共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
UvsW protein belongs to the SF2 helicase family and is one of three helicases found in T4 phage. UvsW governs the transition from origin-dependent to origin-independent replication through the dissociation of R-loops located at the T4 origins of replication. Additionally, in vivo evidence indicates that UvsW plays a role in recombination-dependent replication and/or DNA repair. Here, the biochemical properties of UvsW helicase are described. UvsW is a 3' to 5' helicase that unwinds a wide variety of substrates, including those resembling stalled replication forks and recombination intermediates. UvsW also contains a potent single-strand DNA annealing activity that is enhanced by ATP hydrolysis but does not require it. The annealing activity is inhibited by the non-hydrolysable ATP analog (adenosine 5'-O-(thiotriphosphate)), T4 single-stranded DNA-binding protein (gp32), or a small 8.8-kDa polypeptide (UvsW.1). Fluorescence resonance energy transfer experiments indicate that UvsW and UvsW.1 form a complex, suggesting that the UvsW helicase may exist as a heterodimer in vivo. Fusion of UvsW and UvsW.1 results in a 68-kDa protein having nearly identical properties as the UvsW-UvsW.1 complex, indicating that the binding locus of UvsW.1 is close to the C terminus of UvsW. The biochemical properties of UvsW are similar to the RecQ protein family and suggest that the annealing activity of these helicases may also be modulated by protein-protein interactions. The dual activities of UvsW are well suited for the DNA repair pathways described for leading strand lesion bypass and synthesis-dependent strand annealing. 相似文献
3.
Bacteriophage T4 UvsW protein is a helicase involved in recombination, repair and the regulation of DNA replication origins. 总被引:7,自引:0,他引:7
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Bacteriophage T4 UvsW protein is involved in phage recombination, repair and the regulation of replication origins. Here, we provide evidence that UvsW functions as a helicase. First, expression of UvsW allows growth of an (otherwise inviable) Escherichia coli recG rnhA double mutant, consistent with UvsW being a functional analog of the RecG helicase. Second, UvsW contains helicase sequence motifs, and a substitution (K141R) in the Walker 'A' motif prevents growth of the E.coli recG rnhA double mutant. Third, UvsW, but not UvsW-K141R, inhibits replication from a T4 origin at which persistent RNA-DNA hybrids form and presumably trigger replication initiation. Fourth, mutations that inactivate UvsW and endonuclease VII (which cleaves DNA branches) synergistically block repair of double-strand breaks. These in vivo results are consistent with a model in which UvsW is a DNA helicase that catalyzes branch migration and dissociation of RNA-DNA hybrids. In support of this model, a partially purified GST/UvsW fusion protein, but not a GST/UvsW-K141R fusion, displays ssDNA-dependent ATPase activity and is able to unwind a branched DNA substrate. 相似文献
4.
The UvsW protein of bacteriophage T4 is involved in many aspects of phage DNA metabolism, including repair, recombination, and recombination-dependent replication. UvsW has also been implicated in the repression of origin-dependent replication at late times of infection, when UvsW is normally synthesized. Two well-characterized T4 origins, ori(uvsY) and ori(34), are believed to initiate replication through an R-loop mechanism. Here we provide both in vivo and in vitro evidence that UvsW is an RNA-DNA helicase that catalyzes the dissociation of RNA from origin R-loops. Two-dimensional gel analyses show that the replicative intermediates formed at ori(uvsY) persist longer in a uvsW mutant infection than in a wild-type infection. In addition, the inappropriate early expression of UvsW protein results in the loss of these replicative intermediates. Using a synthetic origin R-loop, we also demonstrate that purified UvsW functions as a helicase that efficiently dissociates RNA from R-loops. These and previous results from a number of studies provide strong evidence that UvsW is a molecular switch that allows T4 replication to progress from a mode that initiates from R-loops at origins to a mode that initiates from D-loops formed by recombination proteins. 相似文献
5.
