. 2014 20