Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Repair of quercetin-induced single-strand breaks of DNA was studied in vitro using cell free extract from GM 00637D cells, a human cell line. Single-strand breaks were introduced into DNA by the treatment of closed circular, superhelical form of pBR322 plasmid DNA with quercetin and Cu(II). Repair of these breaks was demonstrated by agarose gel electrophoresis after incubating damaged DNA with cell extract, DNA polymerase I (klenow fragment of DNA polymerase), four deoxynucleotide triphosphates, ATP and T4 DNA ligase. The present results suggested that 1) the exonuclease is involved in the initiation of repair of quercetin-induced single-strand breaks by removing the 3' ends of quercetin damaged DNA and 2) oxygen free radicals are involved in quercetin-induced DNA strand scission.
Biochem Mol Biol Int 1994 Feb
PMID:Repair of quercetin-induced single-strand breaks by a cell free system. 801 39

We have used in vitro selection to investigate the sequence requirements for efficient template-directed ligation of oligonucleotides at 0 degrees C using a water-soluble carbodiimide as condensing agent. We find that only 2 bp at each side of the ligation junction are needed. We also studied chemical ligation of substrate ensembles that we have previously selected as optimal for ligation by RNA ligase or by DNA ligase. As anticipated, we find that substrates selected with DNA ligase ligate efficiently with a chemical ligating agent, and vice versa. Substrates selected using RNA ligase are not ligated by the chemical condensing agent and vice versa. The implications of these results for prebiotic chemistry are discussed.
J Mol Evol 1994 Jun
PMID:In vitro selection of optimal DNA substrates for ligation by a water-soluble carbodiimide. 808 82

Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements. When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs). Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences. Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays. This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding. Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR. The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA. The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold. Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE. HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences. The A- or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding. DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure. It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form. The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA.
Mol Cell Biol 1994 May
PMID:The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences. 816 86

A 28.5 kDa catalytic fragment of the uracil-DNA glycosylase DNA repair enzyme from Herpes simplex virus type 1 (HSV-1) has been crystallized using protein from a highly expressing Escherichia coli clone of the Herpes simplex virus type 1 UL2 gene. The protein crystallizes at 12 mg/ml from 11% (w/v) polyethylene glycol 8000 at pH values in the range 6.8 to 7.0, in the presence of (NH4)2SO4. Long trigonal rods (0.08 mm x 0.08 mm x > 0.5 mm) diffract beyond 3.0 A using a laboratory source. The enzyme crystallizes in P3(1) (or P3(2)) a = 65.3 A, c = 49.0 A with a single molecule in the asymmetric unit and an estimated solvent content of 41% by volume.
J Mol Biol 1993 Dec 05
PMID:Crystallization and preliminary X-ray analysis of the uracil-DNA glycosylase DNA repair enzyme from herpes simplex virus type 1. 825 88

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.
Mol Cell Biol 1994 Jan
PMID:An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. 826 37

A 248 residue C-terminal fragment of rat DNA polymerase beta (335 amino acid residues), a eukaryotic DNA repair enzyme, has been crystallized from polyethylene glycol 6000 solution. The crystals are orthorhombic, space group P2(1)2(1)2 with cell dimensions a = 120.3 A, b = 64.2 A, c = 39.4 A, and contain a single 31 kDa fragment in an asymmetric unit. The crystals diffract to 2.8 A resolution with laboratory X-ray source, and to 2.3 A resolution with synchrotron X-ray source, and are suitable for detailed structural analysis.
J Mol Biol 1994 Jan 28
PMID:Crystallization of 31 kDa C-terminal fragment of rat DNA polymerase beta. 830 96

Recent evidence indicates that 5-fluorouracil (5-FlUra) is incorporated into DNA and is removed by the DNA repair enzyme uracil DNA glycosylase. Synthetic oligonucleotides containing either a single uracil or 5-FlUra residue were constructed to examine the mechanisms by which human cells remove 5-FlUra from DNA. The human uracil DNA glycosylase excised uracil in a manner similar to that observed for the bacterial enzyme. In contrast, a significant difference was observed in their abilities to remove 5-FlUra. In particular, both the bacterial and normal human enzymes displayed 13-17-fold increases in their apparent Km values but the apparent Vmax values remained virtually constant. These results demonstrate that normal human cells possess a defined capacity to remove 5-FlUra incorporated into DNA. However, specific kinetic differences may exist that affect their capacity to remove 5-FlUra formed in DNA after treatment with this cancer chemotherapeutic agent.
Mol Pharmacol 1993 Jun
PMID:Mechanisms of excision of 5-fluorouracil by uracil DNA glycosylase in normal human cells. 831 18

Protein kinase activity was revealed in complex forms of rat liver DNA polymerase alpha containing 3'-5'-exonuclease, primase, helicase, DNA ligase. Protein kinase (mol. mass about 200 kDa) has been partially purified from a specimen of high molecular mass DNA polymerase alpha of nuclear membrane of regenerating liver. The protein kinase activity of the complex form of DNA polymerase alpha was maximal in the cytosol in normal rat liver cells and in the nuclear membrane in dividing cells (40 h after partial hepatectomy). The main phosphokinase properties of this enzyme were determined.
Mol Biol (Mosk)
PMID:[Isolation of protein phosphokinase from a complex form of DNA polymerase alpha from rat liver]. 831 39

To understand the influence of different factors on the efficiency of T4 DNA ligase-catalyzed connection of oligonucleotides, a comparative study of polycondensation of AT-containing concatemer octanucleotides (some of them potentially can form "parallel" duplexes) has been undertaken. It has been shown that the size of substrate duplex is the most significant factor, while the sequence of oligonucleotides influences the enzymatic reaction indirectly, by determining the thermal stability of double-stranded complexes. In practice, none of the octanucleotides under study does form "parallel" duplexes.
Mol Biol (Mosk)
PMID:[Features of connecting short oligonucleotides with phage T4 DNA ligase]. 831 45

The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.
Mol Cell Biol 1993 Sep
PMID:Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding. 835 88


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