Webb MR Plank JL Long DT Hsieh TS Kreuzer KN 《The Journal of biological chemistry》2007,282(47):34401-34411
The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair. 相似文献
6.
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
7.
Liu Z Macias MJ Bottomley MJ Stier G Linge JP Nilges M Bork P Sattler M 《Structure (London, England : 1993)》1999,7(12):1557-1566
BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent manner. However, the specific functional roles of these proteins in DNA recombination and replication are not known. An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function. RESULTS: We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids. We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases. Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p. CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism. In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions. 相似文献
8.
Talavera MA Matthews EE Eliason WK Sagi I Wang J Henn A De La Cruz EM 《Journal of molecular biology》2006,355(4):697-707
The Escherichia coli DEAD-box protein A (DbpA) belongs to the highly conserved superfamily-II of nucleic acid helicases that play key roles in RNA metabolism. A central question regarding helicase activity is whether the process of coupling ATP hydrolysis to nucleic acid unwinding requires an oligomeric form of the enzyme. We have investigated the structural and functional properties of DbpA by multi-angle laser light-scattering, size-exclusion chromatography, analytical ultracentrifugation, chemical cross-linking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 microM and over the temperature range of 4 degrees C to 22 degrees C. Binding of neither nucleotide (ATP or ADP) nor peptidyl transferase center (PTC) RNA, the presumed physiological RNA substrate, favor oligomerization. The hydrodynamic parameters were used together with hydrodynamic bead modeling and structural homology in conjunction with ab initio structure prediction methods to define plausible shapes of DbpA. Collectively, the results favor models where DbpA functions as an active monomer that possesses two distinct RNA binding sites, one in the helicase core domain and the other in the carboxyl-terminal domain that recognizes 23S rRNA and interacts specifically with hairpin 92 of the PTC. 相似文献
9.
Gajewski S Webb MR Galkin V Egelman EH Kreuzer KN White SW 《Journal of molecular biology》2011,405(1):65-76
Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates. 相似文献
10.
11.
The budding yeast Srs2 protein possesses 3′ to 5′ DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR). 相似文献
12.
RPA alleviates the inhibitory effect of vinylphosphonate internucleotide linkages on DNA unwinding by BLM and WRN helicases 总被引:3,自引:1,他引:2
Garcia PL Bradley G Hayes CJ Krintel S Soultanas P Janscak P 《Nucleic acids research》2004,32(12):3771-3778
Bloom (BLM) and Werner (WRN) syndrome proteins are members of the RecQ family of SF2 DNA helicases. In this paper, we show that restricting the rotational DNA backbone flexibility, by introducing vinylphosphonate internucleotide linkages in the translocating DNA strand, inhibits efficient duplex unwinding by these enzymes. The human single-stranded DNA binding protein replication protein A (RPA) fully restores the unwinding activity of BLM and WRN on vinylphosphonate-containing substrates while the heterologous single-stranded DNA binding protein from Escherichia coli (SSB) restores the activity only partially. Both RPA and SSB fail to restore the unwinding activity of the SF1 PcrA helicase on modified substrates, implying specific interactions of RPA with the BLM and WRN helicases. Our data highlight subtle differences between SF1 and SF2 helicases and suggest that although RecQ helicases belong to the SF2 family, they are mechanistically more similar to the SF1 PcrA helicase than to other SF2 helicases that are not affected by vinylphosphonate modifications. 相似文献
13.
Unwinding of unnatural substrates by a DNA helicase 总被引:6,自引:0,他引:6
Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined. Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding. The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA). PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts. Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates. The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates. 相似文献
14.
Nishino T Komori K Tsuchiya D Ishino Y Morikawa K 《Structure (London, England : 1993)》2005,13(1):143-153
DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the "thumb" domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family. 相似文献
15.
