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)

Werner syndrome (WS) is a human premature aging disorder characterized by the early onset of age-related clinical features and an elevated incidence of cancer. The Werner protein (WRN) belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. Potential cooperation between RecQ helicases and topoisomerases in many aspects of DNA metabolism, such as the progression of replication forks, transcription, recombination, and repair, has been reported. Here, we show a physical and functional interaction between WRN and topoisomerase I (topo I). WRN colocalizes and interacts directly with topo I. WRN stimulates the ability of topo I to relax negatively supercoiled DNA and specifically stimulates the religation step of the relaxation reaction. Moreover, cell extracts from WS fibroblasts exhibit a decrease in the relaxation activity of negatively supercoiled DNA. We have identified two regions of WRN that mediate functional interaction with topo I, and they are located at the NH(2) and COOH termini of the WRN protein. In a reciprocal functional interaction, topo I inhibits the ATPase activity of WRN. Our data provide new insight into the interrelationship between RecQ helicases and topoisomerases in the maintenance of genomic integrity and prevention of tumorigenesis.
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PMID:Werner protein stimulates topoisomerase I DNA relaxation activity. 1461 7

Werner syndrome (WS) is a genetic premature aging disorder in which patients appear much older than their chronological age. The gene mutated in WS encodes a nuclear protein (WRN) which possesses 3'-5' exonuclease and ATPase-dependent 3'-5' helicase activities. The genomic instability associated with WS cells and the biochemical characteristics of WRN suggest that WRN plays a role in DNA metabolic pathways such as transcription, replication, recombination and repair. Recently we have identified poly(ADP-ribose) polymerase-1 (PARP-1) as a new WRN interacting protein. In this paper, we further mapped the interacting domains. We found that PARP-1 bound to the N-terminus of WRN and to the C-terminus containing the RecQ-conserved (RQC) domain. WRN bound to the N-terminus of PARP-1 containing DNA binding and BRCA1 C-terminal (BRCT) domains. We show that unmodified PARP-1 inhibited both WRN exonuclease and helicase activities, and to our knowledge is the only known WRN protein partner that inactivates both of the WRN's catalytic activities suggesting a biologically significant regulation. Moreover, this dual inhibition seems to be specific for PARP-1, as PARP-2 did not affect WRN helicase activity and only slightly inhibited WRN exonuclease activity. The differential effect of PARP-1 and PARP-2 on WRN catalytic activity was not due to differences in affinity for WRN or the DNA substrate. Finally, we demonstrate that the inhibition of WRN by PARP-1 was influenced by the poly(ADP-ribosyl)ation state of PARP-1. The biological relevance of the specific modulation of WRN catalytic activities by PARP-1 are discussed in the context of pathways in which these proteins may function together, namely in the repair of DNA strand breaks.
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PMID:Poly(ADP-ribose) polymerase 1 regulates both the exonuclease and helicase activities of the Werner syndrome protein. 1529 49

Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A, is involved in transcription and RNA processing. Here, we report that NDH II interacts with the Werner syndrome helicase WRN, an enzyme associated with premature aging and predisposition to tumorigenesis. NDH II was co-purified with WRN, DNA polymerase delta, and replication protein A (70 kDa) during several steps of conventional column chromatography. Co-immunoprecipitations revealed an association between NDH II, WRN, and polymerase delta. We demonstrate a direct protein-protein interaction between WRN and NDH II that is mediated by the N-terminal double-strand RNA-binding domain II and C-terminal RGG box of NDH II and the N-terminal exonuclease domain of WRN. WRN inhibited the DNA-dependent NTPase and DNA helicase activities of NDH II. On the other hand, the 3' --> 5' exonuclease activity of WRN was increased by the presence of NDH II. NDH II directly stimulated the exonuclease domain of WRN, whereas the exonuclease domain of WRN suppressed the DNA-dependent (but not RNA-dependent) ATPase activity of NDH II. These results suggest that the double-strand RNA-binding domain II and RGG box of NDH II together form a protein-protein interaction surface that contacts the exonuclease domain of WRN. Furthermore, NDH II enhanced the degradation of D-loop DNA by the WRN exonuclease. Taken together, these results suggest that NDH II plays a role in promoting the DNA processing function of WRN, which in turn might be necessary for maintaining genomic stability.
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PMID:Nuclear DNA helicase II (RNA helicase A) interacts with Werner syndrome helicase and stimulates its exonuclease activity. 1599 49

Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by developmental abnormalities, UV sensitivity, and premature aging. The CS group B (CSB) protein belongs to the SNF2-family of DNA-dependent ATPases and is implicated in transcription elongation, transcription coupled repair, and base excision repair. It is a DNA stimulated ATPase and remodels chromatin in vitro. We demonstrate for the first time that full-length CSB positively cooperates in ATP hydrolysis as a function of protein concentration. We have investigated the quaternary structure of CSB using a combination of protein-protein complex trapping experiments and gel filtration, and found that CSB forms a dimer in solution. Chromatography studies revealed that enzymatically active CSB has an apparent molecular mass of approximately 360 kDa, consistent with dimerization of CSB. Importantly, in vivo protein cross-linking showed the presence of the CSB dimer in the nucleus of HeLa cells. We further show that dimerization occurs through the central ATPase domain of the protein. These results have implications for the mechanism of action of CSB, and suggest that other SNF2-family members might also function as dimers.
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PMID:The Cockayne syndrome group B protein is a functional dimer. 1612 1

Werner syndrome (WS) is a premature aging disorder characterized by genomic instability and increased cancer risk (Martin, 1978). The WRN gene product defective in WS belongs to the RecQ family of DNA helicases (Yu et al., 1996). Mutations in RecQ family members BLM and RecQ4 result in two other disorders associated with elevated chromosomal instability and cancer, Bloom syndrome and Rothmund-Thomson syndrome, respectively (for review see Opresko et al., 2004a). RecQ helicase mutants display defects in DNA replication, recombination, and repair, suggesting a role for RecQ helicases in maintaining genomic integrity. The WRN gene encodes a 1,432 amino acid protein that has several catalytic activities (Brosh and Bohr, 2002) (Fig. 1). WRN is a DNA-dependent ATPase and utilizes the energy from ATP hydrolysis to unwind double-stranded DNA. WRN is also a 3' to 5' exonuclease, consistent with the presence of three conserved exonuclease motifs homologous to the exonuclease domain of Escherichia coli DNA polymerase I and RNase D. Most recently, WRN (Machwe et al., 2005) and other human RecQ helicases (Garcia et al., 2004; Machwe et al., 2005; Sharma et al., 2005) have been reported to possess an intrinsic single-strand annealing activity. In addition to its catalytic activities, WRN interacts with a number of proteins involved in various aspects of DNA metabolism. To understand the role of WRN in the maintenance of genome stability, a number of laboratories have undertaken a thorough characterization of its molecular and cellular functions. Here, we describe methods and approaches used for the functional and mechanistic analysis of WRN helicase or exonuclease activity. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding or exonuclease activity of WRN protein are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of WRN or related helicases or nucleases, which would serve as a platform for further investigation of its molecular and cellular functions.
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PMID:Enzymatic mechanism of the WRN helicase/nuclease. 1679 95

alpha-klotho was identified as a gene associated with premature aging-like phenotypes characterized by short lifespan. In mice, we found the molecular association of alpha-Klotho (alpha-Kl) and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) and provide evidence for an increase of abundance of Na+,K+-ATPase at the plasma membrane. Low concentrations of extracellular free calcium ([Ca2+]e) rapidly induce regulated parathyroid hormone (PTH) secretion in an alpha-Kl- and Na+,K+-ATPase-dependent manner. The increased Na+ gradient created by Na+,K+-ATPase activity might drive the transepithelial transport of Ca2+ in cooperation with ion channels and transporters in the choroid plexus and the kidney. Our findings reveal fundamental roles of alpha-Kl in the regulation of calcium metabolism.
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PMID:alpha-Klotho as a regulator of calcium homeostasis. 1756 64

The premature aging and cancer-prone disease Werner syndrome stems from loss of WRN protein function. WRN deficiency causes replication abnormalities, sensitivity to certain genotoxic agents, genomic instability and early replicative senescence in primary fibroblasts. As a RecQ helicase family member, WRN is a DNA-dependent ATPase and unwinding enzyme, but also possesses strand annealing and exonuclease activities. RecQ helicases are postulated to participate in pathways responding to replication blockage, pathways possibly initiated by fork regression. In this study, a series of model replication fork substrates were used to examine the fork regression capability of WRN. Our results demonstrate that WRN catalyzes fork regression and Holliday junction formation. This process is an ATP-dependent reaction that is particularly efficient on forks containing single-stranded gaps of at least 11-13 nt on the leading arm at the fork junction. Importantly, WRN exonuclease activity, by digesting the leading daughter strand, enhances regression of forks with smaller gaps on the leading arm, thus creating an optimal structure for regression. Our results suggest that the multiple activities of WRN cooperate to promote replication fork regression. These findings, along with the established cellular consequences of WRN deficiency, strongly support a role for WRN in regression of blocked replication forks.
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PMID:Replication fork regression in vitro by the Werner syndrome protein (WRN): holliday junction formation, the effect of leading arm structure and a potential role for WRN exonuclease activity. 1771 3

