- Email: [email protected]
Homologous Recombination: A ring for a warhead
DAvID MJ LILLEY
HOMOLOGOUS RECOMBINATION
A ring for a warhead The active 'warhead' RuvB of the Escherichia coli protein that catalyzes the branch-migration step of homologous recombination isa helicase that binds DNA as a double-ring oligomer. From being almost the exclusive province of the geneticist, recombination is now getting a solid biochemical and mechanistic foundation. This is particularly true in Escherichia coli, where specific proteins that act at each stage are now becoming identified. Recombination brings about a reconnection of homologous DNA molecules, and involves branched species as important intermediates (Fig. 1). These must first be formed, requiring the action of nucleases such as RecBCD, and the strand-exchange protein RecA (reviewed in [1]). The result of the action of these proteins is the formation of a four-way (or Holliday) junction [2]. The four-way junction is then manipulated and ultimately resolved by other proteins: it undergoes an exchange of base-pairing by the homologous strands in a branch migration process, followed by a resolution event that regenerates two duplex DNA molecules. Both events require the intervention of proteins that are specific for DNA containing the fourway junction. The four-way junction adopts a precise three-dimensional structure in solution (reviewed in [3]), and the proteins mediating the latter stages of homologous recombination - the events that follow formation of the junction - exhibit a high structural selectivity for the junction. The major enzymes of E. coli that manipulate the fourway junction are products of the ruv locus [4]. This locus is required for recombinational DNA repair, and ruv mutants are sensitive to DNA damage. The ruv locus contains two operons. The ruvAB operon encodes RuvA and RuvB, which together catalyze branch migration, while RuvC is a resolving enzyme encoded by the other operon. In addition to these proteins, branch migration may also be catalyzed by the RecG protein [5], and resolution by the newly discovered Rus enzyme [6]. Thus, the latter stages of homologous recombination appear to be degenerate in E. coli.
Junction-specific proteins are somewhat akin to structureseeking guided missiles; they require a delivery vehicle that can locate the target - the four-way junction and a warhead that carries out the requisite reaction upon arrival. In the case of junction-resolving enzymes, they can be regarded as the functional fusion of nuclease and binding activities, and the two can be separated genetically. It is perhaps less obvious what activity would be required by the warhead of an enzyme that promotes branch migration. However, recent experiments make it clear that the spontaneous process is slow [7] and this is consistent with the folded structure of the four-way junction. To facilitate the exchange of base-pairing, it seems that the structure must be severely disrupted and thus an enzyme that dissociates the two DNA strands may be required. Such activity is characteristic of the helicases. The RuvAB complex accelerates the rate of branch migration in the presence of ATP and Mg2 + ions. It promotes the dissociation of circular DNA containing a four-way junction ('(i structures'; it does this much more efficiently than RecA) [8], the reabsorption of cruciform structures in supercoiled DNA [9] and the dissociation of synthetic four-way DNA junctions into frayed duplex structures [10]. The complex appears to be directed selectively to the structure of the four-way junction by RuvA, which forms a complex with synthetic junctions [10,11] that exhibits retarded mobility in polyacrylamide gel electrophoresis. No shift was observed using RuvB alone [11]. Thus RuvA is the delivery vehicle of this complex. Upon delivery to the junction by RuvA, the main player facilitating branch migration is RuvB, which has all the characteristics of a helicase: this is the warhead. The strand-dissociation reactions promoted by the RuvAB complex [8] can be achieved by RuvB alone at a high concentration [12]. The sequence of RuvB contains motifs associated with E. coli helicase proteins,
Fig. 1. Genetic recombination in E. coli. A simple model for the mechanism of genetic recombination, emphasizing the latter stages in which the four-way junction is manipulated by structure-selective enzymes.
