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1.
Yang B Zhang J Cai D Li D Chen W Jiang H Hu Y 《Biochemical and biophysical research communications》2006,342(4):1188-1196
The non-structural (NS) proteins of parvoviruses are involved in essential steps of the viral life cycle. Various biochemical functions, such as ATP binding, ATPase, site-specific DNA binding and nicking, and helicase activities, have been assigned to the protein NS1. Compared with the non-structural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been well characterized. Here, we describe the biochemical properties of NS1 of Periplaneta fuliginosa densovirus (PfDNV). We have expressed and purified NS1 using a baculovirus system and analyzed its enzymatic activity. The purified recombinant NS1 protein possesses ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg(2+) or Mn(2+) as a cofactor. The ATPase activity of NS1 can be efficiently stimulated by single-stranded DNA. The ATPase coupled helicase activity was detected on blunt-ended double-stranded oligonucleotide substrate. Using South-Western and Dot-spot assays, we identified a DNA fragment that is recognized specifically by the recombinant NS1 protein. The fragment consists of (CAC)(4) and is located on the hairpin region of the terminal palindrome. The domain for DNA binding was defined to the amino-terminal region (amino acids 1-250). In addition, we found that NS1 can form oligomeric complexes in vivo and in vitro. Mutagenesis analysis showed that ATP binding is necessary for oligomerization. Based on these results, it seems that PfDNV NS1, a multifunctional protein, plays an important role in viral DNA replication comparable to those of vertebrate parvovirus initiator proteins. 相似文献
2.
Wu Y Sommers JA Loiland JA Kitao H Kuper J Kisker C Brosh RM 《The Journal of biological chemistry》2012,287(26):21699-21716
The Q motif, conserved in a number of RNA and DNA helicases, is proposed to be important for ATP binding based on structural data, but its precise biochemical functions are less certain. FANCJ encodes a Q motif DEAH box DNA helicase implicated in Fanconi anemia and breast cancer. A Q25A mutation of the invariant glutamine in the Q motif abolished its ability to complement cisplatin or telomestatin sensitivity of a fancj null cell line and exerted a dominant negative effect. Biochemical characterization of the purified recombinant FANCJ-Q25A protein showed that the mutation disabled FANCJ helicase activity and the ability to disrupt protein-DNA interactions. FANCJ-Q25A showed impaired DNA binding and ATPase activity but displayed ATP binding and temperature-induced unfolding transition similar to FANCJ-WT. Size exclusion chromatography and sedimentation velocity analyses revealed that FANCJ-WT existed as molecular weight species corresponding to a monomer and a dimer, and the dimeric form displayed a higher specific activity for ATPase and helicase, as well as greater DNA binding. In contrast, FANCJ-Q25A existed only as a monomer, devoid of helicase activity. Thus, the Q motif is essential for FANCJ enzymatic activity in vitro and DNA repair function in vivo. 相似文献
3.
Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2, 4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex. Biochemical analyses of these mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated. 相似文献
4.
J Yanagisawa M Seki T Kohda T Enomoto M Ui 《The Journal of biological chemistry》1992,267(6):3644-3649
In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771). 相似文献
5.
DNA helicase and nucleoside-5'-triphosphatase activities of polyoma virus large tumor antigen 总被引:15,自引:0,他引:15
Polyoma virus large tumor antigen (PyV T antigen) has been purified to near homogeneity by immunoaffinity column chromatography. We have detected DNA helicase and ATPase (nucleoside-5'-triphosphatase) activities in the purified PyV T antigen fraction and characterized these activities. The ATPase activity was stimulated about 2-fold by poly(dT), which was the most effective stimulator among the synthetic polynucleotides tested. Natural nucleic acids, such as calf thymus native and heat-denatured DNA, and single-stranded circular fd DNA were also effective, but the degree of stimulation was less than 1.5-fold. The basal and poly(dT)-stimulated ATPase activities showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optima. The preference for nucleoside 5'-triphosphates was ATP, dATP greater than CTP, UTP much greater than GTP. The only difference observed between the two activities was salt sensitivity. The basal ATPase activity was resistant to KC1 up to 300 mM. In contrast, poly-(dT)-stimulated activity was reduced to the level of basal activity at 300 mM KC1. DNA helicase activity required divalent cations and was dependent on hydrolysis of ATP. The activity showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optimum as the two ATPase activities, and the salt sensitivity of DNA helicase activity was similar to that of poly(dT)-stimulated ATPase activity. The helicase activity was inhibited competitively by the addition of single-stranded or double-stranded DNA, and a relatively high inhibitory activity was observed with poly [d(A-T)]. The PyV T antigen helicase was found to migrate in the 3' to 5' direction along the DNA strand to which the protein bound. 相似文献
6.
