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

E. coli Rep protein is a 3' to 5' SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required.
J Mol Biol 2001 Jul 06
PMID:E. coli Rep oligomers are required to initiate DNA unwinding in vitro. 1142 93

The UV-sensitive V-H1 cell line has a T46I substitution mutation in the Walker A box in both alleles of XPD and lacks DNA helicase activity. We characterized three partial revertants that curiously display intermediate UV cytotoxicity (2- to 2.5-fold) but normal levels of UV-induced hprt mutations. In revertant RH1-26, the efficient removal of pyrimidine (6-4) pyrimidone photoproducts from both strands of hprt suggests that global-genomic nucleotide excision repair is normal, but the pattern of cyclobutane pyrimidine dimer removal suggests that transcription-coupled repair (TCR) is impaired. To explain the intermediate UV survival and lack of RNA synthesis recovery in RH1-26 after 10 J of UV/m(2), we propose a defect in repair-transcription coupling, i.e., the inability of the cells to resume or reinitiate transcription after the first TCR event within a transcript. All three revertants carry an R658H suppressor mutation, in one allele of revertants RH1-26 and RH1-53 and in both alleles of revertant RH1-3. Remarkably, the R658H mutation produces the clinical phenotype of trichothiodystrophy (TTD) in several patients who display intermediate UV sensitivity. The XPD(R658H) TTD protein, like XPD(T46I/R658H), is codominant when overexpressed in V-H1 cells and partially complements their UV sensitivity. Thus, the suppressing R658H substitution must restore helicase activity to the inactive XPD(T46I) protein. Based on current knowledge of helicase structure, the intragenic reversion mutation may partially compensate for the T46I mutation by perturbing the XPD structure in a way that counteracts the effect of this mutation. These findings have implications for understanding the differences between xeroderma pigmentosum and TTD and illustrate the value of suppressor genetics for studying helicase structure-function relationships.
Mol Cell Biol 2001 Nov
PMID:Restoration of nucleotide excision repair in a helicase-deficient XPD mutant from intragenic suppression by a trichothiodystrophy mutation. 1158 17

Biogenesis of small nucleolar RNA-protein complexes (snoRNPs) consists of synthesis of the snoRNA and protein components, snoRNP assembly, and localization to the nucleolus. Recently, two nucleoplasmic proteins from mice were observed to bind to a model box C/D snoRNA in vitro, suggesting that they function at an early stage in snoRNP biogenesis. Both proteins have been described in other contexts. The proteins, called p50 and p55 in the snoRNA binding study, are highly conserved and related to each other. Both have Walker A and B motifs characteristic of ATP- and GTP-binding and nucleoside triphosphate-hydrolyzing domains, and the mammalian orthologs have DNA helicase activity in vitro. Here, we report that the Saccharomyces cerevisiae ortholog of p50 (Rvb2, Tih2p, and other names) is required for production of C/D snoRNAs in vivo and, surprisingly, H/ACA snoRNAs as well. Point mutations in the Walker A and B motifs cause temperature-sensitive or lethal growth phenotypes and severe defects in snoRNA accumulation. Notably, depletion of p50 (called Rvb2 in this study) also impairs localization of C/D and H/ACA core snoRNP proteins Nop1p and Gar1p, suggesting a defect(s) in snoRNP assembly or trafficking to the nucleolus. Findings from other studies link Rvb2 orthologs with chromatin remodeling and transcription. Taken together, the present results indicate that Rvb2 is involved in an early stage of snoRNP biogenesis and may play a role in coupling snoRNA synthesis with snoRNP assembly and localization.
Mol Cell Biol 2001 Nov
PMID:A well-connected and conserved nucleoplasmic helicase is required for production of box C/D and H/ACA snoRNAs and localization of snoRNP proteins. 1160 9