Werner syndrome (WS) is a premature aging disorder caused by mutations in the WS gene (WRN). Although WRN has been suggested to play an important role in DNA metabolic pathways, such as recombination, replication and repair, its precise role still remains to be determined. WRN possesses ATPase, helicase and exonuclease activities. Previous studies have shown that the WRN exonuclease is inhibited in vitro by certain lesions induced by oxidative stress and positioned in the digested strand of the substrate. The presence of the 70/86 Ku heterodimer (Ku), participating in the repair of double-strand breaks (DSBs), alleviates WRN exonuclease blockage imposed by the oxidatively induced DNA lesions. The current study demonstrates that WRN exonuclease is inhibited by several additional oxidized bases, and that Ku stimulates the WRN exonuclease to bypass these lesions. Specific lesions present in the non-digested strand were shown also to inhibit the progression of the WRN exonuclease; however, Ku was not able to stimulate WRN exonuclease to bypass these lesions. Thus, this study considerably broadens the spectrum of lesions which block WRN exonuclease progression, shows a blocking effect of lesions in the non-digested strand, and supports a function for WRN and Ku in a DNA damage processing pathway.
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PMID:WRN Exonuclease activity is blocked by specific oxidatively induced base lesions positioned in either DNA strand. 1865 45

Cockayne syndrome (CS) is a complex, progressive disease that involves neurological and developmental impairment and premature aging. The majority of CS patients have mutations in the CSB gene. The CSB protein is involved in multiple DNA repair pathways and CSB mutated cells are sensitive to a broad spectrum of genotoxic agents. We tested the hypothesis that sensitivity to such genotoxins could be mediated by mitochondrial dysfunction as a consequence of the CSB mutation. mtDNA from csb(m/m) mice accumulates oxidative damage including 8-oxoguanine, and cells from this mouse are hypersensitive to the mitochondrial oxidant menadione. Inhibitors of mitochondrial complexes and the glycolysis inhibitor 2-deoxyglucose kill csb(m/m) cells more efficiently than wild-type cells, via a mechanism that does not correlate with mtDNA damage formation. Menadione depletes cellular ATP, and recovery after depletion is slower in csb(m/m) cells. The bioenergetic alteration in csb(m/m) cells parallels the simpler organization of supercomplexes consisting of complexes I, III and IV in addition to partially disassembled complex V in the inner mitochondrial membrane. Exposing wild-type cells to DNA intercalating agents induces complex alterations, suggesting a link between mtDNA integrity, respiratory complexes and mitochondrial function. Thus, mitochondrial dysfunction may play a role in the pathology of CS.
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PMID:Accumulation of mitochondrial DNA damage and bioenergetic dysfunction in CSB defective cells. 1938 14

Cockayne syndrome complementation group B (CSB) protein is engaged in transcription-coupled repair (TCR) of UV induced DNA damage and its deficiency leads to progressive multisystem degeneration and premature aging. Here, we show that human CSB-deficient cells are hypersensitive to physiological concentrations (1-10 microM) of a lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE), and in response to HNE they develop a higher level of sister chromatid exchanges (SCEs) in comparison to the wild-type cells. HNE-DNA adducts block in vitro transcription by T7 RNA polymerase, as well as by HeLa cell-free extracts. Treatment of wild-type cells with 1-20 microM HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity necessary for TCR. However, high HNE concentrations (100-200 microM) inhibit in vitro CSB ATPase activity as well as the transcription machinery in HeLa cell-free extracts. Cell lines expressing CSB protein mutated in different ATPase domains exhibit different sensitivities to HNE. The motif II mutant, which binds ATP, but is defective in ATP hydrolysis was as sensitive to HNE as CSB-null cells. In contrast, motif V mutant cells were as sensitive to HNE as were the cells bearing wild-type protein, while motif VI mutant cells showed intermediate sensitivity to HNE. These mutants exhibit decreased ATP binding, but retain residual ATPase activity. Homology modeling suggested that amino acids mutated in motifs II and VI are localized closer to the ATP binding site than amino acids mutated in ATPase motif V. These results suggest that HNE-DNA adducts are extremely toxic endogenous DNA lesion, and that their processing involves CSB. When these lesions are not removed from the transcribed DNA strand due to CSB gene mutation or CSB protein inactivation by high, pathological HNE concentrations, they may contribute to accelerated aging.
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PMID:Cockayne syndrome group B protein is engaged in processing of DNA adducts of lipid peroxidation product trans-4-hydroxy-2-nonenal. 1948 76


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