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)

The Rad2, Rad3, Rad4, and Ss12 proteins are required for nucleotide excision repair in yeast cells and are homologs of four human proteins which are involved in a group of hereditary repair-defective diseases. We have previously shown that Rad3 protein is one of the five subunits of purified RNA polymerase II basal transcription initiation factor b (TFIIH) and that Ss12 protein physically associates with factor b (W.J. Feaver, J.Q. Svejstrup, L. Bardwell, A.J. Bardwell, S. Buratowski, K.D. Gulyas, T.F. Donahue, E.C. Friedberg, and R.D. Kornberg, Cell 75:1379-1387, 1993). Here we show that the Rad2 and Rad4 proteins interact with purified factor b in vitro. Rad2 (a single-stranded DNA endonuclease) specifically interacts with the Tfb1 subunit of factor b, and we have mapped a limited region of the Rad2 polypeptide which is sufficient for this interaction. Rad2 also interacts directly with Ss12 protein (a putative DNA helicase). The binding of Rad2 and Rad4 proteins to factor b may define intermediates in the assembly of the nucleotide excision repair repairosome. Furthermore, the loading of factor b (or such intermediates) onto promoters during transcription initiation provides a mechanism for the preferential targeting of repair proteins to actively transcribing genes.
Mol Cell Biol 1994 Jun
PMID:Yeast nucleotide excision repair proteins Rad2 and Rad4 interact with RNA polymerase II basal transcription factor b (TFIIH). 819 2

The human ERCC3 gene, which corrects specifically the nucleotide excision repair defect in human xeroderma pigmentosum group B and cross-complements the repair deficiency in rodent UV-sensitive mutants of group 3, encodes a presumed DNA helicase that is identical to the p89 subunit of the general transcription factor TFIIH/BTF2. To examine the significance of the postulated functional domains in ERCC3, we have introduced mutations in the ERCC3 cDNA by means of site-specific mutagenesis and have determined the repair capacity of each mutant to complement the UV-sensitive phenotype of rodent group 3 cells. A conservative substitution of arginine for the invariant lysine residue in the ATPase motif (helicase domain I), six deletion mutations in the other helicase domains, and a deletion in the potential helix-turn-helix DNA-binding motif fail to complement the ERCC3 excision repair defect of rodent group 3 mutants, which implies that the helicase domains as well as the potential DNA-binding motif are required for the repair function of ERCC3. Analysis of carboxy-terminal deletions suggests that the carboxy-terminal exon may comprise a distinct determinant for the DNA repair function. In addition, we show that a functional epitope-tagged version of ERCC3 accumulates in the nucleus. Deletion of the putative nuclear location signal impairs neither the nuclear location nor the repair function, indicating that other sequences may (also) be involved in translocation of ERCC3 to the nucleus.
Mol Cell Biol 1994 Jun
PMID:Mutational analysis of ERCC3, which is involved in DNA repair and transcription initiation: identification of domains essential for the DNA repair function. 819 50

In mammalian cells, the incision step of DNA excision repair triggers a dramatic metabolic response in chromatin. The reaction starts with the binding of a zinc-finger protein, i.e. poly(ADP-ribose)polymerase to DNA nicks, activation of four resident catalytic activities leading to poly(ADP-ribose) synthesis, conversion of the polymerase into a protein modified with up to 28 variably sized ADP-ribose polymers, and rapid degradation of polymerase-bound polymers by poly(ADP-ribose)glycohydrolase. This automodification cycle catalyzes a transient and reversible dissociation of histones from DNA. Shuttling of histones on the DNA allows selected other proteins, such as DNA helicase A and topoisomerase I, to gain access to DNA. Histone shuttling in vitro mimics nucleosomal unfolding/refolding in vivo that accompanies the postincisional steps of DNA excision repair. Suppression of the automodification cycle in mammalian cells prevents nucleosomal unfolding and nucleotide excision repair.
Environ Mol Mutagen 1993
PMID:Histone shuttle driven by the automodification cycle of poly(ADP-ribose)polymerase. 822 11

DNA-binding antibiotics such as intercalators, narrow groove binders, and other substances modify duplex DNA, making it an altered substrate for DNA helicases. The intercalators daunorubicin, actinomycin D, echinomycin, and elsamicin, the narrow groove binders distamycin and mithramycin, and the plant toxin teniposide, each representing a different chemical class, block SV40 large T antigen DNA helicase action with IC50 values ranging from 4 x 10(-8) to 2 x 10(-6) M. A partially purified human HeLa cell DNA helicase is also potently blocked by daunorubicin, distamycin, and teniposide. Because eukaryotic cells contain helicases of varying abundance, specificity, and type, this site of action for DNA-binding antibiotics may help explain antibiotic potency and specificity for DNA or RNA inhibition. The antihelicase effect of the antibiotic-double-stranded DNA complex may be central to the anticancer activities of these substances. An additional interesting correlation is the antihelicase action of DNA-intercalating antibiotics and their DNA-binding preference for G-C base pair sites. The G-C base pair binding preference of the intercalating antibiotics may result from evolutionary selection because of the higher G-C binding stability, compared with A-T binding stability. The combination of the higher base pair stability at G-C regions and increased duplex DNA stability induced by intercalating antibiotic yields a total additive stability of the intercalator-G-C base pair complex that resists helicase action.
Mol Pharmacol 1993 Nov
PMID:Antihelicase action of DNA-binding anticancer agents: relationship to guanosine-cytidine intercalator binding. 824 9

