Crystallographic and NMR Analyses of UvsW and UvsW.1 from Bacteriophage T4

Abstract

The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3′-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7Å and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases. The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3′-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7Å and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases. Bacteriophage T4 remains an ideal model system in which to study the fundamental mechanisms of nucleic acid metabolism. DNA replication is particularly well suited for study in T4 because the viral genome encodes all the necessary replicative enzymes and accessory proteins that exist in higher organisms (1Miller E.S. Kutter E. Mosig G. Arisaka F. Kunisawa T. Rüger W. Microbiol. Mol. Biol. Rev. 2003; 67: 86-156Crossref PubMed Scopus (543) Google Scholar). During the early stage of the T4 infection cycle, DNA synthesis is initiated by a classic origin-dependent mechanism in which replication complexes are assembled onto persistent R-loops (RNA-DNA hybrids) (2Nossal N.G. Dudas K.C. Kreuzer K.N. Mol. Cell. 2001; 7: 31-41Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). However, this mechanism is quickly abandoned, and during the later stages of infection the switch is made to the more efficient recombination-dependent replication (RDR) 6The abbreviations used are: RDR, recombination-dependent replication; PDB, Protein Data Bank; R.m.s.d., root mean square deviation; HJ, Holliday junctions; ss, single-stranded; ds, double-stranded. 6The abbreviations used are: RDR, recombination-dependent replication; PDB, Protein Data Bank; R.m.s.d., root mean square deviation; HJ, Holliday junctions; ss, single-stranded; ds, double-stranded. to synthesize the bulk of the DNA (3Kreuzer K.N. Annu. Rev. Microbiol. 2005; 59: 43-67Crossref PubMed Scopus (77) Google Scholar). RDR produces a complex concatemeric network of T4 DNA that is ultimately resolved into the circularly permuted and terminally redundant genome that exists within the mature virus particles. The initiation stage of RDR is a recombination reaction in which the 3′-end of a single-stranded region of T4 DNA invades a homologous region of DNA to generate a displacement (D) loop intermediate. The T4 replication machinery is then assembled within the D loop to begin replication from the invading 3′-end (3Kreuzer K.N. Annu. Rev. Microbiol. 2005; 59: 43-67Crossref PubMed Scopus (77) Google Scholar). Homologous recombination is a key process in DNA metabolism that remains poorly understood at the molecular level, and T4 is an ideal system in which to study the mechanism due to its central role in RDR and T4 replication. The T4 proteins UvsX and UvsY perform equivalent functions to the recombination proteins Rad51 and Rad52, respectively, in eukaryotic systems (4Beernink H.T. Morrical S.W. Trends Biochem. Sci. 1999; 24: 385-389Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). UvsX and UvsY are both members of the uvsWXY system, which is associated with susceptibility to DNA damage because of UV radiation (hence the name). This is consistent with the roles that recombination and RDR are now known to play in the repair of DNA double-strand breaks (5Klein H.L. Kreuzer K.N. Mol. Cell. 2002; 9: 471-480Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In addition, it has been known for some time that stalled and collapsed replication forks can lead to DNA double-strand breaks (6Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (866) Google Scholar), and RDR has a pivotal role in repairing the lesions generated by these potentially lethal events (7McGlynn P. Lloyd R.G. Nat. Rev. Mol. Cell Biol. 2002; 3: 859-870Crossref PubMed Scopus (360) Google Scholar). Thus, T4 has also emerged as a model system for studying the molecular basis of the DNA repair processes that serve to maintain genome stability. UvsW is a T4-encoded protein that was first identified through mutations that have multiple and seemingly diverse effects on T4 DNA metabolism (8Derr L.K. Drake J.W. Mol. Gen. Genet. 1990; 222: 257-264Crossref PubMed Scopus (15) Google Scholar, 9Derr L.K. Kreuzer K.N. J. Mol. Biol. 1990; 214: 643-656Crossref PubMed Scopus (23) Google Scholar). The uvsW gene is the third member of the uvsWXY system, and the multiple roles of UvsW were rationalized by in vitro and in vivo studies which revealed that it can catalyze a variety of nucleic acid processing functions as an ATP-dependent helicase (10Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (66) Google Scholar). Primary sequence analyses (10Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (66) Google Scholar) revealed the presence of seven conserved helicase motifs that are involved in substrate binding and hydrolysis (11Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1026) Google Scholar, 12Hall M.C. Matson S.W. Mol. Microbiol. 