Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bloom's syndrome is a rare autosomal recessive disorder characterized by genomic instability and predisposition to cancer. BLM, the gene defective in Bloom's syndrome, encodes a 159-kDa protein possessing DNA-stimulated ATPase and ATP-dependent DNA helicase activities. We have examined mechanistic aspects of the catalytic functions of purified recombinant BLM protein. Through analyzing the effects of different lengths of DNA cofactor on ATPase activity, we provide evidence to suggest that BLM translocates along single-stranded DNA in a processive manner. The helicase reaction catalyzed by BLM protein was examined as a function of duplex DNA length. We show that BLM catalyzes unwinding of short DNA duplexes (</=71 base pairs (bp)) but is severely compromised on longer DNA duplexes (>/=259-bp). The presence of the human single-stranded DNA-binding protein (human replication protein A (hRPA)) stimulates the BLM unwinding reaction on the 259-bp partial duplex DNA substrate. Heterologous single-stranded DNA-binding proteins fail to stimulate similarly the helicase activity of BLM protein. This is the first demonstration of a functional interaction between BLM and another protein. Consistent with a functional interaction between hRPA and the BLM helicase, we demonstrate a direct physical interaction between the two proteins mediated by the 70-kDa subunit of RPA. The interactions between BLM and hRPA suggest that the two proteins function together in vivo to unwind DNA duplexes during replication, recombination, or repair.
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PMID:Replication protein A physically interacts with the Bloom's syndrome protein and stimulates its helicase activity. 1082 62

The meiosis-specific MER3 protein of Saccharomyces cerevisiae is required for crossing over, which ensures faithful segregation of homologous chromosomes at the first meiotic division. The predicted sequence of the MER3 protein contains the seven motifs characteristic of the DExH-box type of DNA/RNA helicases. The purified MER3 protein is a DNA helicase, which can displace a 50-nucleotide fragment annealed to a single-stranded circular DNA. MER3 was found to have ATPase activity, which was stimulated either by single- or double-stranded DNA. The turnover rate, k(cat), of ATP hydrolysis was approximately 500/min in the presence of either DNA. MER3 was able to efficiently displace relatively long 631-nucleotide fragments from single-stranded circular DNA only in the presence of the S. cerevisiae single-stranded DNA-binding protein, RPA (replication protein A). It appears that RPA inhibits re-annealing of the single-stranded products of the MER3 helicase. The MER3 helicase was found to unwind DNA in the 3' to 5' direction relative to single-stranded regions in the DNA substrates. Possible roles for the MER3 helicase in meiotic crossing over are discussed.
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PMID:The MER3 helicase involved in meiotic crossing over is stimulated by single-stranded DNA-binding proteins and unwinds DNA in the 3' to 5' direction. 1137 1

Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in double-strand break (DSB) repair. Here we demonstrate that Rhp55 and Rhp57 proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p and Rad57p. Mutations in the conserved ATP-binding/hydrolysis folds of both the Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell proliferation. However, when combined, ATPase fold mutations in Rhp55p and Rhp57p resulted in severe defects of both functions, characteristic of the deletion mutants. Yeast two-hybrid analysis also revealed other multiple in vivo interactions among S. pombe proteins involved in recombinational DNA repair. Similar to S. cerevisiae Rad51p-Rad54p, S. pombe Rhp51p and Rhp54p were found to interact. Both putative Rad52 homologs in S. pombe, Rad22p and Rti1p, were found to interact with the C-terminal region of Rhp51 protein. Moreover, Rad22p and Rti1p exhibited mutual, as well as self-, interactions. In contrast to the S. cerevisiae interacting pair Rad51p-Rad55p, S. pombe Rhp51 protein strongly interacted with Rhp57 but not with Rhp55 protein. In addition, the Rti1 and Rad22 proteins were found to form a complex with the large subunit of S. pombe RPA. Our data provide compelling evidence that most, but not all, of the protein-protein interactions found in S. cerevisiae DSB repair are evolutionarily conserved.
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PMID:Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe. 1156 Aug 89

Five Rad51-like proteins, referred to as Rad51 paralogs, have been described in vertebrates. We show that two of them, Rad51B and Rad51C, are associated in a stable complex. Rad51B-Rad51C complex has ssDNA binding and ssDNA-stimulated ATPase activities. We also examined the functional interaction of Rad51B-Rad51C with Rad51 and RPA. Even though RPA enhances Rad51-catalyzed DNA joint formation via removal of secondary structure in the ssDNA substrate, it can also compete with Rad51 for binding to the substrate, leading to suppressed reaction efficiency. The competition by RPA for substrate binding can be partially alleviated by Rad51B-Rad51C. This recombination mediator function of Rad51B-Rad51C is likely required for the assembly of the Rad51-ssDNA nucleoprotein filament in vivo.
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PMID:Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange. 1175 36

