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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein. Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E. coli cells containing the overexpression plasmid. Overproduction of helicase II protein at these levels is lethal in E. coli. The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 [20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C]. We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties. The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT). In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions. However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important. On the basis of these observations and similarities between helicase II and the E. coli Rep helicase, which appears to function as a dimer [Chao, K., & Lohman, T. (1991) J. Mol. Biol. 221, 1165-1181], we suggest that the active form of helicase II may also be a dimer or larger oligomer.
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PMID:Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II). 838 Jul 1

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.
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PMID:Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I. 838 20

An Escherichia coli recBCD deletion mutant was transformed with plasmids containing the Bacillus subtilis add genes. The transformants had relatively high ATP-dependent exonuclease- and ATP-dependent helicase activities, and their viability, the ability to repair u.v.-damaged DNA and the recombination in conjugation were nearly completely restored. The B. subtilis Add enzyme did not show Chi-activity in phage lambda recombination. The individual B. subtilis Add proteins were not able to form an enzymatically active complex with the E. coli RecB,C,D proteins, and they could not complement the recB,C,D deficiency. Evidence is presented that only two subunits are involved in the B. subtilis ATP-dependent exonuclease. This is in contrast to E. coli in which the RecBCD enzyme consists of three subunits.
Mol Microbiol 1993 Mar
PMID:The Bacillus subtilis addAB genes are fully functional in Escherichia coli. 838 45

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

The Escherichia coli recB2109CD enzyme displays a defect in homologous recombination. In vitro, it possesses significant levels of non-specific nuclease activity but is deficient in chi-dependent nicking activity. To determine whether an alteration in helicase activity contributes further to its in vivo defect, the ability of recB2109CD enzyme to unwind dsDNA was examined. The mutant enzyme is able to unwind DNA but has a kcat which is one-third that of the wild-type enzyme. While the Km for DNA ends of the wild-type and mutant enzymes at low NaCl concentrations are essentially equivalent, the Km for ATP of recB2109CD enzyme is nearly six times greater. The processivity of unwinding (i.e. the average length of DNA unwound before recB2109CD enzyme dissociates from the DNA substrate) at 1 mM-Mg2+ ion and 1 mM-ATP is approximately 13 kb/end, whereas that of wild-type recBCD enzyme is 30 kb/end. In an assay which requires the co-ordinate actions of the recBCD, recA, and SSB proteins, joint molecule formation in the presence of recB2109CD enzyme is up to sixfold slower and proceeds to a lower extent than that mediated by the wild-type enzyme. We conclude that although the reduced helicase activity of the mutant recBCD enzyme may contribute to its recombination deficiency, its defect in the chi-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E. coli strains bearing the recB2109 mutation.
J Mol Biol 1993 Jun 05
PMID:The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro. 839 May 78

We have sequenced and genetically characterized comF, a Bacillus subtilis competence locus, previously identified by Tn917 transposon insertion mutagenesis. Expression of the locus, in which three open reading frames (ORFs) were found, is driven by a single sigma A-like promoter in front of comFORF1 and is dependent on early regulatory competence genes and only expressed in competence medium. The predicted amino acid sequences of two of the ORFs showed similarities to known proteins in the GenBank and SwissProt databases: ComFORF1 is similar to an extensive family of ATP-dependent RNA/DNA helicases with closer similarity to the DEAD protein subfamily and to the PriA protein in Escherichia coli. The latter is a DNA translocase/helicase required for primosome assembly at the replication fork of phage phi X174. ComFORF3 is 22% identical to Com101, a protein required for genetic competence in Haemophilus influenzae, a naturally competent Gram-negative bacterium. In-frame comFORF1 deletions were 1000-fold deficient in transformability compared to the wild-type, whereas disruptions of the other two ORFs were only five- to 10-fold lower. These observations allow us to hypothesize that the ComFORF1 late gene product plays an essential role during the binding and uptake events involved in Bacillus subtilis transformation.
Mol Microbiol 1993 Jul
PMID:comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases. 841 57

eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eIF-4B, it exhibits duplex RNA helicase activity. eIF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of eIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduced eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity. Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B.
Mol Cell Biol 1993 Nov
PMID:The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. 841 73

Cloning and sequence analysis of a new open reading frame from Bacillus cereus reveals the relationship to a recently identified family of putative eukaryotic transcription activators similar to the yeast SNF2 gene product. As a result of comparative analysis of sequence features conserved in all members of this family, a gene from a chilo iridescent virus, as well as a putative helicase from Escherichia coli (hepA), can also be grouped into this family. The unexpected presence of prokaryotic and viral sequences in the previously purely eukaryotic SNF2 family suggests a defined subgroup of DNA helicases present in all species, with specific function in transcription activation.
J Mol Biol 1993 Mar 20
PMID:Prokaryotic members of a new family of putative helicases with similarity to transcription activator SNF2. 846 78

The traY gene product (TraYp) from the Escherichia coli F factor has previously been purified and shown to bind a DNA fragment containing the F plasmid oriT region (E. E. Lahue and S. W. Matson, J. Bacteriol. 172:1385-1391, 1990). To determine the precise nucleotide sequence bound by TraYp, DNase I footprinting was performed. The TraYp-binding site is near, but not coincident with, the site that is nicked to initiate conjugative DNA transfer. In addition, a second TraYp binding site, which is coincident with the mRNA start site at the traYI promoter, is described. The Kd for each binding site was determined by a gel mobility shift assay. TraYp exhibits a fivefold higher affinity for the oriT binding site compared with the traYI promoter binding site. Hydrodynamic studies were performed to show that TraYp is a monomer in solution under the conditions used in DNA binding assays. Early genetic experiments implicated the traY gene product in the site- and strand-specific endonuclease activity that nicks at oriT (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980; S. McIntire and N. Willetts, Mol. Gen. Genet. 178:165-172, 1980). As this activity has recently been ascribed to helicase I, it was of interest to see whether TraYp had any effect on this reaction. Addition of TraYp to nicking reactions catalyzed by helicase I showed no effect on the rate or efficiency of oriT nicking. Roles for TraYp in conjugative DNA transfer and a possible mode of binding to DNA are discussed.
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PMID:Characterization of the Escherichia coli F factor traY gene product and its binding sites. 846 82

The DnaC protein is required for loading the DnaB helicase at oriC. Thus DnaC promotes the formation of the pre-replication complex, but must leave the complex in order for the DnaB protein to function as a helicase. In vitro, a slight excess of DnaC inhibits the movement of replication forks by inhibiting DnaB helicase activity (Allen and Kornberg, 1991). Here we show that inhibition of DNA replication by excess DnaC also occurs in vivo. The rate of replication-fork movement was measured by flow cytometry. Initiation of replication was inhibited with rifampicin and the rate of fork movement monitored during replication runout by measuring the increase in the fraction of the cell population with fully replicated chromosomes. The replication rate was inversely related to the amount of excess DnaC protein. Initiation of replication was also inhibited. Co-overexpression of DnaB protein alleviated the inhibition of replication caused by moderate excess of DnaC. The results show that DnaC interacts with replication forks during elongation in vivo, probably by binding to DnaB and inhibiting its helicase activity. Therefore, the ratio of DnaC to DnaB and the affinity of DnaC for a helicase hexamer at an established replication fork are of great importance for the rate of replication fork movement also in vivo.
Mol Microbiol 1995 Sep
PMID:The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. 859 32


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