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

Aphidicolin inhibits purified DNA polymerases-a and -d in vitro and inhibits mitosis in animal cells. The Chinese hamster V79 cell mutant, Aphr-4-2, was selected for its ability to form colonies in cultured medium supplemented with 1.0 microM aphidicolin. At this concentration, the parental wild-type V79 cells (clone 743x) have a survival rate of less than 10(-7). The mutant DNA polymerase-a is resistant to aphidicolin at concentrations that are inhibitory to the wild-type V79 DNA polymerase-a. The apparent Km for dCTP of the mutant DNA polymerase-a is consistently lower than that of the wild-type DNA polymerase-a. This mutant exhibits slow growth, mutator activity, hypersensitivity, and hypermutability to UV. We wanted to know the basis of UV hypersensitivity in this mutant. Using the antisera (UV2) raised against UV-induced thymidine dimers and a sensitive immunofluorescence assay to measure UV-induced thymidine dimers and with detection in ACAS 570 Workstation, we observed that 50% of the thymidine dimers disappeared within 5 h after irradiation and more than 80% of the dimers were removed within 24 h in both cell lines. These results indicate that the recognition, incision, and excision steps in nucleotide excision repair pathway are normal in the mutant. In order to know if there is a difference in DNA polymerase-a or -d activities in the parental V79(wt) and Aphr-4-2 cells, DNA polymerases were partially purified from the parental and the mutant cells using sequential centrifugation and column chromatographies on DEAE-cellulose (DE23 and DE52) to remove DNA polymerases-beta and -gamma. More than 90% of the enzymatic activities from both cells showed characteristics of DNA polymerase-a type on the basis of these criteria: sensitivity to butyl phenyl dGTP (1 microM) and to IgG raised against DNA polymerase-a (SJK 132-20). The results indicate that DNA replication involving a mutant DNA polymerase-a with altered affinity for dCTP may be responsible for the UV sensitivity and mutability of the mutant.
Somat Cell Mol Genet 1990 Jan
PMID:On the DNA polymerase-a mutant: immunofluorescence assay of UV-induced thymidine dimers in Aphr-4-2 cells. 210 26

Spontaneous mutants of mouse FM3A cells (AC1, AC2, and AC3), highly resistant to aphidicolin (3000-, 2500-, and 300-fold increase in resistance, respectively), were isolated by multistep selection. The DNA synthesizing activity in permeabilized cells of all three mutants was substantially resistant to aphidicolin, like that in intact cells. The DNA polymerase activity in nuclear extracts in AC1 and AC3, but not AC2, was resistant to aphidicolin. Partially purified DNA polymerase alpha from AC3, but not from AC1 or AC2, showed resistance to aphidicolin. The apparent Ki value for aphidicolin of AC3 polymerase alpha was three to four times that of the enzyme from the parent cells, but the apparent Km values of the enzyme for dCTP and dTTP were normal. All the mutants showed cross-resistance to both arabinofuranosyladenine and arabinofuranosylcytosine. The AC3 mutant had expanded deoxyribonucleoside triphosphate pools. On two-dimensional polyacrylamide gel electrophoresis, AC1 gave a new protein (mol wt 40 kDa). The aphidicolin-resistance trait was reversible in AC2, unlike in AC1 and AC3. These results show that in mammalian cells there are at least two mechanisms of aphidicolin-resistance that involve an altered DNA polymerase alpha that is resistant to aphidicolin and simultaneous expansion of the four DNA-precursor pools.
Somat Cell Mol Genet 1990 Sep
PMID:High level of aphidicolin resistance with multiple mutations in mouse FM3A cell mutants. 212 28

