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

We have recently described a new helicase, the Dna2 helicase, that is essential for yeast DNA replication. We now show that the yeast FEN-1 (yFEN-1) nuclease interacts genetically and biochemically with Dna2 helicase. FEN-1 is implicated in DNA replication and repair in yeast, and the mammalian homolog of yFEN-1 (DNase IV, FEN-1, or MF1) participates in Okazaki fragment maturation. Overproduction of yFEN-1, encoded by RAD27/RTH1, suppresses the temperature-sensitive growth of dna2-1 mutants. Overproduction of Dna2 suppresses the rad27/rth1 delta temperature-sensitive growth defect. dna2-1 rad27/rth1 delta double mutants are inviable, indicating that the mutations are synthetically lethal. The genetic interactions are likely due to direct physical interaction between the two proteins, since both epitope-tagged yFEN-1 and endogenous yFEN-1 coimmunopurify with tagged Dna2. The simplest interpretation of these data is that one of the roles of Dna2 helicase is associated with processing of Okazaki fragments.
Mol Cell Biol 1997 Apr
PMID:A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function. 912 62

The large T antigen of simian virus 40 (SV40) is a multifunctional regulatory protein, responsible for both the control of viral infection and the required alterations of cellular processes. T antigen is the only viral protein required for viral DNA replication. It binds specifically to the viral origin and as a helicase unwinds the SV40 DNA bidirectionally. The functional complex is a double hexameric oligomer. In the absence of DNA, but in the presence of ATP or a non-hydrolyzable analog, T antigen assembles into hexamers, which are active as a helicase when a partially single-stranded (3') entry site exists on the substrate. We have used negative staining electron microscopy, single particle image processing and three-dimensional reconstruction with a new algebraic reconstruction techniques (ART) algorithm to study the structure of these hexameric particles in the presence of different nucleotide cofactors (ATP, ADP, and the non-hydrolyzable analogs ATPgammaS and AMP-PNP). In every case a strong 6-fold structure was found, with the six density maxima arranged in a ring-like particle around a channel, and a well-defined vorticity. Because these structural features have recently been found in other prokaryotic helicases, they seem to be strongly related to the activity of the protein, which suggests a general functional model conserved through evolution.
J Mol Biol 1997 Apr 25
PMID:Six molecules of SV40 large T antigen assemble in a propeller-shaped particle around a channel. 914 37

To investigate the role that the individual subunits play in the ATP-dependent helicase activity of the RecBCD protein we have investigated the ability of the RecB, RecC and RecD proteins to displace various 20-mer oligonucleotides annealed to either end or to the centre of an oligonucleotide 60 bases long. The results show that the only subunit which can displace the 20-mers in the absence of the other subunits is the RecB protein. Moreover, the 20-mer is displaced only if it is annealed to the 60-mer at the 5' end or the middle, suggesting that the RecB protein translocates along the 60-mer in the 3' to 5' direction, displacing annealed 20-mers as it proceeds. We have shown that reconstituted RecBC and RecBCD complexes displace the 20-mers but, unlike RecB, they do not require a 3'-ended single-stranded region for helicase action, but can displace the 20-mers from either end of the 60-mer. The level of helicase activity of the RecBC complex is considerably greater than that of RecB alone, and the activity of the RecBCD complex appears to be greater still. This hierarchy of activity is also shown by DNA binding studies, but is not reflected in the ATPase activities of the enzymes. We have also shown that the ability of trypsin to cleave various sites on the RecB molecule is modified by the presence of ATP or ATP-gamma-S, suggesting that conformational changes may be induced in RecB upon ATP binding. We discuss a model for the ATP-driven, unidirectional motion of the RecB translocase along single-stranded DNA. In this model, the RecB molecule binds to single-stranded DNA and then translocates along it, one base at a time, in the 3' to 5' direction, by a 'ratchet' mechanism in which repeated stretching and contraction of the protein is coupled to ATP hydrolysis. The RecC protein in the RecBC complex is proposed to act as a 'sliding clamp' which increases processivity by preventing dissociation.
Mol Gen Genet 1997 Apr 16
PMID:The RecB protein of Escherichia coli translocates along single-stranded DNA in the 3' to 5' direction: a proposed ratchet mechanism. 915 Feb 67

Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.
Microbiol Mol Biol Rev 1997 Jun
PMID:Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription. 918 11

The mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means. Tandem repeats 624 bp long were introduced into the lacZ gene of E. coli and the efficiency of deletion of one repeat was compared in different recombination mutants. No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected. Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways. A new mutant in which RecA-independent recombination is increased 15-fold was isolated. The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133. Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA-independent recombination. It is proposed that tandem-repeat recombination occurs by a replication slippage mechanism. RecA-independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.
Mol Microbiol 1997 Jun
PMID:Isolation of a dnaE mutation which enhances RecA-independent homologous recombination in the Escherichia coli chromosome. 921 71

The PriA protein of Escherichia coli provides a vital link between recombination and DNA replication. To establish the molecular basis for this link, we investigated the ability of PriA to target DNA substrates modelled on D-loops, the intermediates formed during the early stages of RecA-mediated recombination. We show that PriA binds D-loops and unwinds the DNA in reactions that rely on its ability to function as a helicase. The minimal structure that binds PriA is a duplex DNA molecule with unpaired single strands at one end, an arrangement likely to occur at a D-loop. It resembles features of the stem-loop formed by primosome assembly site (PAS) sequences in the DNA of bacteriophage phiX174 and plasmid ColE1, and which enable PriA to assemble active primosomes for the initiation of lagging strand synthesis. We suggest that PAS sequences may have evolved to mimic the natural D-loop target for PriA formed in the chromosome of E. coli during recombination and DNA repair. Genetic studies have revealed an interaction between PriA and RecG, a DNA helicase that drives branch migration of recombination intermediates. We therefore compared PriA and RecG for their ability to bind and unwind DNA. RecG, like PriA, binds D-loops and unwinds the DNA. However, it prefers branched structures with at least two duplex components. The possibility that it competes with PriA for binding recombination intermediates is discussed.
J Mol Biol 1997 Jul 11
PMID:The DNA replication protein PriA and the recombination protein RecG bind D-loops. 923 23