The xeroderma pigmentosum group D (XPD) protein is a subunit of transcription factor TFIIH with DNA helicase activity. TFIIH has two functions, in basal transcription and nucleotide excision repair. Mutations in XPD that affect DNA repair but not transcription result in the skin cancer-prone disorder, xeroderma pigmentosum (XP). If transcription is also affected, the result is the multi-system disorder trichothiodystrophy (TTD), in which there is no skin cancer predisposition, or in rare cases, XP combined with Cockayne syndrome. Up till now there have been no reports of combined clinical features of XP and TTD. We have now identified two patients with some features of both these disorders. One of these, XP189MA, a 3-year-old girl with sun sensitivity, mental and physical developmental delay, has XPD mutations not previously reported, and barely detectable levels of nucleotide excision repair. The other, XP38BR, a 28-year-old woman with sun sensitivity, pigmentation changes and skin cancers typical of XP, has a mutation that has been identified previously, but only in TTD patients with no features of XP. The level of repair of UV damage in XP38BR is substantially higher than that in other patients with the same mutation. With both patients, polarized light microscopy revealed a 'tiger-tail' appearance of the hair, and amino acid analysis of the hair shafts show levels of sulfur-containing proteins intermediate between those of normal and TTD individuals. Our findings highlight the complexities of genotype-phenotype relationships in the XPD gene.
Hum Mol Genet 2001 Oct 15
PMID:Two individuals with features of both xeroderma pigmentosum and trichothiodystrophy highlight the complexity of the clinical outcomes of mutations in the XPD gene. 1170 41

Removal of a telomere from yeast chromosome VII in a strain having two copies of this chromosome often results in its loss. Here we show that there are three pathways that can stabilize this broken chromosome: homologous recombination, nonhomologous end joining, and de novo telomere addition. Both in a wild-type and a recombination deficient rad52 strain, most stabilization events were due to homologous recombination, whereas nonhomologous end joining was exceptionally rare. De novo telomere addition was relatively rare, stabilizing <0.1% of broken chromosomes. Telomere addition took place at a very limited number of sites on chromosome VII, most occurring close to a 35-base pair stretch of telomere-like DNA that is normally approximately 50 kb from the left telomere of chromosome VII. In the absence of the Pif1p DNA helicase, telomere addition events were much more frequent and were not concentrated near the 35-base pair tract of telomere-like DNA. We propose that internal tracts of telomere-like sequence recruit telomerase by binding its anchor site and that Pif1p inhibits telomerase by dissociating DNA primer-telomerase RNA interactions. These data also show that telomeric DNA is essential for the stable maintenance of linear chromosomes in yeast.
Mol Biol Cell 2001 Dec
PMID:Repair of chromosome ends after telomere loss in Saccharomyces. 1173 2

We highlight selected contributions of Dr. Richard Setlow that contributed to our earlier understanding of excision repair processes and set the stage for dissecting nucleotide excision repair (NER) in mammalian cells through molecular genetics. More than 20 years ago, large-scale screens for UV-sensitive mutants of hamster CHO cells isolated approximately 200 mutants, many of which were assigned to the XPD/ERCC2 complementation group, but the nature of the mutations was not determined. The XPD protein performs not only an essential viability function as a structural component of transcription initiation factor TFIIH, but also an NER function as a 5' to 3' DNA helicase within TFIIH that unwinds DNA on the 3' side of bulky lesions. Alterations in these XPD functions are responsible for three UV-sensitivity genetic disorders that have distinguishable clinical features. In this study, we sequenced six UV-sensitive ICR170-induced Chinese hamster ovary (CHO) cell mutants that previously were assigned to the XPD complementation group to determine whether they carry frameshift mutations. All six mutants show 3- to 5-fold increased hypersensitivity to UV irradiation, similar to the XPD mutant prototype UV5. Even though ICR170 is a strong frameshift mutagen, all six cell lines contain base substitution mutations, five of which are unique among all mutations identified so far in human and rodent cells. The sixth mutation was identical to the R75W mutation previously found in CHO UVL-1. The results presented here contribute to a mutation database that should prove useful in structure-function studies of this unique DNA-structure-specific helicase and its complex mutant phenotypes.
Environ Mol Mutagen 2001
PMID:Identification of ICR170-induced XPD mutations in UV-sensitive CHO cells. 1174 43

Crossing over is regulated to occur at least once per each pair of homologous chromosomes during meiotic prophase to ensure proper segregation of chromosomes at the first meiotic division. In a mer3 deletion mutant of Saccharomyces cerevisiae, crossing over is decreased, and the distribution of the crossovers that occur is random. The predicted Mer3 protein contains seven motifs characteristic of the DExH box type of DNA/RNA helicases. The mer3G166D and the mer3K167A mutation, amino acid substitutions of conserved residues in a putative nucleotide-binding domain of the helicase motifs caused a defect in the transition of meiosis-specific double-strand breaks to later intermediates, decreased crossing over, and reduced crossover interference. The purified Mer3 protein was found to have DNA helicase activity. This helicase activity was reduced by the mer3GD mutation to <1% of the wild-type activity, even though binding of the mutant protein to single- and double-strand DNA was unaffected. The mer3KA mutation eliminated the ATPase activity of the wild-type protein. These results demonstrate that Mer3 is a DNA helicase that functions in meiotic crossing over.
Mol Cell Biol 2002 May
PMID:Saccharomyces cerevisiae Mer3 is a DNA helicase involved in meiotic crossing over. 1197 62