1152
© Current Biology 1994, Vol 4 No 12
DISPATCH
such as GxGKT (ATP-binding motif I) and DExH (using the single-letter amino-acid code) [13]. RuvB is also a DNA-dependent ATPase, requiring ATP or dATP but not ATP-yS or ADP. Hydrolysis of ATP is required by helicase proteins as a source of free energy to drive the vectorial process, thereby causing a very large rate enhancement relative to a random walk. ATP is not required for the binding of RuvA to junctions [1 1]. The RuvAB complex provides a clear example of the divisibility of targeting and catalytic functions. In all known resolving enzymes, binding and catalytic functions are part of the same protein molecule, and this is also true of the alternative E. coli branch-migration enzyme RecG. Stoichiometric measurements suggested that RuvB would probably function as a multimeric protein [13], and this has been confirmed by recent electron microscopic structural studies. Egelman and co-workers [14] obtained uranyl acetate-stained images of RuvB bound to relaxed, circular DNA (Fig. 2), in which the protein has the appearance of a double ring. The rings are aligned, suggesting that the DNA passes through the centre, and the contour length appears to exclude wrapping of the DNA. Mass measurements by scanning transmission electron microscopy indicated that each ring contains six RuvB subunits; 800 images of the doublering structure were aligned and averaged, revealing a structure with bipolar symmetry. Each of the rings has six-fold rotational symmetry (symmetry group C6), and the two rings are related by two-fold axes perpendicular to the six-fold ring axis (thus generating D6 symmetry for the dodecamer). Using two projections of the structure, a three-dimensional reconstruction of the double-ring structure was achieved at 30 A resolution (Fig. 3). The complex contains a central hole of 20-25 A in diameter, through which the DNA is assumed to pass.
Fig. 2. Electron micrograph showing double rings of RuvB on a DNA duplex. The inset shows selected top views of the rings. The bar indicates 400 A. (Reproduced from [14] with permission.) The structure of RuvB is likely to have much in common with other helicases. The E. coli Rho protein, which plays a role in the termination of transcription, acts as an ATPdependent DNA-RNA helicase; it also appears to form rings composed of six monomers (arranged in this case as
Fig. 3. Reconstruction of the three-dimensional structure of the RuvB double-ring. Note the apparently hollow central channel, which is assumed to be occupied by unstained DNA. (Reproduced from [141 with permission.)
1153
1154
Current Biology 1994, Vol 4 No 12 a trimer of dimers, with three-fold symmetry) [15]. The large-T antigen of SV40 - a multifunctional protein possessing strong ATP-dependent helicase activity that is important in viral replication - is reported to be constructed as a two-ringed structure with each ring comprising six subunits [16]. Thus six-fold symmetry appears to be a recurrent theme in the structures of helicases in general. It is clear that helicases play vital roles in many fundamental processes, including DNA replication, transcription, RNA splicing and translation. To these we must now add genetic recombination. Acknowledgements: I gratefully thank Ed Egelman, Steve West and Bob Lloyd for discussion, and Ed Egelman for provision of Figures 2 and 3.
References 1. Kowalczykowski SC, Eggleston AK: Homologous pairing and DNA strand-exchange proteins. Annu Rev Biochem 1994, 63:991-1043. 2. Holliday R: A mechanism for gene conversion in fungi. Genet Res 1964, 5:282-304. 3. Lilley DMJ, Clegg RM: The structure of the four-way junction in DNA. Annu Rev Biophys Biomol Struct 1993, 22:299-328. 4. Sharples GJ, Benson FE, Iling CT, Lloyd RG: Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination. Mol Gen Genet 1990, 221:219-226. 5. Lloyd RG, Sharples GJ: Dissociation of synthetic Holliday junctions by E coli RecG protein. EMBO J 1993, 12:17-22. 6. Sharples GJ, Chan SN, Mahdi AA, Whitby MC, Lloyd RG: Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions. EMBO J 1994, 24:in press.