Repair of DNA interstrand cross-links is a challenging problem for cells. Many human gene products influence sensitivity to DNA cross-linking agents, but the mechanisms of cross-link repair are unknown. In Drosophila melanogaster, the mus308 mutation leads to marked sensitivity to DNA cross-linking agents. The C-terminal portion of the Mus308 polypeptide encodes a DNA polymerase, whereas a putative DNA helicase is encoded by the N-terminal portion. As a step toward isolating proteins involved in DNA cross-link repair, we searched for mammalian genes similar to the DNA helicase portion of Mus308. Human and mouse homologs were isolated from cDNA expression libraries and designated HEL308. Human HEL308 is on chromosome 4q21 and encodes a polypeptide of 1101 amino acids. The protein was expressed in insect cells and purified. HEL308 is a single-stranded DNA-dependent ATPase and DNA helicase. Mutation of a highly conserved lysine to methionine in helicase domain I eliminated both activities. The protein readily displaces 20- to 40-mer duplex oligonucleotides. Displacement of longer substrates was less efficient but was stimulated by the single-stranded DNA-binding protein RPA. Activity was supported by ATP or dATP but not other nucleotide triphosphates. The enzyme translocates on DNA with 3' to 5' polarity and behaves as a multimer upon gel filtration.
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PMID:A human DNA helicase homologous to the DNA cross-link sensitivity protein Mus308. 1175 61

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by growth deficiency, skin and skeletal abnormalities, and a predisposition to cancer. Mutations in the RECQ4 gene, one of five human homologs of the E. coli recQ gene, have been identified in a subset of RTS patients. Cells derived from RTS patients show high levels of chromosomal instability, implicating this protein in the maintenance of genomic integrity. However, RECQ4 is the least characterized of the RecQ helicase family with regard to its molecular and catalytic properties. We have expressed the human RECQ4 protein in E. coli and purified it to near homogeneity. We show that RECQ4 has an ATPase function that is activated by DNA, with ssDNA being much more effective than dsDNA in this regard. We have determined that a DNA length of 60 nucleotides is required to maximally activate ATP hydrolysis by RECQ4, while the minimal site size for ssDNA binding by RECQ4 is between 20 and 40 nucleotides. Interestingly, RECQ4 possesses a single-strand DNA annealing activity that is inhibited by the single-strand DNA binding protein RPA. Unlike the previously characterized members of the RecQ family, RECQ4 lacks a detectable DNA helicase activity.
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PMID:Biochemical characterization of the RECQ4 protein, mutated in Rothmund-Thomson syndrome. 1621 24

Yeast Dna2 helicase/nuclease is essential for DNA replication and assists FEN1 nuclease in processing a subset of Okazaki fragments that have long single-stranded 5' flaps. It is also involved in the maintenance of telomeres. DNA2 is a gene conserved in eukaryotes, and a putative human ortholog of yeast DNA2 (ScDNA2) has been identified. Little is known about the role of human DNA2 (hDNA2), although complementation experiments have shown that it can function in yeast to replace ScDNA2. We have now characterized the biochemical properties of hDna2. Recombinant hDna2 has single-stranded DNA-dependent ATPase and DNA helicase activity. It also has 5'-3' nuclease activity with preference for single-stranded 5' flaps adjacent to a duplex DNA region. The nuclease activity is stimulated by RPA and suppressed by steric hindrance at the 5' end. Moreover, hDna2 shows strong 3'-5' nuclease activity. This activity cleaves single-stranded DNA in a fork structure and, like the 5'-3' activity, is suppressed by steric hindrance at the 3'-end, suggesting that the 3'-5' nuclease requires a 3' single-stranded end for activation. These biochemical specificities are very similar to those of the ScDna2 protein, but suggest that the 3'-5' nuclease activity may be more important than previously thought.
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PMID:Biochemical analysis of human Dna2. 1659

