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1.
Bacillus subtilis bacteriophage SPP1 hexameric DNA helicase,G40P,interacts with forked DNA
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SPP1-encoded replicative DNA helicase gene 40 product (G40P) is an essential product for phage replication. Hexameric G40P, in the presence of AMP-PNP, preferentially binds unstructured single-stranded (ss)DNA in a sequence-independent manner. The efficiency of ssDNA binding, nucleotide hydrolysis and the unwinding activity of G40P are affected in a different manner by different nucleotide cofactors. Nuclease protection studies suggest that G40P protects the 5′ tail of a forked molecule, and the duplex region at the junction against exonuclease attack. G40P does not protect the 3′ tail of a forked molecule from exonuclease attack. By using electron microscopy we confirm that the ssDNA transverses the centre of the hexameric ring. Our results show that hexameric G40P DNA helicase encircles the 5′ tail, interacts with the duplex DNA at the ss–double-stranded DNA junction and excludes the 3′ tail of the forked DNA. 相似文献
2.
Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities 总被引:7,自引:6,他引:1
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Xiaohuai Zhou Irene Zolotukhin Dong-Soo Im Nicholas Muzyczka 《Journal of virology》1999,73(2):1580-1590
The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl2. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The Km values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement. 相似文献
3.
Shuang Wang Wei Qin Jing-Hua Li Ying Lu Ke-Yu Lu Da-Guan Nong Shuo-Xing Dou Chun-Hua Xu Xu-Guang Xi Ming Li 《Nucleic acids research》2015,43(7):3736-3746
There are lines of evidence that the Bloom syndrome helicase, BLM, catalyzes regression of stalled replication forks and disrupts displacement loops (D-loops) formed during homologous recombination (HR). Here we constructed a forked DNA with a 3′ single-stranded gap and a 5′ double-stranded handle to partly mimic a stalled DNA fork and used magnetic tweezers to study BLM-catalyzed unwinding of the forked DNA. We have directly observed that the BLM helicase may slide on the opposite strand for some distance after duplex unwinding at different forces. For DNA construct with a long hairpin, progressive unwinding of the hairpin is frequently interrupted by strand switching and backward sliding of the enzyme. Quantitative study of the uninterrupted unwinding length (time) has revealed a two-state-transition mechanism for strand-switching during the unwinding process. Mutational studies revealed that the RQC domain plays an important role in stabilizing the helicase/DNA interaction during both DNA unwinding and backward sliding of BLM. Especially, Lys1125 in the RQC domain, a highly conserved amino acid among RecQ helicases, may be involved in the backward sliding activity. We have also directly observed the in vitro pathway that BLM disrupts the mimic stalled replication fork. These results may shed new light on the mechanisms for BLM in DNA repair and homologous recombination. 相似文献
4.
Andreas Sturzenegger Kamila Burdova Radhakrishnan Kanagaraj Maryna Levikova Cosimo Pinto Petr Cejka Pavel Janscak 《The Journal of biological chemistry》2014,289(39):27314-27326
The 5′-3′ resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5′-3′ DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells. 相似文献
5.
RecQ5 is one of five RecQ helicase homologs identified in humans. Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms. To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity. However, little is known about other members of the RecQ family. Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases. Drosophila RECQ5 helicase is capable of unwinding 3′ Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5′ Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM. 相似文献
6.
Anuradha Kumari Irina G. Minko Rebecca L. Smith R. Stephen Lloyd Amanda K. McCullough 《The Journal of biological chemistry》2010,285(28):21313-21322
UvrD (DNA helicase II) has been implicated in DNA replication, DNA recombination, nucleotide excision repair, and methyl-directed mismatch repair. The enzymatic function of UvrD is to translocate along a DNA strand in a 3′ to 5′ direction and unwind duplex DNA utilizing a DNA-dependent ATPase activity. In addition, UvrD interacts with many other proteins involved in the above processes and is hypothesized to facilitate protein turnover, thus promoting further DNA processing. Although UvrD interactions with proteins bound to DNA have significant biological implications, the effects of covalent DNA-protein cross-links on UvrD helicase activity have not been characterized. Herein, we demonstrate that UvrD-catalyzed strand separation was inhibited on a DNA strand to which a 16-kDa protein was covalently bound. Our sequestration studies suggest that the inhibition of UvrD activity is most likely due to a translocation block and not helicase sequestration on the cross-link-containing DNA substrate. In contrast, no inhibition of UvrD-catalyzed strand separation was apparent when the protein was linked to the complementary strand. The latter result is surprising given the earlier observations that the DNA in this covalent complex is severely bent (∼70°), with both DNA strands making multiple contacts with the cross-linked protein. In addition, UvrD was shown to be required for replication of plasmid DNAs containing covalent DNA-protein complexes. Combined, these data suggest a critical role for UvrD in the processing of DNA-protein cross-links. 相似文献
7.
