Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A ribonuclease H which degrades RNA specifically in RNA-DNA hybrids and, moreover, stimulates its homologous DNA-polymerase-primase complex was purified from calf thymus. The enzyme consists of a single polypeptide of molecular mass 78 kDa. It requires divalent cations for activity, and prefers Mg2+ over Mn2+. Ribonuclease H is optimally active at neutral pH and in 75 mM potassium acetate and is strongly sensitive to N-ethylmaleimide. [3H]Poly(rA).poly(dT), [3H]poly(rC).poly(dI), and [3H]RNA.M13-DNA are degraded to 3-9-mer oligoribonucleotides with similar kinetics, whereas double- or single-stranded DNA, and double- and single-stranded RNA remain unaffected. The enzyme stimulates in vitro DNA synthesis by the immunoaffinity-purified calf-thymus DNA-polymerase-alpha-primase complex threefold. When ribonuclease H is present in a three-fold molar excess to the polymerase-primase complex, twice as much primer is formed as in the absence of ribonuclease H. Ribonuclease H also stimulates the elongation rate of DNA polymerase alpha by a factor of 2-3, independent of whether primase-primed DNA templates or templates primed with oligonucleotides are used. Our results suggest that this form of ribonuclease H is a likely candidate for a genuine primer-removing enzyme in mammalian cells.
 ...
PMID:A distinct form of ribonuclease H from calf thymus stimulates its homologous DNA-polymerase-alpha-primase complex. 255 72

A simple method for generating cDNA libraries has been described (1) in which RNase H-DNA polymerase I-mediated second-strand cDNA synthesis primes from an RNA oligonucleotide derived from the 5' (capped) end of mRNA. The size of this oligonucleotide and the fate of the information corresponding to the RNA during subsequent cloning have not been established. We show here that the 5'-most RNA primer varies in length from 8 to 21 nucleotides, and that information corresponding to the length of the RNA primer is normally lost during cloning. A modification of the second-strand cDNA synthesis procedure is described which allows cloning of all, or almost all, of the primer sequence information. In addition, we show that the presence of E. coli DNA ligase during second-strand cDNA synthesis can increase the length of the cDNA clones obtained from long RNAs. Cloning by addition of linkers provides the greatest chance of obtaining near full-length cDNA clones from long mRNAs.
 ...
PMID:Second-strand cDNA synthesis with E. coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E. coli DNA ligase. 283 25

The replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.
 ...
PMID:Complete enzymatic synthesis of DNA containing the SV40 origin of replication. 284 39

A series of antisera directed against amino acid sequences from different segments of the duck hepatitis B virus (DHBV) P-gene were shown to immunoprecipitate DHBV DNA molecules that were covalently linked to the DHBV DNA terminal protein. Restriction analysis and sizing after protease treatment demonstrated that the P-gene proteins were bound to the 5'-end of the DHBV DNA minus-strand which was mapped to a G-residue in the centre of the repeat sequence DR1. Resistance to alkali treatment indicated a phosphodiester linkage to tyrosine between protein and DNA. Limited protease treatment prior to immunoprecipitation cleaved C-terminal P-proteins from the viral DNA, indicating that the terminal protein forms a separate domain encoded in the N-terminal part of the P-gene. Functional analysis of a deletion mutant confirmed the notion that a non-essential spacer separates the terminal protein from the polymerase domain residing in the C-terminal half of the P-gene. Thus, the major proteins required for hepadnaviral reverse transcription, namely the primer, DNA polymerase, and possibly also RNase H, appear to be synthesized as a polyprotein precursor which is at least initially linked as such to its first DNA product.
 ...
PMID:The amino-terminal domain of the hepadnaviral P-gene encodes the terminal protein (genome-linked protein) believed to prime reverse transcription. 285 56

Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase H, which has been shown to be absolutely required for in vitro initiation of ColE1 DNA replication, is dispensable in vivo, and (ii) ColE1 replication in the absence of RNase H is not dependent on "stable DNA replication," which has been reported to be an alternative mode of chromosomal DNA replication. Another class of bacterial mutations was also isolated. These mutations, named herB, suppressed cer-6 replication in rnh+ bacteria. herB mutations mapped close to the polA gene on the E. coli chromosome and increased the activity of DNA polymerase I. These findings suggest that when the DNA polymerase I has an opportunity to initiate DNA synthesis before RNase H acts, the replication-defective cer-6 mutant or the wild-type ColE1 replicates in E. coli.
 ...
PMID:RNase H and replication of ColE1 DNA in Escherichia coli. 287 52