Binding and melting of D-loops by the Bloom syndrome helicase 总被引:10,自引:0,他引:10
Bloom syndrome is a rare autosomal disorder characterized by predisposition to cancer and genomic instability. BLM, the structural gene mutated in individuals with the disorder, encodes a DNA helicase belonging to the RecQ family of helicases. These helicases have been established to serve roles in both promoting and preventing recombination. Mounting evidence has implicated a function for BLM during DNA replication; specifically, BLM might be involved in rescuing stalled or collapsed replication forks by a recombination-based mechanism. We have tested this idea by examining the binding and melting activity of BLM on oligonucleotide substrates containing D-loops, DNA structures that model the presumed initial intermediate formed during homologous recombination. We find that BLM preferentially melts those D-loops that are formed more favorably by the strand exchange protein Rad51, but whose polarity could be less favorable for enabling restoration of an active replication fork. We propose a model in which BLM selectively dissociates recombination intermediates likely to be unfavorable for recombination-promoted replication. 相似文献
16.
Liu D Windsor WT Wyss DF 《Protein science : a publication of the Protein Society》2003,12(12):2757-2767
The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function. 相似文献
17.
Gupta R Sharma S Sommers JA Jin Z Cantor SB Brosh RM 《The Journal of biological chemistry》2005,280(27):25450-25460
We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination. 相似文献
18.
《Cell cycle (Georgetown, Tex.)》2013,12(12):2317-2329
Helicases catalytically unwind structured nucleic acids in a nucleoside-triphosphate-dependent and directionally specific manner, and are essential for virtually all aspects of nucleic acid metabolism. ATPase-driven helicases which translocate along nucleic acids play a role in damage recognition or unwinding of a DNA tract containing the lesion. Although classical biochemical experiments provided evidence that bulky covalent adducts inhibit DNA unwinding catalyzed by certain DNA helicases in a strand-specific manner (i.e., block to DNA unwinding restricted to adduct residence in the strand the helicase translocates), recent studies suggest more complex arrangements that may depend on the helicase under study, its assembly in a protein complex, and the type of structural DNA perturbation. Moreover, base and sugar phosphate backbone modifications exert effects on DNA helicases that suggest specialized tracking mechanisms. As a component of the replication stress response, the single-stranded DNA binding protein Replication Protein A (RPA) may serve to enable eukaryotic DNA helicases to overcome certain base lesions. Helicases play important roles in DNA damage signaling which also involve their partnership with RPA. In this review, we will discuss our current understanding of mechanistic and biological aspects of helicase action on damaged DNA. 相似文献
19.
Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function. 相似文献
20.
Bloom syndrome is a familial genetic disorder associated with sunlight sensitivity and a high predisposition to cancers. The mutated gene, Bloom protein (BLM), encodes a DNA helicase that functions in genome maintenance via roles in recombination repair and resolution of recombination structures. We designed substrates representing illegitimate recombination intermediates formed when a displaced DNA flap generated during maturation of Okazaki fragments escapes cleavage by flap endonuclease-1 and anneals to a complementary ectopic DNA site. Results show that displaced, replication protein A (RPA)-coated flaps could readily bind and ligate at the complementary site to initiate recombination. RPA also displayed a strand-annealing activity that hastens the rate of recombination intermediate formation. BLM helicase activity could directly disrupt annealing at the ectopic site and promote flap endonuclease-1 cleavage. Additionally, BLM has its own strand-annealing and strand-exchange activities. RPA inhibited the BLM strand-annealing activity, thereby promoting helicase activity and complex dissolution. BLM strand exchange could readily dissociate invading flaps, e.g. in a D-loop, if the exchange step did not involve annealing of RPA-coated strands. Use of ATP to activate the helicase function did not aid flap displacement by exchange, suggesting that this is a helicase-independent mechanism of complex dissociation. When RPA could bind, it displayed its own strand-exchange activity. We interpret these results to explain how BLM is well equipped to deal with alternative recombination intermediate structures. 相似文献