The Saccharomyces cerevisiae Pif1p DNA helicase is the prototype member of a helicase subfamily conserved from yeast to humans. S. cerevisiae has two PIF1-like genes, PIF1 itself and RRM3, that have roles in maintenance of telomeric, ribosomal, and mitochondrial DNA. Here we describe the isolation and characterization of pfh1+, a Schizosaccharomyces pombe gene that encodes a Pif1-like protein. Pfh1p was the only S. pombe protein with high identity to Saccharomyces Pif1p. Unlike the two S. cerevisiae Pif1 subfamily proteins, the S. pombe Pfh1p was essential. Like Saccharomyces Pif1p, a truncated form of the S. pombe protein had 5' to 3' DNA helicase activity. Point mutations in an invariant lysine residue in the ATP binding pocket of Pfh1p had the same phenotype as deleting pfh1+, demonstrating that the ATPase/helicase activity of Pfh1p was essential. Although mutant spores depleted for Pfh1p proceeded through S phase, they arrested with a terminal cellular phenotype consistent with a postinitiation defect in DNA replication. Telomeric DNA was modestly shortened in the absence of Pfh1p. However, genetic analysis demonstrated that maintenance of telomeric DNA was not the sole essential function of S. pombe Pfh1p.
Mol Biol Cell 2002 Jun
PMID:Schizosaccharomyces pombe pfh1+ encodes an essential 5' to 3' DNA helicase that is a member of the PIF1 subfamily of DNA helicases. 1205 79

The DNA helicase PcrA is found in gram-positive bacteria and belongs to the superfamily 1 (SF1) of helicases, together with Rep and UvrD helicases from Escherichia coli. These helicases have been extensively studied in vitro and their mode of unwinding are well characterised. However, little is known about the putative cellular partners of such helicases. To identify PcrA-interacting factors, PcrA was used as a bait in a genome-wide yeast two-hybrid screen of a Bacillus subtilis library. Three proteins with unknown functions - YxaL, YwhK and YerB - were found to interact specifically with PcrA. The yxaL gene was cloned, the product was overexpressed and purified, and its effect on the PcrA activity was investigated in vitro. YxaL enhanced the processivity of the PcrA helicase. A comparison of the amino acid sequence of YxaL with other proteins from data banks suggests that YxaL belongs to a family of proteins with a repeated domain, which adopt a typical three-dimensional structure designated as a "beta-propeller". This raises the possibility that YxaL acts as a connector protein between PcrA and another cellular component.
Mol Genet Genomics 2002 May
PMID:The beta-propeller protein YxaL increases the processivity of the PcrA helicase. 1207 41

DNA helicases are molecular motors that use the energy from NTP hydrolysis to drive the process of duplex DNA strand separation. Here, we measure the translocation and energy coupling efficiency of a replicative DNA helicase from bacteriophage T7 that is a member of a class of helicases that assembles into ring-shaped hexamers. Presteady state kinetics of DNA-stimulated dTTP hydrolysis activity of T7 helicase were measured using a real time assay as a function of ssDNA length, which provided evidence for unidirectional translocation of T7 helicase along ssDNA. Global fitting of the kinetic data provided an average translocation rate of 132 bases per second per hexamer at 18 degrees C. While translocating along ssDNA, T7 helicase hydrolyzes dTTP at a rate of 49 dTTP per second per hexamer, which indicates that the energy from hydrolysis of one dTTP drives unidirectional movement of T7 helicase along two to three bases of ssDNA. One of the features that distinguishes this ring helicase is its processivity, which was determined to be 0.99996, which indicated that T7 helicase travels on an average about 75kb of ssDNA before dissociating. We propose that the ability of T7 helicase to translocate unidirectionally along ssDNA in an efficient manner plays a crucial role in DNA unwinding.
J Mol Biol 2002 Aug 30
PMID:T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA. 1220 63


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