1999; 34: 867-877Crossref PubMed Scopus (270) Google Scholar). The motifs specifically suggested that UvsW belongs to the SF2 family of helicases, and this was confirmed by the 2.3-Å crystal structure of a stable N-terminal fragment of the inactive UvsW point mutant K141R (UvsWNF) (13Sickmier E.A. Kreuzer K.N. White S.W. Structure. 2004; 12: 583-592Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). The fragment a N-terminal that is homologous to the of the T4 E.A. White S.W. Mol. Microbiol. 2002; PubMed Scopus (29) Google Scholar), and the first RecA-like motor that has been in all monomeric helicase to Annu. Rev. Biochem. PubMed Scopus Google Scholar). UvsW is also involved in the switch from early origin-dependent to replication during the T4 infection cycle, and specifically the of R-loops that as at replication K.C. Kreuzer K.N. Mol. Cell Biol. 2001; PubMed Scopus Google Scholar). During structural analysis of identified an error in the T4 genome sequence that in a UvsW open reading frame and the of a open reading frame that encodes a acid protein now to as UvsW.1. This open reading frame is conserved in and this that the function of UvsW.1 is to that of was shown that exists a and UvsW and UvsW.1 S.W. S.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the crystal structure of UvsW and the NMR solution structure of UvsW.1. the region of structure the RecA-like motor and the structural to UvsW is Rad54. UvsW.1 has a bundle structure that has structural to the in RecQ helicases. and was by Kreuzer The gene was into the the and to a for Escherichia coli were with the and at in of and the was initiated by of and the were for a at were by at for and in of A and two The was at for and in an DNA from the was to the to a of by of and the was at for to The was to on by for and the proteins were by at for and in and The solution was of and through a The was to a and the with of and were an from to and were by were and an The involved a with D was to and at Data and were used to at The was with a of and of of protein and of The were and these of was by the in and and a was from a crystal at the at the Data were in a of and and and the W. 1997; PubMed Scopus Google Scholar). to with a The structure was determined by molecular the J. 1997; Scopus Google Scholar), and the N-terminal fragment as the A solution was with an of and a of and the was in the and and P. K. Biol. 2004; PubMed Scopus Google Scholar) and S.W. PubMed Scopus Google Scholar). of model and P. J. T. Biol. PubMed Scopus Google Scholar) the structure with and of and The structure was then and the J. 1993; Google Scholar). and are shown in and of is the and the all in to the region in the as by is the and the all to the region in the as by J. 1993; Google Scholar). in a gene was by with from T4 DNA by and into the and that a was at the and UvsW.1 was in of the were in with of and of with and of the were a and UvsW.1 was from the a The was from the protein a and the was through a for a time to protein that the The involved a with UvsW.1 was by to and at NMR were a through the analysis of and and a NMR at and by the The were through the analysis of and were through the analysis of and which were an NMR with a F. G. J. J. PubMed Scopus Google Scholar) and were used for NMR and and and structure were the J. Mol. Biol. 1997; PubMed Scopus Google Scholar) as with the structure P. J. T. Biol. PubMed Scopus Google Scholar). A of and were used for the structure to the of and J. J. 2001; PubMed Scopus Google Scholar), for UvsW.1 were used to generate the G. F. J. 1999; PubMed Scopus Google Scholar). structure was with generated The were used for the T. J. 2005; PubMed Scopus Google Scholar). The structural was The was by model to a of The generated by were first for This was by a the was to and then to K. The were then and the J. PubMed Scopus Google Scholar). are shown in of UvsW.1 determined by NMR of of from mean structure residues of region in of by in of by in in in in in a of the UvsW.1 was by and by the of in of The were with a of a of by a a the first G. T. PubMed Scopus Google Scholar). The of the for was as the of in the and perform the UvsW.1 in at was and then in to of that the A inactive point mutant for is more and this mutant was used for the structural studies of the stable N-terminal fragment that to as (13Sickmier E.A. Kreuzer K.N. White S.W. Structure. 2004; 12: 583-592Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). was also used to generate the of the and an was at the from the in that to were from and the structure was determined by molecular the structure as a The of and the model is residues that were in the residues to the region that the two RecA-like motor domains. The structure of reveals that the a N-terminal two RecA-like and Annu. Rev. Biochem. PubMed Scopus Google Scholar) and a from from The of and of and are to in the structure (13Sickmier E.A. Kreuzer K.N. White S.W. Structure. 