RNA-Stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3
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Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviviridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3'-to-5' direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5' and 3' overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain. 相似文献
7.
Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding. 相似文献
8.
DNA helicase from calf thymus. Purification to apparent homogeneity and biochemical characterization of the enzyme 总被引:9,自引:0,他引:9
We have purified a DNA helicase from calf thymus to apparent homogeneity by monitoring the activity with a strand displacement assay. DNA helicase followed the DNA polymerase alpha-primase complex through chromatography on phosphocellulose and hydroxylapatite. Separation from DNA polymerase alpha-primase complex as well as from the bulk of another DNA-dependent ATPase was achieved on heparin-Sepharose. Further purification steps included ATP-agarose and fast protein liquid chromatography-Mono S. A 47-kDa polypeptide cosedimented with the DNA helicase activity in a glycerol gradient as well as in gel filtration on Superose 6. The calf thymus DNA helicase had a sedimentation coefficient of 4-7 S and Stokes radius of about 45 A suggesting that the enzyme might be monomer in its functional form. DNA helicase activity requires a divalent cation with Mg2+ being more efficient than Mn2+ or Ca2+. Hydrolysis of ATP is required since the two nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenylyl (beta, gamma-methylene)diphosphonate cannot substitute for ATP or dATP in the displacement reaction. Calf thymus DNA helicase is able to use ATP, dATP, dideoxy-ATP, CTP, and dCTP with Km for ATP and dATP of 0.2 and 0.25 mM, respectively. The enzyme can displace a fragment of 24 bases completely in an enzyme concentration- and time-dependent manner. The DNA helicase appears to bind to single-stranded DNA and to move to single-strand double-strand transition. The directionality of unwinding is 3'----5' with respect to the single-stranded DNA to which the enzyme is bound. 相似文献
9.
Borowski P Niebuhr A Mueller O Bretner M Felczak K Kulikowski T Schmitz H 《Journal of virology》2001,75(7):3220-3229
The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH(2) terminus by microsequencing, and a binding assay with 5'-[(14)C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with k(cat) values of 133 and 5.5 x 10(-3) s(-1), respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg(2+) requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5'-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine and O(6)-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value. 相似文献
10.
The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding. 总被引:13,自引:3,他引:10
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Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent. 相似文献
11.
The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)(8) oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in other DNA and RNA helicases, their role in the mechanism of unwinding of double-stranded nucleic acid is discussed. 相似文献
12.
Prakash R Krejci L Van Komen S Anke Schürer K Kramer W Sung P 《The Journal of biological chemistry》2005,280(9):7854-7860
The MPH1 (mutator pHenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of elevated spontaneous mutation rates of haploid cells deleted for this gene. Further studies showed that MPH1 functions to channel DNA lesions into an error-free DNA repair pathway. The Mph1 protein contains the seven conserved motifs of the superfamily 2 (SF2) family of nucleic acid unwinding enzymes. Genetic analyses have found epistasis of the mph1 deletion with mutations in the RAD52 gene group that mediates homologous recombination and DNA repair by homologous recombination. To begin dissecting the biochemical functions of the MPH1-encoded product, we have expressed it in yeast cells and purified it to near homogeneity. We show that Mph1 has a robust ATPase function that requires single-stranded DNA for activation. Consistent with its homology to members of the SF2 helicase family, we find a DNA helicase activity in Mph1. We present data to demonstrate that the Mph1 DNA helicase activity is fueled by ATP hydrolysis and has a 3' to 5' polarity with respect to the DNA strand on which this protein translocates. The DNA helicase activity of Mph1 is enhanced by the heterotrimeric single-stranded DNA binding protein replication protein A. These results, thus, establish Mph1 as an ATP-dependent DNA helicase, and the availability of purified Mph1 should facilitate efforts at deciphering the role of this protein in homologous recombination and mutation avoidance. 相似文献
13.
