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)

The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in the trp A gene. Screening of the resulting clones allowed selection of non-interactive mutant alpha subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: alpha 126 valine (GTG)----glutamic acid (GAG) and alpha 128 valine (GTT)----aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as alpha 66 asparagine (AAC)----aspartic acid (GAC); alpha 109 lysine (AAA)----arginine (AGA); alpha 118 cysteine (TGC)----arginine (CGC). Where possible, we individually assessed the importance of these residues in alpha beta interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.
Mol Gen Genet 1992 May
PMID:Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit. 160 55

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.
J Mol Biol 1992 Jun 20
PMID:Telomere end-replication problem and cell aging. 161 1

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.
J Mol Biol 1992 Jul 05
PMID:Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity. 161 61

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.
Mol Cell Biol 1991 Sep
PMID:Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site. 165 64

Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD') that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at approximately 180 copies per uninduced cell and was measured at approximately 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with approximately 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.
Mol Gen Genet 1991 Sep
PMID:Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage. 165 3

(+/-)-(1 alpha,2 beta,3 alpha)-9-[2,3-Bis(hydroxymethyl)cyclobutyl] guanine [(+/-)-BHCG] is a nucleoside analog with potent in vitro activity against herpesviruses [Tetrahedron Lett. 30:6453-6456 (1989)]. The two enantiomers have been synthesized, and their biochemical characterization is reported here. [1S(1 alpha,2 beta,3 alpha)]-9-[2,3-Bis(hydroxymethyl)cyclobutyl]guanine [(S)-BHCG] was phosphorylated by herpes simplex virus type 1 (HSV-1) thymidine kinase (Vmax = 8 nmol/hr/micrograms of enzyme), whereas [1R(1 alpha,2 beta,3 alpha)]-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine [(R)-BHCG] was a poor substrate for the viral thymidine kinase under these conditions. The triphosphate of each enantiomer was enzymatically synthesized, and both enantiomers competitively inhibited HSV-1 DNA polymerase with respect to dGTP. However, the potency of (R)-BHCG-TP was 4 orders of magnitude greater than that of (S)-BHCG-TP. (R)-BHCG-TP inhibited HeLa DNA polymerase alpha, but the inhibition constant was 30-fold higher than that for the viral DNA polymerase. In comparison, (S)-BHCG-TP was a very poor inhibitor of DNA polymerase alpha. (R)-[3H]BHCG-TP could be incorporated into a synthetic DNA template by HSV-1 DNA polymerase at 80% the extent of dGTP under the assay conditions used and, therefore, could act as an alternative substrate. Incorporation of (R)-BHCG-TP was similar to that observed for acyclovir triphosphate and ganciclovir triphosphate, based on maximal velocities. In contrast, HSV-1 DNA polymerase did not incorporate (S)-BHCG-TP into DNA. Compared with dGTP, only limited extension (10%) of the DNA primer by HSV-1 DNA polymerase occurred after incorporation of (R)-BHCG-TP and, therefore, (R)-BHCG-TP acts as a nonobligate chain terminator.
Mol Pharmacol 1991 Oct
PMID:Inhibition of herpes simplex virus type 1 DNA polymerase by [1R(1 alpha,2 beta,3 alpha)]-9-[2,3-bis(hydroxymethyl)cyclobutyl] guanine. 165 94

Sequence-Independent, Single-Primer Amplification (SISPA) is a primer initiated technique that requires target sequence modification to achieve the non-selective logarithmic amplification of heterogeneous DNA populations. The method contrasts with the polymerase chain reaction (PCR), and its modified approaches, that have as their objective the amplification of unique or homologous sequences. SISPA requires the directional ligation of an asymmetric linker/primer oligonucleotide onto the target population of blunt ended DNA molecules. The common end sequence allows one strand of the double-stranded linker/primer to be used in repeated rounds of annealing, extension and denaturation in the presence of thermostable Taq DNA polymerase. The linker/primers contain restriction endonuclease sites to facilitate the molecular cloning of as little as 1 pg of starting material after amplification. SISPA is especially useful when the nucleotide sequence of the desired molecule is both unknown and present in limited amounts making its recovery by standard cloning procedures technically difficult. These conditions are present in the initial isolation and cloning of previously uncharacterized viral genomes. The application and quantitation of SISPA is described, together with its utility in the cloning and recovery of low abundance genetic sequences, as illustrated here with the hepatitis C virus.
Mol Cell Probes 1991 Dec
PMID:Sequence-independent, single-primer amplification (SISPA) of complex DNA populations. 166 49