7. Panyutin IG, Hsieh P: The kinetics of spontaneous DNA branch migration. Proc Natl Acad Sci USA 1994, 91:2021-2025. 8. Tsaneva IR, Muller B, West SC: ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coil. Cell 1992, 69:1171-1180. 9. Shiba T, Iwasaki H, Nakata A, Shinagawa H: SOS-inducible DNA repair proteins RuvA and RuvB, of Escherichia coil: Functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA. Proc Nat/ Acad Sci USA 1991, 88:8445-8449. 10. Parsons CA, Tsaneva I, Lloyd RG, West SC: Interaction of Escherichia coil RuvA and RuvB proteins with synthetic Holliday junctions. Proc Natl Acad Sci USA 1992, 89:5452-5456. 11. Parsons CA, West SC: Formation of a RuvAB-Holliday junction complex in vitro. J Mol Biol 1993, 232:397-405. 12. Muller, B, Tsaneva, IR, West SC: Branch migration of Holliday junctions promoted by the Escherichia coil RuvA and RuvB proteins.1. Comparison of RuvAB-mediated and RuvB-mediated reactions. J Biol Chem 1993, 268:17179-17184. 13. Tsaneva IR, Muller B, West SC: RuvA and RuvB proteins of Escherichia coil exhibit DNA helicase activity in vitro. Proc Natl Acad Sci USA 1993, 90:1315-1319. 14. Stasiak A, Tsaneva IR, West SC, Benson CJB, Yu X, Egelman EH: The Escherichia coil RuvB branch migration protein forms double hexameric rings around DNA. Proc Natl Acad Sci USA 1994, 91:7618-7622. 15. Gogol EP, Seifried SE, von Hippel PH: Structure and assembly of the Escherichia coil transcription factor Rho and its interaction with RNA. I. Cryoelectron microscopic studies. J Mo/ Biol 1991, 221:1127-1138. 16. Mastrangelo IA, Hough PVC, Wall S, Dodson M, Dean FB, Hurwitz J: ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication. Nature 1989, 338:658-662.
David M.J. Lilley, CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University, Dundee DD1 4HN, UK.
HOMOLOGOUS RECOMBINATION
A ring for a warhead The active 'warhead' RuvB of the Escherichia coli protein that catalyzes the branch-migration step of homologous recombination isa helicase that binds DNA as a double-ring oligomer. From being almost the exclusive province of the geneticist, recombination is now getting a solid biochemical and mechanistic foundation. This is particularly true in Escherichia coli, where specific proteins that act at each stage are now becoming identified. Recombination brings about a reconnection of homologous DNA molecules, and involves branched species as important intermediates (Fig. 1). These must first be formed, requiring the action of nucleases such as RecBCD, and the strand-exchange protein RecA (reviewed in [1]). The result of the action of these proteins is the formation of a four-way (or Holliday) junction [2]. The four-way junction is then manipulated and ultimately resolved by other proteins: it undergoes an exchange of base-pairing by the homologous strands in a branch migration process, followed by a resolution event that regenerates two duplex DNA molecules. Both events require the intervention of proteins that are specific for DNA containing the fourway junction. The four-way junction adopts a precise three-dimensional structure in solution (reviewed in [3]), and the proteins mediating the latter stages of homologous recombination - the events that follow formation of the junction - exhibit a high structural selectivity for the junction. The major enzymes of E. coli that manipulate the fourway junction are products of the ruv locus [4]. This locus is required for recombinational DNA repair, and ruv mutants are sensitive to DNA damage. The ruv locus contains two operons. The ruvAB operon encodes RuvA and RuvB, which together catalyze branch migration, while RuvC is a resolving enzyme encoded by the other operon. In addition to these proteins, branch migration may also be catalyzed by the RecG protein [5], and resolution by the newly discovered Rus enzyme [6]. Thus, the latter stages of homologous recombination appear to be degenerate in E. coli.