Recently we cloned a novel human cDNA homologous to yeast SKI2, reported a partial cDNA sequence, and mapped the gene to human chromosome 6p21 (Lee et al., 1995). It was a member of the DEAD/DExH family gene with seven conserved helicase domains; thus, it was named DDX13 consequently. We determined the complete genomic organization of the DDX13 gene. It consisted of 28 exons distributed over 11 kb of genomic DNA. An Alu element was present in introns 17 and 18, respectively. The major transcription start site was located 390 bp upstream from the translation initiation codon. The DDX13 gene was located in the class III region of the MHC between the genes coding for two other nuclear proteins, RD and RP1. The RD and DDX13 genes were oppositely oriented, and their first exons were overlapped. The distance between their first methionine codons was only 745 bp. It was of note that DDX13 and RD are in such proximity that their 5' regulatory regions overlap. The RP1 gene was located immediately downstream from the DDX13 gene in the same transcriptional orientation, and the distance between the stop codon of DDX13 and the translation initiation codon of RP1 was 2,272 bp.
Mol Cells 1997 Jun 30
PMID:Genomic organization of the human DDX13 gene located between RD and RP1 in the class III MHC complex. 926 31

Bloom's syndrome (BS), a human recessive disorder associated with an increased risk of malignancy, arises through mutations in both alleles of the BLM gene, which was recently identified as a member of the RecQ helicase family. BS cells are characterized by an increased rate of sister chromatid exchange (SCE). However, a subpopulation of lymphocytes exhibiting a normal level of SCE is observed in some patients. It has been proposed that reversion to a low-SCE phenotype involves an intragenic crossing over between the paternal and maternal BLM alleles, generating a wild-type allele. In this study we characterize a new BLM mutation in a BS patient leading to the replacement, in the C-terminal region of Blm, of a highly conserved cysteine by a phenylalanine in codon 1036. Moreover, our data show that this patient also inherited a BLM allele carrying a mutation affecting its expression and that a somatic intragenic crossing over was involved in reversion to the low-SCE phenotype. Further, we show that both topoisomerase II alpha mRNA and protein levels are decreased in the high-SCE cells derived from this patient, whereas they are normal in the corresponding low-SCE cells. Altogether, our data led us to propose that besides its putative helicase activity, Blm could be involved in transcription regulation.
Hum Mol Genet 1997 Sep
PMID:Characterization of a new BLM mutation associated with a topoisomerase II alpha defect in a patient with Bloom's syndrome. 928 78

Many DNA helicases utilise the energy derived from nucleoside triphosphate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB protein (a hexameric ring helicase), promotes the branch migration of Holliday junctions during genetic recombination and DNA repair. To analyse the relationship between ATP-dependent DNA helicase activity and branch migration, a site-directed mutation was introduced into the helicase II motif of RuvB. Over-expression of RuvBD113N in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical properties of RuvBD113N were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major alterations to the biochemical activities of RuvB, such that RuvBD113N was defective in DNA binding and ATP hydrolysis, while retaining the ability to form hexameric rings and interact with RuvA. RuvBD113N formed heterohexamers with wild-type RuvB, and could inhibit RuvB function by affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not all subunits of the ring need to be catalytically competent.
J Mol Biol 1997 Sep 05
PMID:Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities. 929 21

The gene 59 protein (gp59) of bacteriophage T4 is an important accessory protein of the phage-encoded replicative DNA helicase, gp41. The properties of this 26 kDa protein include selective binding to ssDNA, and specific interactions with both gp41 and gp32, the T4-encoded ssDNA- binding protein. gp59 stimulates many of the DNA-dependent activities of the gp41 enzyme by promoting its assembly onto gp32-ssDNA complexes. Direct interactions between gp59 and gp32-ssDNA complexes are essential for helicase assembly, and gp59-gp32 protein-protein interactions have been shown to play a central role. Presumably, the ssDNA-binding activity of gp59 is also important for helicase assembly; however, to date this activity has been poorly characterized. In this study, we present the first detailed biochemical investigation of the interactions of gp59 with single-stranded polynucleotides. Using etheno-DNA fluorescence enhancement and quantitative ssDNA-cellulose methods, we demonstrate the following: (1) gp59 binds to single-stranded polynucleotides with a binding site size of nine to ten nucleotide residues per monomer; (2) gp59 exhibits relative affinities towards four different ssDNA lattices used in this study according to the heirarchy: ssDNA (random sequence) > epsilonDNA (random sequence) > poly(dA) > poly(depsilonA); (3) gp59 exhibits two or more different polynucleotide binding modes distinguished by their cooperativities of binding, and modulated by salt and/or lattice effects; (4) gp59-ssDNA binding is characterized by a large salt effect on the association constant, consistent with multiple ionic contacts between protein and ssDNA phosphate residues and with the displacement of anions from the protein. The implications of our findings for the mechanism of action of gp59 in helicase-ssDNA assembly are discussed.
J Mol Biol 1997 Sep 26
PMID:Interactions of the bacteriophage T4 gene 59 protein with single-stranded polynucleotides: binding parameters and ion effects. 932 92


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