A site-specific lysine to methionine mutation has been engineered at the invariant Lys35 residue in the ATPase A binding site of the Escherichia coli uvrD gene encoding DNA helicase II. The mutant protein (UvrDK35M) has been purified to apparent homogeneity and characterized. The kcat for DNA-dependent ATP hydrolysis was less than 0.5% that of the wild-type enzyme with no change in the apparent Km for ATP. No unwinding of partial duplex DNA substrates could be detected using the mutant protein. Moreover, the mutant protein inhibited the unwinding reaction catalyzed by the wild-type protein at ratios of mutant enzyme to wild-type enzyme < 1. We conclude that the K35M mutation renders helicase II catalytically inactive as a DNA helicase with little or no effect on the ability of the enzyme to bind ATP, DNA, or other proteins. In vivo complementation assays indicate that the mutant protein cannot substitute for the wild-type protein in methyl-directed mismatch repair, suggesting that the ATPase and/or helicase activity of helicase II is required in this repair pathway. Additional genetic characterization of the uvrDK35M allele, supplied on a plasmid, suggests that expression of the mutant protein, at levels equivalent to that of the wild-type protein, results in a dominant negative phenotype. Expression of lower levels of the mutant protein, both in the presence and absence of wild-type helicase II, results in a constitutive induction of the cellular SOS response and extensive filamentation of cells. This induction of the SOS response is not due to a defect in methyl-directed mismatch repair. Taken together, these data are consistent with the notion that E. coli helicase II may have a role in DNA replication.
J Mol Biol 1994 Jan 14
PMID:A dominant negative allele of the Escherichia coli uvrD gene encoding DNA helicase II. A biochemical and genetic characterization. 828 72

DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.
 ...
PMID:Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I. 838 20

The Rad3 protein from Saccharomyces cerevisiae is a DNA helicase which participates in the repair of ultraviolet-irradiated DNA and is inhibited in the presence of DNA containing thymine dimers. This protein is also involved in mitotic recombination and spontaneous mutagenesis and is essential for cell viability in the absence of DNA damage. Furthermore, the Rad3 protein also exhibits a DNA:RNA helicase activity in which there is a significant preference for a partial DNA:RNA hybrid rather than a partial duplex DNA substrate, which suggests that this protein might be involved in DNA repair within transcriptionally active genes. Finally, the Rad3 protein contains the DEAH motif and shares high amino acid sequence similarity with the DEAD family of RNA helicase proteins, suggesting that it might also possess an RNA helicase activity.
Mol Microbiol 1993 Mar
PMID:The Rad3 protein from Saccharomyces cerevisiae: a DNA and DNA:RNA helicase with putative RNA helicase activity. 838 43

A crude DNA polymerase fraction partially purified from a low salt extract of HeLa cells was fractionated on a hydroxylapatite column by an elution with a linear gradient of potassium phosphate. By this procedure, DNA polymerase alpha, delta and epsilon were separated from each other. DNA helicase activities were detected in the DNA polymerase alpha and delta fractions but not in the epsilon fraction. Characterization of DNA helicases after further purification on heparin column revealed that the DNA helicase in the DNA polymerase alpha fraction required ATP (or dCTP) in addition to ATP (or dATP). Both DNA helicases translocated on single-stranded DNA in the same direction of 3' to 5'. By a repeated gel-filtration on Superose 6 (SMART system), activities of DNA polymerase alpha and delta were eluted at positions of approx. 600 kDa and 400 kDa, respectively, and the activities of DNA helicases were well associated with those of corresponding DNA polymerases. These results strongly suggest that the DNA helicases described here are physically associated with DNA polymerase alpha and delta to make large complexes.
Biochem Mol Biol Int 1993 Mar
PMID:DNA helicases associated with DNA polymerases from human cells. 838 69

RecQ protein of Escherichia coli is a DNA helicase implicated in the RecF pathway of genetic recombination. To gain insight into the mode of its action, the effect of single-stranded DNA-binding proteins (SSBs) on the RecQ-mediated unwinding reaction was investigated. When the unwinding of M13-based, circular partially duplex substrates was measured as a function of the enzyme dose, a markedly sigmoidal relation was revealed, with relatively large amounts of the enzyme being necessary for substantial unwinding to occur. For instance, unwinding 50% of a 71 base-pair (bp) partial duplex substrate in ten minutes required an enzyme-to-substrate molar ratio of about 60. However, these features, indicating the enzyme's "inefficiency", were reversed by SSBs: in the presence of a saturating amount of E. coli SSB the sigmoidal relation was converted to a typically hyperbolic one, and the enzyme-to-substrate molar ratio at 50% unwinding of the 71 bp substrate was reduced to as low as 0.5. Phage T4 gene 32 protein also showed similar stimulatory activity. Further, the single-stranded DNA-dependent ATPase activity of RecQ was found to be relatively insensitive to E. coli SSB; its large excess brought about only a 60% inhibition. It is postulated that RecQ helicase is highly adapted to an SSB-rich environment, where the strand exchange reaction mediated by RecA protein, perhaps coupled closely with the RecQ reaction, should also take place.
J Mol Biol 1993 Apr 20
PMID:RecQ DNA helicase of Escherichia coli. Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein. 838 4

RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain. The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced approximately 2 to 3-fold relative to the wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme. Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at chi sites, which are hotspots for genetic recombination. Since this interaction with chi significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.
J Mol Biol 1993 Jun 05
PMID:Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chi-specific, but not non-specific, nuclease activity. 839 May 77


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