Junction-specific proteins are somewhat akin to structureseeking guided missiles; they require a delivery vehicle that can locate the target - the four-way junction and a warhead that carries out the requisite reaction upon arrival. In the case of junction-resolving enzymes, they can be regarded as the functional fusion of nuclease and binding activities, and the two can be separated genetically. It is perhaps less obvious what activity would be required by the warhead of an enzyme that promotes branch migration. However, recent experiments make it clear that the spontaneous process is slow [7] and this is consistent with the folded structure of the four-way junction. To facilitate the exchange of base-pairing, it seems that the structure must be severely disrupted and thus an enzyme that dissociates the two DNA strands may be required. Such activity is characteristic of the helicases. The RuvAB complex accelerates the rate of branch migration in the presence of ATP and Mg2 + ions. It promotes the dissociation of circular DNA containing a four-way junction ('(i structures'; it does this much more efficiently than RecA) [8], the reabsorption of cruciform structures in supercoiled DNA [9] and the dissociation of synthetic four-way DNA junctions into frayed duplex structures [10]. The complex appears to be directed selectively to the structure of the four-way junction by RuvA, which forms a complex with synthetic junctions [10,11] that exhibits retarded mobility in polyacrylamide gel electrophoresis. No shift was observed using RuvB alone [11]. Thus RuvA is the delivery vehicle of this complex. Upon delivery to the junction by RuvA, the main player facilitating branch migration is RuvB, which has all the characteristics of a helicase: this is the warhead. The strand-dissociation reactions promoted by the RuvAB complex [8] can be achieved by RuvB alone at a high concentration [12]. The sequence of RuvB contains motifs associated with E. coli helicase proteins,
Fig. 1. Genetic recombination in E. coli. A simple model for the mechanism of genetic recombination, emphasizing the latter stages in which the four-way junction is manipulated by structure-selective enzymes.
1152
© Current Biology 1994, Vol 4 No 12
DISPATCH
such as GxGKT (ATP-binding motif I) and DExH (using the single-letter amino-acid code) [13]. RuvB is also a DNA-dependent ATPase, requiring ATP or dATP but not ATP-yS or ADP. Hydrolysis of ATP is required by helicase proteins as a source of free energy to drive the vectorial process, thereby causing a very large rate enhancement relative to a random walk. ATP is not required for the binding of RuvA to junctions [1 1]. The RuvAB complex provides a clear example of the divisibility of targeting and catalytic functions. In all known resolving enzymes, binding and catalytic functions are part of the same protein molecule, and this is also true of the alternative E. coli branch-migration enzyme RecG. Stoichiometric measurements suggested that RuvB would probably function as a multimeric protein [13], and this has been confirmed by recent electron microscopic structural studies. Egelman and co-workers [14] obtained uranyl acetate-stained images of RuvB bound to relaxed, circular DNA (Fig. 2), in which the protein has the appearance of a double ring. The rings are aligned, suggesting that the DNA passes through the centre, and the contour length appears to exclude wrapping of the DNA. Mass measurements by scanning transmission electron microscopy indicated that each ring contains six RuvB subunits; 800 images of the doublering structure were aligned and averaged, revealing a structure with bipolar symmetry. Each of the rings has six-fold rotational symmetry (symmetry group C6), and the two rings are related by two-fold axes perpendicular to the six-fold ring axis (thus generating D6 symmetry for the dodecamer). Using two projections of the structure, a three-dimensional reconstruction of the double-ring structure was achieved at 30 A resolution (Fig. 3). The complex contains a central hole of 20-25 A in diameter, through which the DNA is assumed to pass.
Fig. 2. Electron micrograph showing double rings of RuvB on a DNA duplex. The inset shows selected top views of the rings. The bar indicates 400 A. (Reproduced from [14] with permission.) The structure of RuvB is likely to have much in common with other helicases. The E. coli Rho protein, which plays a role in the termination of transcription, acts as an ATPdependent DNA-RNA helicase; it also appears to form rings composed of six monomers (arranged in this case as
Fig. 3. Reconstruction of the three-dimensional structure of the RuvB double-ring. Note the apparently hollow central channel, which is assumed to be occupied by unstained DNA. (Reproduced from [141 with permission.)