Changes in the expression pattern of the DNA polymerase beta gene during inhibition of spermatogenesis by busulphan and by temperature (artificial cryptorchidism) have been studied. Transient arrest of spermatogenesis in two-month-old rats after injection of a single dose of busulphan (10 mg/kg) resulted in parallel but transient decrease in the 1.4 kb of beta-pol mRNA level to an undetectable value, followed by its reappearance after resumption of spermatogenesis. An artificial cryptorchidism also caused a drastic decrease of beta-pol mRNA level. Both results as well as morphological examination of testis after busulphan injection and artificial cryptorchidism revealed that spermatocytes and spermatids represent the testicular cell fraction containing the elevated amount of beta-pol mRNA. Involvement of DNA polymerase beta in meiotic recombination is discussed.
Mol Biol Rep 1991 Feb
PMID:Effect of busulphan treatment and elevated temperature on the expression of the beta-pol gene in rat testis. 167 54

2',3'-Dideoxyuridine (ddUrd) exhibits poor if any anti-human immunodeficiency virus (HIV) activity in ATH8 and MT-4 cells. This is in agreement with the failure of ddUrd to be efficiently anabolized intracellularly to its 5'-triphosphate metabolite. However, 2',3'-dideoxyuridine-5'-triphosphate (ddUTP) proved to be a potent and selective inhibitor of the reverse transcriptase of HIV (Ki, 0.05 microM) and avian myeloblastosis virus (Ki, 1.0 microM). Bacterial DNA polymerase I, mammalian DNA polymerase alpha, terminal deoxyribonucleotidyl transferase, and Moloney murine leukemia virus reverse transcriptase were resistant to ddUTP. ddUTP is incorporated into the growing DNA chain principally at dTTP sites and inhibits further elongation. The potential of ddUTP as an anti-HIV therapeutic agent merits further investigation. However, to achieve this goal, it will be necessary to resort to techniques capable of delivering preformed phosphorylated ddUrd to the susceptible cells.
Mol Pharmacol 1990 Feb
PMID:Potent DNA chain termination activity and selective inhibition of human immunodeficiency virus reverse transcriptase by 2',3'-dideoxyuridine-5'-triphosphate. 168 52

The enzymes of DNA polymerization and DNA precursor synthesis are assembled in the replitase complex during the S phase of the cell cycle. Cross-inhibition is a phenomenon shown by enzymes of the replitase complex, in which inhibition of one enzyme of the complex leads to inhibition of a second, unrelated enzyme. This inhibition occurs only in vivo and only during S phase. The second enzyme shows no inhibition in vitro. In this study, using Chinese hamster embryo fibroblast cells, we have shown that direct allosteric interactions, i.e., structural interaction from a remote site within the replitase complex, is the cause of cross-inhibition of thymidylate synthase activity by the inhibitors of ribonucleotide reductase and DNA polymerase, because disruptions of the deoxynucleotide pools, which would be predicted for alternative explantations, do not occur. Cross-inhibition of DNA polymerase by hydroxyurea is demonstrated by the cessation of DNA synthesis when ribonucleotide reductase block is circumvented by the provision of all four deoxynucleosides. In addition to the cross-inhibition for thymidylate synthase and DNA polymerase, we have also presented evidence, on the basis of alterations of the in vivo conversion of deoxyuridine to dUMP, that cross-inhibition also occurs for the enzyme thymidine kinase. This conclusion is further supported by the lack of inhibition of the similar process in RNA synthesis, because enzymes of RNA synthesis are not included in the replitase complex. To facilitate the measurements, we have introduced a novel method of distinguishing between thymidine and deoxyuridine derivatives, making use of the fact that a tritium label placed in the 5'-position of deoxyuridine is removed on conversion to thymidine by methylation, whereas a tritium placed in the 6'-position is not.
Mol Pharmacol 1990 Jul
PMID:Allosteric interaction of components of the replitase complex is responsible for enzyme cross-inhibition. 169 15


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