Tresa George Qin Wen Richard Griffiths Anil Ganesh Mark Meuth Cyril M. Sanders 《Nucleic acids research》2009,37(19):6491-6502
Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity. 相似文献
8.
Katrina N. Koc Joseph L. Stodola Peter M. Burgers Roberto Galletto 《Nucleic acids research》2015,43(8):4179-4190
The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo− to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities. 相似文献
9.
The Bloom's syndrome helicase promotes the annealing of complementary single-stranded DNA 总被引:14,自引:7,他引:7
The product of the gene mutated in Bloom's syndrome, BLM, is a 3′–5′ DNA helicase belonging to the highly conserved RecQ family. In addition to a conventional DNA strand separation activity, BLM catalyzes both the disruption of non-B-form DNA, such as G-quadruplexes, and the branch migration of Holliday junctions. Here, we have characterized a new activity for BLM: the promotion of single-stranded DNA (ssDNA) annealing. This activity does not require Mg2+, is inhibited by ssDNA binding proteins and ATP, and is dependent on DNA length. Through analysis of various truncation mutants of BLM, we show that the C-terminal domain is essential for strand annealing and identify a 60 amino acid stretch of this domain as being important for both ssDNA binding and strand annealing. We present a model in which the ssDNA annealing activity of BLM facilitates its role in the processing of DNA intermediates that arise during repair of damaged replication forks. 相似文献
10.
Bypass of heterology during strand transfer by Saccharomyces cerevisiae Rad51 protein 总被引:1,自引:1,他引:0
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Victor F. Holmes Kirsten R. Benjamin Nancy J. Crisona Nicholas R. Cozzarelli 《Nucleic acids research》2001,29(24):5052-5057
During recombination-mediated repair of DNA double-strand breaks, strand transfer proteins must distinguish a homologous repair template from closely related genomic sequences. However, some tolerance by strand transfer proteins for sequence differences is also critical: too much stringency will prevent recombination between different alleles of the same gene, but too much tolerance will lead to illegitimate recombination. We characterized the heterology tolerance of Saccharomyces cerevisiae Rad51 by testing bypass of small heterologous inserts in either the single- or double-stranded substrate of an in vitro strand transfer reaction that models the early steps of homologous recombination. We found that the yeast protein is rather stringent, only tolerating heterologies up to 9 bases long. The efficiency of heterology bypass depends on whether the insert is in the single- or double-stranded substrate, as well as on the location of the insert relative to the end of the double-stranded linear substrate. Rad51 is distinct in that it can catalyze strand transfer in either the 3′→5′ or 5′→3′ direction. We found that bypass of heterology was independent of the polarity of strand transfer, suggesting that the mechanism of 5′→3′ transfer is the same as that of 3′→5′ transfer. 相似文献
11.
Wei Qin Na-Nv Liu Lijun Wang Min Zhou Hua Ren Elisabeth Bugnard Jie-Lin Liu Lin-Hu Zhang Jeremie Vend?me Jin-Shan Hu Xu Guang Xi 《Journal of bacteriology》2014,196(24):4216-4228
RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg2+-dependent DNA helicase activity with a 3′→5′ polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3′-overhang but is inactive on blunt-ended dsDNA and 5′-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5′-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]''s possible functions in preserving genomic stability. 相似文献
12.
Yuriy Chaban Jonathan A. Stead Ksenia Ryzhenkova Fiona Whelan Ekaterina P. Lamber Alfred Antson Cyril M. Sanders Elena V. Orlova 《Nucleic acids research》2015,43(17):8551-8563
Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases. 相似文献
13.
DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3′-5′ direction and shows ATP-dependent 3′-5′, and surprisingly also, 5′-3′ helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved β-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD’s 3′-5′ and 5′-3′ helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5′-3′ helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo. 相似文献
14.
Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements
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The flap endonuclease (FEN) of the hyperthermophilic archaeon Methanococcus jannaschii was expressed in Escherichia coli and purified to homogeneity. FEN retained activity after preincubation at 95°C for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5′ end adjacent to the single-strand–duplex junction. Deletion of the free 3′ end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3′ end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5′ end failed to block cleavage, although this substrate was refractory to cleavage by the 5′-3′ exonuclease activity of Taq DNA polymerase. For verification, the free 5′ end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for Taq DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5′ single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion X174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme. 相似文献
15.
Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer 总被引:4,自引:0,他引:4
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Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ~17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex. 相似文献
16.
Xin Ee Yong V Raghuvamsi Palur Ganesh S Anand Thorsten Wohland Kamal K Sharma 《Nucleic acids research》2021,49(10):5832
By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5′-3′ panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5′-3′ panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication. 相似文献
17.
Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase. 相似文献
18.
Human RECQ5beta helicase promotes strand exchange on synthetic DNA structures resembling a stalled replication fork
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The role of the human RECQ5β helicase in the maintenance of genomic stability remains elusive. Here we show that RECQ5β promotes strand exchange between arms of synthetic forked DNA structures resembling a stalled replication fork in a reaction dependent on ATP hydrolysis. BLM and WRN can also promote strand exchange on these structures. However, in the presence of human replication protein A (hRPA), the action of these RecQ-type helicases is strongly biased towards unwinding of the parental duplex, an effect not seen with RECQ5β. A domain within the non-conserved portion of RECQ5β is identified as being important for its ability to unwind the lagging-strand arm and to promote strand exchange on hRPA-coated forked structures. We also show that RECQ5β associates with DNA replication factories in S phase nuclei and persists at the sites of stalled replication forks after exposure of cells to UV irradiation. Moreover, RECQ5β is found to physically interact with the polymerase processivity factor proliferating cell nuclear antigen in vitro and in vivo. Collectively, these findings suggest that RECQ5β may promote regression of stalled replication forks to facilitate the bypass of replication-blocking lesions by template-switching. Loss of such activity could explain the elevated level of mitotic crossovers observed in RECQ5β-deficient cells. 相似文献
19.
Stimulation of MCM helicase activity by a Cdc6 protein in the archaeon Thermoplasma acidophilum 总被引:2,自引:1,他引:1
Replicative DNA helicases are ring-shaped hexamers that play an essential role in chromosomal DNA replication. They unwind the two strands of the duplex DNA and provide the single-stranded (ss) DNA substrate for the polymerase. The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in eukarya and archaea. The proteins of only a few archaeal organisms have been studied and revealed that although all have similar amino acid sequences and overall structures they differ in their biochemical properties. In this report the biochemical properties of the MCM protein from the archaeon Thermoplasma acidophilum is described. The enzyme has weak helicase activity on a substrate containing only a 3′-ssDNA overhang region and the protein requires a forked DNA structure for efficient helicase activity. It was also found that the helicase activity is stimulated by one of the two T.acidophilum Cdc6 homologues. This is an interesting observation as it is in sharp contrast to observations made with MCM and Cdc6 homologues from other archaea in which the helicase activity is inhibited when bound to Cdc6. 相似文献
20.
Christopher Capp Jianhong Wu Tao-shih Hsieh 《The Journal of biological chemistry》2009,284(45):30845-30852
Members of the RecQ family of proteins are highly conserved DNA helicases that have important functions in the maintenance of genomic stability. Deficiencies in RecQ4 have been linked to human diseases including Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes, all of which are characterized by developmental defects, tumor propensity, and genetic instability. However, there are conflicting results shown in the literature regarding the DNA helicase activity of RecQ4. We report here the expression of Drosophila melanogaster RecQ4 with a baculoviral vector and its purification to near homogeneity. The purified protein has a DNA-dependent ATPase activity and is a 3′-5′ DNA helicase dependent on hydrolysis of ATP. The presence of 5′-adenylyl-β,γ-imidodiphosphate (AMPPNP), a nonhydrolyzable ATP analog, promotes stable complex formation between RecQ4 and single-stranded DNA. Drosophila RecQ4 can also anneal complementary single strands; this activity was reduced in the presence of AMPPNP, possibly because of the stable protein-DNA complex formed under such conditions. A point mutation of the highly conserved lysine residue in the helicase domain, although retaining the wild type level of annealing activity, inactivated ATPase and helicase activities and eliminated stable complex formation. These results suggest that the helicase domain alone is responsible for the DNA unwinding action of the Drosophila enzyme. We generated a null recq4 mutant that is homozygous lethal, which we used to test the genetic function of the helicase-dead mutant in flies. Complementation tests showed that the helicase-dead mutant recq4 transgenes are incapable of rescuing the null mutation, demonstrating that the helicase activity has an essential biological function. 相似文献