Both thymidine kinase (TK) and DNA polymerase (DNAp) are present in measurable amounts in human serum. Even though the use of TK as a clinical marker is rapidly increasing there has been no attempt to characterize the serum TK in a wider extent, i.e.; with respect to Mw or other biochemical parameters. Therefore sera with high TK or DNAp activities derived from patients with cytomegalovirus (CMV) infection, B12-deficiency and leukaemia were fractionated by gel exclusion chromatography. The TK activity eluted as two peaks, one major TK activity with an apparent molecular weight (Mw) or 730 kD and one minor TK activity corresponding to a Mw of 58 kD. The amount of TK activity at 58 kD varied between 7 and 23% of total activity, depending on the serum fractionated. The DNAp activity in sera from patients with malignant disease and B12 deficiency eluted as a single peak corresponding to a Mw of 240 kD. A DNAp with a different Mw (greater than 1000 kD) was recovered from 1 of 3 investigated immunosuppressed patients with CMV infection. A similar pattern of enzyme forms was observed when sera were separated by glycerol gradient centrifugation. The effect of high salt and various reaction solution components on the enzymes were studied. The only condition found that affected the molecular forms of TK was the state of reduction. Incubation of sera with high concentrations of dithioerythritol (DTE) (400 mM) prior to separation transferred all serum TK to the 58 kD form, it also converted most of the serum DNAp from the 240 kD form to a smaller form (56 kD) without affecting the total recovery of enzymatic activity. The reaction product from both TK forms was exclusively monophosphate and none of the TK forms could efficiently utilize cytidine triphosphate as phosphate donor. The substrate kinetics of the small serum TK fraction was identical with those of an enzyme with similar size purified from proliferating HeLa cells, indicating that both serum TK activities are forms of TK 1, the proliferation associated cellular isozyme.
Mol Cell Biochem 1990 Jan 18
PMID:Molecular forms in human serum of enzymes synthesizing DNA precursors and DNA. 215 79

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1). CEDU is preferentially phosphorylated by HSV-infected (Vero) cells, as compared with mock-infected cells or cells infected with a thymidine kinase-deficient strain of HSV-1. The end product of this phosphorylation process, CEDU 5'-triphosphate, is a competitive inhibitor of HSV-1 DNA polymerase activity and, to a lesser extent, of cellular DNA polymerase alpha activity. However, in the absence of the natural substrate dTTP, CEDU 5'-triphosphate also serves as an alternative substrate for viral and cellular DNA polymerase. When exposed to HSV-1-infected cells, [2-14C]CEDU was incorporated into both viral and cellular DNA. The extent to which [2-14C]CEDU was incorporated remained approximately constant over a concentration range of 0.5 to 50 microM. Within this concentration range, CEDU effected a concentration-dependent inhibition of viral DNA synthesis that closely paralleled the inhibition of viral progeny formation. It is postulated that CEDU owes (i) its selectivity as an antiviral agent to its preferential phosphorylation by the virus-infected cell and (ii) its antiviral potency to an inhibition of viral DNA synthesis at the level of the viral DNA polymerization reaction.
Mol Pharmacol 1990 May
PMID:Mechanism of action of 5-(2-chloroethyl)-2'-deoxyuridine, a selective inhibitor of herpes simplex virus replication. 216 59

beta-Polymerase is a vertebrate cellular DNA polymerase involved in gap-filling synthesis during some types of genomic DNA repair. We report that a cloned human beta-polymerase promoter in a transient expression assay is activated by p21v-rasH expression in NIH 3T3 cells. A decanucleotide palindromic element, GTGACGTCAC, at positions -49 to -40 in the promoter is required for this ras-mediated stimulation.
Mol Cell Biol 1990 Jul
PMID:Transfected human beta-polymerase promoter contains a ras-responsive element. 219 67

By using T4 DNA polymerase rather than S1 or Bal31 nuclease to clone yeast telomeres, very little telomeric DNA is lost. These clones were used to determine the DNA sequence of virtually the entire telomeric tract. Our results demonstrated that a slightly modified version, C2-3A(CA)1-6, of the consensus derived from sequence analysis of more-internal regions (J. Shampay, J. W. Szostak, and E. H. Blackburn, Nature [London] 310:154-157, 1984) extends to the very end of the chromosome. The sequence analysis also suggests that yeast telomeres consist of two domains: the proximal 120 to 150 base pairs, which appear to be protected from processes such as recombination, degradation, and elongation, and the distal portion of the telomere, which is more susceptible to these events.
Mol Cell Biol 1990 Aug
PMID:Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains. 219 53