2004; 12: 583-592Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). A of onto of the and with of and The in two loop and and are in residues an of the and and the structure and the associated structure of the that the structure now the which from that (13Sickmier E.A. Kreuzer K.N. White S.W. Structure. 2004; 12: 583-592Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of is to that of equivalent RecA-like of monomeric as in the structure Structure. Full Text Full Text PDF PubMed Scopus Google Scholar). The structure is a in which the is and to are the The two and that are and and a and This an it as to with sequence of The sequence is shown in the and the structure elements are by the The SF2 helicase motifs are shown in and are that the structure elements are and to and to consistent with structural studies of monomeric a of the crystal structure in which the of key residues within the SF2 helicase motifs as by and Matson M.C. Matson S.W. Mol. Microbiol. 1999; 34: 867-877Crossref PubMed Scopus (270) Google Scholar) are can that the motifs within the two are well and an of and is to the motifs to the architecture that is of the monomeric Annu. Rev. Biochem. PubMed Scopus Google Scholar). We that the crystal structure a of the the structure this has been to the and it can that the residues now in the of the the shown in the basis of Functional seven SF2 helicase motifs are in the in monomeric helicase Annu. Rev. Biochem. PubMed Scopus Google Scholar), and within the UvsW structure are in that the of from that on sequence analysis (10Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (66) Google Scholar). and at the and are involved in binding and and motifs and have roles in binding the nucleic acid substrate M.C. Matson S.W. Mol. Microbiol. 1999; 34: 867-877Crossref PubMed Scopus (270) Google Scholar, Annu. Rev. Biochem. PubMed Scopus Google Scholar). J. P. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), an has been identified that to interact with the of and this is in UvsW as within We used the structure of the helicase to to generate a model of a complex (13Sickmier E.A. Kreuzer K.N. White S.W. Structure. 2004; 12: 583-592Abstract Full Text Full Text PDF PubMed Scopus Google Scholar), and this model can now to the UvsW structure and in and the and are to the DNA from and are within the sequence in UvsW that to the in EMBO J. PubMed Scopus Google Scholar). and within and and within can all interact with DNA from from Sequence is now for a of and all UvsW that a of The motifs and residues are all The model shown in well with the of the and SF2 that DNA substrates and is consistent with the mechanism that has been suggested for the mechanism on structural studies of the helicase P. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). a more and mechanism has been on structural studies of the helicase W. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). also reveals that may have a role in the with and in the which has been in and in are both that with due to the point at the and respectively, of the these are equivalent in of the of the RecA-like and to perform functions in binding the The and mechanism an of and that a binding and of the and in the of the with to the DNA may have an role in this The sequence also reveals two of UvsW that are conserved and for to loop The first is a loop that has been and is an of (13Sickmier E.A. Kreuzer K.N. White S.W. Structure. 2004; 12: 583-592Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). The is of and and This loop is well resolved in the and a short and that is by the of and a and two with associated and a the motor that the UvsW DNA substrates UvsW can it is the E. coli helicase and the that RecG to and its branched DNA substrates. the UvsW can perform equivalent with a structure that the of DNA of the W. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) and has a role to the that has been identified in RecG Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). UvsW has of these that the at the of in which may perform an equivalent UvsW and the J. Mol. Biol. 1993; PubMed Scopus Google Scholar), it was that the UvsW and the equivalent of Rad54. of have been in the open Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) that to crystal structure of UvsW and a in a Nat. Struct. Mol. Biol. 2005; 12: PubMed Scopus Google Scholar) that the model shown in the two the of UvsW and are and and of UvsW to the and respectively, of Nat. Struct. Mol. Biol. 2005; 12: PubMed Scopus Google Scholar). an in an equivalent both and with to the to in UvsW that may have a role in DNA also an and that a short and the region of UvsW that is in may an equivalent in the and UvsW.1 and an residues at the of the and The structure and structure are shown in The of the solution of UvsW.1 is shown in and a of the structure is shown in UvsW.1 is of and and into a bundle with well and is from this and to the and two of that it is are and are in the for residues in of are to in the This that on the time in to