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

During repair of a double-strand break (DSB) by gene conversion, one or both 3' ends of the DSB invade a homologous donor sequence and initiate new DNA synthesis. The use of the invading DNA strand as a primer for new DNA synthesis requires that any nonhomologous bases at the 3' end be removed. We have previously shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3. We now report that these four proteins are needed only when the nonhomologous ends of recombining DNA are 30 nucleotides (nt) long or longer. An additional protein, the helicase Srs2, is required for the RAD1-dependent removal of long 3' tails. We suggest that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog. 3' tails shorter than 30 nt are removed by another mechanism that depends at least in part on the 3'-to-5' proofreading activity of DNA polymerase delta.
Mol Cell Biol 1997 Nov
PMID:Two pathways for removal of nonhomologous DNA ends during double-strand break repair in Saccharomyces cerevisiae. 934 41

Nuclear extracts prepared from Drosophila preblastoderm embryos contain a nucleoside 5'-triphosphate-dependent helicase that unwinds circular or linear partial duplex DNA substrates and moves along the DNA in 3' to 5' direction. The helicase reaction is supported with both nucleoside and deoxynucleoside 5'-triphosphates and requires divalent cations. Optimum activity in vitro is achieved with dATP or ATP and with MgCl2. In glycerol density gradients, embryonic enzyme migrates as a single peak of around 90kDa and it is the most prominent DNA-unwinding activity detectable in nuclear extracts prepared from embryos 0-2 hours after egg laying. In embryos collected 2-4 hours after egg laying this DNA unwinding activity increases and then gradually decreases in embryos collected 4-8 and 8-16 hours after egg laying.
Biochem Mol Biol Int 1997 Nov
PMID:Characterization of a helicase, DHEL III, from Drosophila melanogaster embryonic nuclei. 938 32

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like "lipid kinase" domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.
Mol Biol Cell 1997 Dec
PMID:Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase. 939 73

It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli. Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD::Tn5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD-mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA, recF, recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD+ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.
Mol Microbiol 1997 Nov
PMID:uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway. 940 25

UV light irradiation increases genetic instability by causing mutations and deletions. The mechanism of UV-induced rearrangements was investigated making use of deletion-prone plasmids. Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide. A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed. In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots. These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate. They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion. Furthermore, UV-induced deletions occurred independently of pBR322 replication. We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone. We propose that the deletion-prone properties of a UV-arrested polymerase depend on the associated helicase.
Mol Microbiol 1997 Nov
PMID:Blocking rolling circle replication with a UV lesion creates a deletion hotspot. 940 26

In plasmid pTF-FC2, three small open reading frames (ORFs) are situated between the repB (primase) gene and the repA (helicase) gene of its IncQ-type replicon. Disruption of each of the three ORFs followed by tests for plasmid stability and host cell growth indicated that the ORFs encoded a poison-antidote plasmid stability system. The three genes were named pasA, pasB and pasC (plasmid addiction system), in which PasA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruption of the pasA gene resulted in two different spontaneous deletions, which inactivated the stability system but did not alter the host range or plasmid copy number. This indicated that the three small ORFs were not involved in plasmid replication. When placed behind a tac promoter, induction of pasB was found to be highly lethal to host cells, which suggests that the Pas system acts by killing plasmid-free host cells rather than by retarding the growth of plasmid-free segregants, as occurs in the ParD system of R1. In spite of this, the presence of the Pas poison-antidote system resulted in a relatively modest threefold stabilization of the pTF-FC2 host replicon and a similar increase in the stabilization of an unstable heterologous R1 plasmid replicon. The Pas system is a poison-antidote plasmid stability module, which appears to have become integrated within the pTF-FC2 replicon module.
Mol Microbiol 1997 Dec
PMID:The poison-antidote stability system of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2. 942 33