The screening of a size-selected cDNA library from the ovary revealed the existence of a second form of PRL receptor in the rat. The polypeptide sequence deduced from cDNAs has a much longer cytoplasmic domain (357 amino acids) than the form previously identified in the liver (57 amino acids). Nucleotide sequence analysis and comparison with rabbit, mouse, and human PRL receptor cDNAs suggests that the two forms of rat PRL receptor result from alternative splicing of a primary transcript. Complementary DNAs encoding the long form of the receptor were also found in a library prepared from estradiol-treated rat liver, although they represent a minor fraction of total PRL receptor cDNAs obtained from this tissue. DNA polymerase chain reaction amplification of cDNA confirmed the presence of the two receptor forms in both the ovary and liver. Northern analysis, using probes that specifically hybridize with either form of mRNA, indicates a major transcript of 1.8 kilobases (kb) in estradiol-treated liver, which encodes the receptor with a short cytoplasmic domain, while the long form of the receptor is encoded by mRNAs of 2.5 and 3 kb. In the ovary, a complex pattern of hybridization to multiple mRNAs (1.8-5.5 kb) is obtained with the probe specific to the long form, and essentially only a 5.5-kb mRNA is obtained with the probe specific to the short form. The predicted size of the mature form of the long PRL receptor (PRL-R2) is 591 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1990 Aug
PMID:Expression of two forms of prolactin receptor in rat ovary and liver. 229 22

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.
J Mol Biol 1990 May 20
PMID:The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site. 234 8

The synthesis of the DNA polymerase of bacteriophage T4 is autogenously regulated. This protein (gp43), the product of gene 43, binds to a segment of its mRNA that overlaps its ribosome binding site, and thereby blocks translation. We have determined the Kd of the gp43-operator interaction to be 1.0 x 10(-9) M. The minimum operator sequence to which gp43 binds consists of 36 nucleotides that include a hairpin (containing a 5 base-pair helix and an 8 nucleotide loop) and a single-stranded segment that contains the Shine-Dalgarno sequence of the ribosome binding site. In the distantly related bacteriophage RB69 there is a remarkable conservation of this hairpin and loop sequence at the ribosome binding site of its DNA polymerase gene. We have constructed phage operator mutants that overproduce gp43 in vivo, yet are unchanged for in vivo replication rates and phage yield. We present data that show that the replicative and autoregulatory functions are mutually exclusive activities of this polymerase, and suggest a model for gp43 synthesis that links autoregulation to replicative demand.
J Mol Biol 1990 Jun 20
PMID:Autogenous translational operator recognized by bacteriophage T4 DNA polymerase. 235 22

Individual mRNA coding for proteins of the alpha-polymerase complex were isolated from replicating hepatocytes. cDNAs were synthesized by reverse transcriptase. Three clones were identified by colony hybridization of the S period cDNA library. The sequences of these clones were complementary to the investigated mRNAs. Two clones named pr12 and pr167 revealed increased expressions in the S period. The level of mRNA pr127 does not change during liver regeneration. The amount of pr127 mRNA was about 1% of the total mRNA population. pr12, pr127 and pr167 mRNAs were isolated by the hybrid-selection method and were translated in a cell-free system. The products of translation were analysed by "activity" gel. It was shown that pr167 mRNA coded for protein 140 kDa with DNA polymerase activity. pr12 protein is an unknown component of the alpha-polymerase complex. We suggested that this protein participates in the initiation of DNA replication.
Mol Biol (Mosk)
PMID:[Increased expression of genes coding for replication proteins during the period of DNA synthesis]. 240 40


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>