1153
1154
Current Biology 1994, Vol 4 No 12 a trimer of dimers, with three-fold symmetry) [15]. The large-T antigen of SV40 - a multifunctional protein possessing strong ATP-dependent helicase activity that is important in viral replication - is reported to be constructed as a two-ringed structure with each ring comprising six subunits [16]. Thus six-fold symmetry appears to be a recurrent theme in the structures of helicases in general. It is clear that helicases play vital roles in many fundamental processes, including DNA replication, transcription, RNA splicing and translation. To these we must now add genetic recombination. Acknowledgements: I gratefully thank Ed Egelman, Steve West and Bob Lloyd for discussion, and Ed Egelman for provision of Figures 2 and 3.
References 1. Kowalczykowski SC, Eggleston AK: Homologous pairing and DNA strand-exchange proteins. Annu Rev Biochem 1994, 63:991-1043. 2. Holliday R: A mechanism for gene conversion in fungi. Genet Res 1964, 5:282-304. 3. Lilley DMJ, Clegg RM: The structure of the four-way junction in DNA. Annu Rev Biophys Biomol Struct 1993, 22:299-328. 4. Sharples GJ, Benson FE, Iling CT, Lloyd RG: Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination. Mol Gen Genet 1990, 221:219-226. 5. Lloyd RG, Sharples GJ: Dissociation of synthetic Holliday junctions by E coli RecG protein. EMBO J 1993, 12:17-22. 6. Sharples GJ, Chan SN, Mahdi AA, Whitby MC, Lloyd RG: Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions. EMBO J 1994, 24:in press.
7. Panyutin IG, Hsieh P: The kinetics of spontaneous DNA branch migration. Proc Natl Acad Sci USA 1994, 91:2021-2025. 8. Tsaneva IR, Muller B, West SC: ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coil. Cell 1992, 69:1171-1180. 9. Shiba T, Iwasaki H, Nakata A, Shinagawa H: SOS-inducible DNA repair proteins RuvA and RuvB, of Escherichia coil: Functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA. Proc Nat/ Acad Sci USA 1991, 88:8445-8449. 10. Parsons CA, Tsaneva I, Lloyd RG, West SC: Interaction of Escherichia coil RuvA and RuvB proteins with synthetic Holliday junctions. Proc Natl Acad Sci USA 1992, 89:5452-5456. 11. Parsons CA, West SC: Formation of a RuvAB-Holliday junction complex in vitro. J Mol Biol 1993, 232:397-405. 12. Muller, B, Tsaneva, IR, West SC: Branch migration of Holliday junctions promoted by the Escherichia coil RuvA and RuvB proteins.1. Comparison of RuvAB-mediated and RuvB-mediated reactions. J Biol Chem 1993, 268:17179-17184. 13. Tsaneva IR, Muller B, West SC: RuvA and RuvB proteins of Escherichia coil exhibit DNA helicase activity in vitro. Proc Natl Acad Sci USA 1993, 90:1315-1319. 14. Stasiak A, Tsaneva IR, West SC, Benson CJB, Yu X, Egelman EH: The Escherichia coil RuvB branch migration protein forms double hexameric rings around DNA. Proc Natl Acad Sci USA 1994, 91:7618-7622. 15. Gogol EP, Seifried SE, von Hippel PH: Structure and assembly of the Escherichia coil transcription factor Rho and its interaction with RNA. I. Cryoelectron microscopic studies. J Mo/ Biol 1991, 221:1127-1138. 16. Mastrangelo IA, Hough PVC, Wall S, Dodson M, Dean FB, Hurwitz J: ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication. Nature 1989, 338:658-662.
David M.J. Lilley, CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University, Dundee DD1 4HN, UK.