Previous work by Sung and colleagues identified unusual salt requirements for hRAD51 strand exchange compared to RecA [S. Sigurdsson, K. Trujillo, B. Song, S. Stratton, P. Sung, Basis for avid homologous DNA strand exchange by human Rad51 and RPA, J. Biol. Chem. 276 (2001) 8798-8806]. Later studies showed that this salt [(NH4)2SO4] appeared to enhance the ability of hRAD51 to distinguish ssDNA from dsDNA [Y. Liu, A.Z. Stasiak, J.Y. Masson, M.J. McIlwraith, A. Stasiak, S.C. West, Conformational changes modulate the activity of human RAD51 protein, J. Mol. Biol. 337 (2004) 817-827]. The mechanism of this salt effect remains enigmatic. Here, we detail the properties of several neutral salts on hRAD51 activities. We found that the cation identity correlated with the stimulatory effect of these neutral salts on hRAD51 ATPase and strand exchange activities. The salt effect appears to be related to the size of the cation, which may be largely mimicked with the cesium ion. These results are consistent with the hypothesis that stimulating cations induce an important conformation and/or transition state in hRAD51. In the presence of an optimal ammonium-based salt (NaNH4HPO4), hRAD51 mediated strand exchange was successfully performed using a simplified protocol. We confirmed and extend the observation that efficient strand exchange correlated with preferential binding of ssDNA over dsDNA. In addition we observed an induced stability of the hRAD51-DNA complex in the presence of ATP that becomes unstable following ATP hydrolysis (the ADP form or nucleotide free form). These salt-induced characteristics of hRAD51 increasingly resemble RecA-mediated recombinase activities, which should help in dissecting the mechanism of these proteins in homologous recombination.
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PMID:Defining the salt effect on human RAD51 activities. 1664 92

Human ChlR1 (hChlR1), a member of the DEAD/DEAH subfamily of helicases, was shown to interact with components of the cohesin complex and play a role in sister chromatid cohesion. In order to study the biochemical and biological properties of hChlR1, we purified the protein from 293 cells and demonstrated that hChlR1 possesses DNA-dependent ATPase and helicase activities. This helicase translocates on single-stranded DNA in the 5' to 3' direction in the presence of ATP and, to a lesser extent, dATP. Its unwinding activity requires a 5'-singlestranded region for helicase loading, since flush-ended duplex structures do not support unwinding. The helicase activity of hChlR1 is capable of displacing duplex regions up to 100 bp, which can be extended to 500 bp by RPA or the cohesion establishment factor, the Ctf18-RFC (replication factor C) complex. We show that hChlR1 interacts with the hCtf18-RFC complex, human proliferating cell nuclear antigen, and hFen1. The interactions between Fen1 and hChlR1 stimulate the flap endonuclease activity of Fen1. Selective depletion of either hChlR1 or Fen1 by targeted small interfering RNA treatment results in the precocious separation of sister chromatids. These findings are consistent with a role of hChlR1 in the establishment of sister chromatid cohesion and suggest that its action may contribute to lagging strand processing events important in cohesion.
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PMID:Studies with the human cohesin establishment factor, ChlR1. Association of ChlR1 with Ctf18-RFC and Fen1. 1849 58

SMARCAL1 (also known as HARP) is a SWI/SNF family protein with an ATPase activity stimulated by DNA containing both single-stranded and double-stranded regions. Mutations in SMARCAL1 are associated with the disease Schimke immuno-osseous dysplasia, a multisystem autosomal recessive disorder characterized by T cell immunodeficiency, growth inhibition, and renal dysfunction. The cellular function of SMARCAL1, however, is unknown. Here, using Xenopus egg extracts and mass spectrometry, we identify SMARCAL1 as a protein recruited to double-stranded DNA breaks. SMARCAL1 binds to double-stranded breaks and stalled replication forks in both egg extract and human cells, specifically colocalizing with the single-stranded DNA binding factor RPA. In addition, SMARCAL1 interacts physically with RPA independently of DNA. SMARCAL1 is phosphorylated in a caffeine-sensitive manner in response to double-stranded breaks and stalled replication forks. It has been suggested that stalled forks can be stabilized by a mechanism involving caffeine-sensitive kinases, or they collapse and subsequently recruit Rad51 to promote homologous recombination repair. We show that depletion of SMARCAL1 from U2OS cells leads to increased frequency of RAD51 foci upon generation of stalled replication forks, indicating that fork breakdown is more prevalent in the absence of SMARCAL1. We propose that SMARCAL1 is a novel DNA damage-binding protein involved in replication fork stabilization.
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PMID:Identification of SMARCAL1 as a component of the DNA damage response. 1984 79


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