The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length. 相似文献
14.
Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA 总被引:10,自引:0,他引:10
Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA. 相似文献
15.
A DNA helicase from Xenopus laevis ovaries 总被引:5,自引:0,他引:5
A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases. 相似文献
16.
The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6. 相似文献
17.
Manhong Guo Venkatasubramanian Vidhyasagar Hao Ding Yuliang Wu 《The Journal of biological chemistry》2014,289(15):10551-10565
Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding and remodeling of structured DNA or RNA, which is coordinated by conserved helicase motifs. FANCJ is a DNA helicase that is genetically linked to Fanconi anemia, breast cancer, and ovarian cancer. Here, we characterized two Fanconi anemia patient mutations, R251C and Q255H, that are localized in helicase motif Ia. Our genetic complementation analysis revealed that both the R251C and Q255H alleles failed to rescue cisplatin sensitivity of a FANCJ null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, our biochemical assays demonstrated that both purified recombinant proteins abolished DNA helicase activity and failed to disrupt the DNA-protein complex. Intriguingly, R251C impaired DNA binding ability to single-strand DNA and double-strand DNA, whereas Q255H retained higher binding activity to these DNA substrates compared with wild-type FANCJ protein. Consequently, R251C abolished its DNA-dependent ATP hydrolysis activity, whereas Q255H retained normal ATPase activity. Physically, R251C had reduced ATP binding ability, whereas Q255H had normal ATP binding ability and could translocate on single-strand DNA. Although both proteins were recruited to damage sites in our laser-activated confocal assays, they lost their DNA repair function, which explains why they exerted a domain negative effect when expressed in a wild-type background. Taken together, our work not only reveals the structural function of helicase motif Ia but also provides the molecular pathology of FANCJ in related diseases. 相似文献
18.
A cloned cDNA corresponding to OsDMC1 from rice anther tissue was expressed in Escherichia coli. The OsDmc1 protein was largely present in the inclusion bodies of the cell lysatE., which was solubilized by 8.0 M urea containing buffeR., purified to homogeneity by Ni-CAM agarose column chromatography, followed by renaturation to its native state through stepwise dialysis against reduced concentrations of urea. The purified protein cross-reacted with anti-yeast Dmc1 antibodies. The binding efficiency observed with circular single-stranded DNA (ssDNA) was similar to that with circular double-stranded DNA (dsDNA). The binding to either DNA showed no ATP dependencE., but required 5–10 mM Mg2+ in the presence of ATP. Even though the protein binding to dsDNA was as efficient as it was to ssDNA, the former induced no DNA dependent ATPasE., whereas the binding to ssDNA stimulated a significant level of DNA dependent ATPase activity. OsDmc1–ssDNA complex, with its ATPase proficiency, also mediated renaturation of homologous complementary strands as well as assimilation of single strands into homologous supercoiled duplexes leading to D-loop formation. The D-loop formation was lowered by excess of OsDmc1 protein. This D-loop formation activity was promoted by non-hydrolyzable ATP analog, AMP-PNP and was not observed in absence of ATP or presence of ADP/ATP--S. These properties reflected the classical hallmarks of a recombinase and represented the first biochemical characterization of a plant Dmc1 protein. 相似文献
19.
Herpes simplex virus helicase-primase: the UL8 protein is not required for DNA-dependent ATPase and DNA helicase activities. 总被引:13,自引:1,他引:12
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The herpes simplex virus type 1 helicase-primase complex consists of the products of the UL5, UL8 and UL52 genes. We have expressed these proteins in insect cells using baculovirus vectors and studied the requirements for enzymatic activities associated with the DNA unwinding function of the complex. In agreement with a recent report (Dodson, M.S., Crute, J.J., Bruckner, R.C. and Lehman, I.R. 1989, J. Biol. Chem. 264, 20835-20838) we find that DNA-dependent ATPase and DNA helicase activities are assembled in vivo in insect cells triply infected with viruses expressing the UL5, UL8 and UL52 proteins. Moreover, these activities were also detected in cells in which only the UL5 and UL52 products were expressed indicating that the presence of the UL8 protein is essential for neither the ATPase nor helicase activity of the complex. 相似文献
20.
The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity
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The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases. 相似文献