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.
 ...
PMID:Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit. 298 14

The enzymatic domains of the avian retrovirus polymerase (pol) gene have been mapped by the use of peptide antibodies and COOH-terminal amino acid analysis. The processed pol beta polypeptide is cleaved in vivo to yield alpha and pp32. Rabbit antibodies were directed against synthetic peptides whose sequence was deduced from the known pol sequence of Rous sarcoma virus, Prague C (Schwartz, D.E., Tizard, R., and Gilbert, W. (1983) Cell 32, 853-869). The RNase H active site of pol was located in the NH2-terminal region of the alpha DNA polymerase subunit. The COOH terminus of the alpha subunit was found to be immediately adjacent to the NH2 terminus of the pp32 pol protein. COOH-terminal amino acid analysis of pp32 revealed that this protein is also processed. From the deduced amino acid sequence of pol, it appears likely that pol encodes an additional 4100-dalton polypeptide located at its extreme COOH terminus. The enzymatic domains on beta appear to map in the following order: RNase H-DNA polymerase-DNA endonuclease. Hydrophilicity analysis and secondary structure predictions of wild type Rous sarcoma virus pol products and mutated pp32 possessing single amino acid changes permit further structural evaluation of the multifunctional pol protein.
 ...
PMID:Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains. 298 84

The replication of plasmid pBR322 DNA has been reconstituted with purified proteins from Escherichia coli. Initiation of the leading-strand requires RNA polymerase holoenzyme, DNA polymerase I, RNase H, and DNA gyrase. Initiation of the lagging-strand requires the primosomal proteins (the dnaB, dnaC, and dnaG proteins, replication factor Y (protein n') and proteins i, n, and n") and the single-stranded DNA binding protein. DNA polymerase III holoenzyme is required for extensive elongation of the nascent DNA chains. The products of this replication reaction are primarily nonsegregated daughter molecules. However, the addition of small amounts of soluble extract from E. coli results in the completion and segregation of these molecules to give mature form I DNA, suggesting that additional factors are required for this process. Topoisomerase I is necessary to make the replication system specific for pBR322 DNA as a template, indicating that the linking number of the DNA, determined by an equilibrium between the opposing activities of topoisomerase I and DNA gyrase, plays a crucial role in determining the reactivity of the DNA molecule toward initiating DNA replication. The function of the proteins involved in the replication of this closed-circular, double-stranded, superhelical DNA is discussed.
 ...
PMID:Replication of pBR322 DNA in vitro with purified proteins. Requirement for topoisomerase I in the maintenance of template specificity. 299 Dec 40

We determined the complete nucleotide sequence of the intracisternal A-particle gene, IAP-H18, cloned from the normal Syrian hamster liver DNA. IAP-H18 was 7,951 base pairs in length with two identical long terminal repeats of 376 base pairs at both ends. On the coding strand, imperfect open reading frames corresponding to gag and pol of the retrovirus genome were observed, whereas many stop codons were present in the region corresponding to env. The putative H18 gag gene (809 amino acids) had a sequence homologous to the N-terminal half of the mouse mammary tumor virus gag gene and locally to the Rous sarcoma virus gag gene. The putative H18 pol gene (900 residues) was homologous to the Rous sarcoma virus pol gene almost throughout the entire region. Two conserved regions among the retrovirus pol genes have been reported. One presumably corresponds to the DNA polymerase and the RNase H domain, and the other corresponds to the DNA endonuclease domain of the multifunctional protein pol. By the comparison of the deduced amino acid sequences of the putative endonuclease domain of six representative oncovirus genomes, a phylogenetic tree of the oncovirus genomes was constructed, and the intracisternal A-particle (type A) genome was found to be more closely related to the mouse mammary tumor virus (type B) and squirrel monkey retrovirus (type D) genomes.
 ...
PMID:Nucleotide sequence of the Syrian hamster intracisternal A-particle gene: close evolutionary relationship of type A particle gene to types B and D oncovirus genes. 299 63

An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second-strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein.
 ...
PMID:A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein. 299 85


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