Genomic DNA of recombinant AcNPV expressing beta-galactosidase was cotransfected with p143 helicase gene of BmNPV into Sf21 cells. Ac-Bm hybrid viruses capable of replicating in both Bm5 and Sf21 cells were isolated. Ac-Bm hybrid viruses expressing beta-galactosidase either at the highest (Ac-Bm hybrid virus-HE) or lowest (Ac-Bm hybrid virus-LE) level were chosen for the characterization of beta-galactosidase expression in Bm5 and Sf21 cells. Expression level of beta-galactosidase and replication of Ac-Bm hybrid virus-HE in Sf21 cells were nearly identical to those of recombinant AcNPV. Furthermore, replication of Ac-Bm hybrid virus-HE in Bm5 cells was similar to that of wild-type BmNPV, and Ac-Bm hybrid virus-HE clearly expressed beta-galactosidase in Bm5 cells. However, expression of beta-galactosidase by Ac-Bm hybrid virus-HE in Bm5 cells was significantly lower than that expressed in Sf21 cells. The titer of Ac-Bm hybrid virus-HE determined by plaque assays in Bm5 cells was similar to that determined in Sf21 cells, but the plaque size formed by Ac-Bm hybrid virus-HE in Bm5 cells was apparently smaller than that formed in Sf21 cells. In addition, expression levels and virus titers of Ac-Bm hybrid virus-LE in Sf21 and Bm5 were significantly lower than those of Ac-Bm hybrid virus-HE. Therefore, DNA sequences were determined for the region of the p143 gene controlling the host range in Ac-Bm hybrid viruses. The results showed that the deduced amino acid sequences of Ac-Bm hybrid virus-HE were almost identical to those of BmNPV. There were differences only in amino acids at positions 461 and 470, whereas those of Ac-Bm hybrid virus-LE were different at position 461, 470, 514, and 528 from those of BmNPV. In conclusion, our results clearly demonstrated that Ac-Bm hybrid virus-HE has an additional advantage of expanded host range for producing recombinant proteins.
Mol Cells 1997 Dec 31
PMID:Characterization of recombinant Autographa californica nuclear polyhedrosis virus (NPV) expressing the beta-galactosidase gene in both Sf21 and Bm5 cells by Bombyx mori NPV p143 helicase gene. 950 18

Two site-directed mutants of Escherichia coli DNA helicase II (UvrD) were constructed to examine the functional significance of motif VI in a superfamily I helicase. Threonine 604 and arginine 605, representing two of the most highly conserved residues in motif VI, were replaced with alanine, generating the mutant alleles uvrD-T604A and uvrD-R605A. Genetic complementation studies indicated that UvrD-T604A, but not UvrD-R605A, functioned in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. Both mutant enzymes were purified and single-stranded DNA (ssDNA)-stimulated ATP hydrolysis, duplex DNA unwinding, and ssDNA binding were studied in the steady-state and compared to wild-type UvrD. UvrD-T604A exhibited a serious defect in ssDNA binding in the absence of nucleotide. However, in the presence of a non-hydrolyzable ATP analog, DNA binding was only slightly compromised. Limited proteolysis experiments suggested that UvrD-T604A had a "looser" conformation and could not undergo conformational changes normally associated with ATP binding/hydrolysis and DNA binding. UvrD-R605A, on the other hand, exhibited nearly normal DNA binding but had a severe defect in ATP hydrolysis (kcat=0.063 s-1 compared to 162 s-1 for UvrD). UvrD-T604A exhibited a much less severe decrease in ATPase activity (kcat=8.8 s-1). The Km for ATP for both mutants was not significantly changed. The results suggest that residues within motif VI of helicase II are essential for multiple biochemical properties associated with the enzyme and that motif VI is potentially involved in conformational changes related to the coupling of ATPase and DNA binding activities.
J Mol Biol 1998 Mar 27
PMID:Site-directed mutations in motif VI of Escherichia coli DNA helicase II result in multiple biochemical defects: evidence for the involvement of motif VI in the coupling of ATPase and DNA binding activities via conformational changes. 951 60

The SNF2/SWI2 ATPase/helicase family comprises proteins from a variety of species, which serve a number of functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. Several proteins with unknown functions are also included in this family. The number of genes that belong to this family is rapidly expanding, which makes it easier to analyze the common biological functions of the family members. This study was designed to clone the SNF2/SWI2 helicase-related genes from the fission yeast Schizosaccharomyces pombe in the hope that this would help to elucidate the common functions of the proteins in this family. The hrp1+ (helicase-related gene from S. pombe) gene was initially cloned by PCR amplification using degenerate primers based on conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. The hrp1+ ORF codes for an 1373-amino acid polypeptide with a molecular mass of 159 kDa. Like other SNF2/SWI2 family proteins, the deduced amino acid sequence of Hrp1 contains DNA-dependent ATPase/7 helicase domains, as well as a chromodomain and a DNA-binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-binding protein 1), suggesting that Hrp1 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to facilitate gene expression. Northern blot analysis showed that the hrp1+ gene produces a 4.6-kb transcript, which reaches its maximal level just before the cells enter the exponential growth phase, and then decreases gradually. DNA-damaging agents, such as MMS, MNNG and UV, decrease the rate of transcription of hrp1+. Deletion of the hrp1+ gene resulted in accelerated cell growth. On the other hand, overexpression of Hrp1 caused a reduction in growth rate. These results indicate that hrp1+ may act as a negative regulator of cellular growth.
Mol Gen Genet 1998 Feb
PMID:Isolation and characterization of hrp1+, a new member of the SNF2/SWI2 gene family from the fission yeast Schizosaccharomyces pombe. 952 Feb 66

A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.
Mol Cell Biol 1998 Apr
PMID:Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae. 952 57


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