the that to on this time The seven residues at the are on and 6The structure of UvsW.1. NMR solution structure the that the bundle that the of the structure is well the and that are to two were a conserved and The was J. 24: Google Scholar) and with E.A. D.J. 1997; PubMed Scopus Google Scholar). to the PubMed Scopus Google Scholar). The of and of to is the and is the The of UvsW.1 are to with of the of of UvsW.1 of a protein in within A of residues at this time and these by UvsW.1 is on the and well the on a time which the that Thus, the of the bundle of UvsW.1 may as well as has been for proteins with bundle Functional is conserved in and the are and that are consistent with the solution structure and which to the and and at the has been shown that UvsW.1 a complex with UvsW S.W. S.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), and are by conserved is on the of the bundle and is on residues and have been to generate a stable complex for structural analyses and this the the two of the protein is an at the and this is from We have confirmed that UvsW is an ATP-dependent monomeric helicase that belongs to the SF2 and biochemical have suggested that UvsW can on a variety of substrates and a of functions during the T4 infection UvsW was first identified as a T4 that genome and mutations within the UvsW gene the virus particularly to DNA damage due to UV radiation and with (8Derr L.K. Drake J.W. Mol. Gen. Genet. 1990; 222: 257-264Crossref PubMed Scopus (15) Google Scholar, K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (66) Google Scholar). suggested that UvsW has roles in DNA double-strand repair and the rescue of collapsed replication is now understood that recombination a central role in and this is consistent with the of the and within the T4 uvsWXY system (5Klein H.L. Kreuzer K.N. Mol. Cell. 2002; 9: 471-480Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The role of UvsW in replication forks has been were from the of the helicase to rescue E. coli (10Carles-Kinch K. George J.W. Kreuzer K.N. EMBO J. 1997; 16: 4142-4151Crossref PubMed Scopus (66) Google Scholar). RecG is known to rescue stalled forks by a process of are to rescue a recombination-dependent mechanism via a Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, P. Lloyd R.G. Sci. 2001; PubMed Scopus Google Scholar). 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Full Text Full Text PDF PubMed Scopus Google Scholar), and the structure of the complex has the structural to into these enzymes specifically on substrates Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). from the UvsW has structural homology to and the T4 to specifically for this it to a SF2 helicase that can to a variety of processes that are during the T4 infection for and to catalyze is consistent with the key roles that play in T4 T4 has been as an ideal model system for studying fundamental in also in are sequence homologies equivalent proteins J. PubMed Google Scholar). it as that the known structural to UvsW is the eukaryotic SF2 helicase Rad54. This is because the two proteins also a of has emerged as a fundamental in eukaryotic homologous recombination J. 2003; PubMed Scopus Google Scholar) and was as a eukaryotic in a of the 34: PubMed Scopus Google Scholar). In addition, was first identified as a gene in which mutations have a to as and J. PubMed Google Scholar). This is of the uvsWXY system in T4 and may the of and in eukaryotic and T4 respectively, via homologous Thus, studies of the T4 proteins and in on a key repair system in all eukaryotic structural analysis of UvsW.1 may into of SF2 helicases, the RecQ This the and helicases, and and are known to have roles in genome and recombination J. J. D.J. J. Cell. Full Text PDF PubMed Scopus Google Scholar, K. Nat. Genet. 2001; PubMed Scopus Google Scholar). The is in a of RecQ and sequence The known of from E. coli RecQ Structure. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), G. P. Structure. 1999; 7: Full Text Full Text PDF PubMed Scopus Google Scholar), and K. T. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), all a bundle at that of UvsW.1. The role of the is is that it substrate elements to that of the SF2 In a study S.W. S.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), it was shown that UvsW and UvsW.1 a and UvsW.1 to function as a of the In addition, DNA has been in members of the RecQ and it was also that UvsW has DNA that are by UvsW.1 S.W. S.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We have identified a conserved on the of and into its a is and NMR solution studies both that UvsW.1 is dynamic and and this that a binding DNA is necessary to a more In of the homology to the it may that the gene is at the 3′-end of the uvsW suggested by the the is at the of RecQ and UvsW.1 is by a gene in it may have been to an the biochemical as the complex S.W. S.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We Kreuzer and at for the UvsW and We also for