- Email: [email protected]
A ubiquitous structural core
COMMENT
TIBS 25 – APRIL 2000
A ubiquitous structural core Over the last few years, the existence of a huge superfamily of structurally homologous proteins that are active in DNA recombination, replication, repair and transcription has been revealed. The underlying structural homology, with divergence of both sequence and function, suggests that all of these proteins (both eukaryotic and prokaryotic) are derived from some common prokaryotic ancestor. The extent of the functional divergence, however, suggests that the common ancestry might be more important in understanding the evolution of these proteins rather than in deducing their current functions. The bacterial RecA protein had been intensively studied for many years as the key catalyst in prokaryotic homologous recombination1–4. Remarkably, this single protein was capable of promoting strand exchange reactions in vitro involving: (i) the formation of joint molecules between two DNA substrates sharing common sequences, and (ii) the exchange of strands between these molecules. Within the bacterial cell, RecA plays a pivotal role in both recombination and repair, establishing that recombination occurs in many circumstances as a tool for the repair of DNA and the maintenance of genome stability. The possibility, long surmised, that RecA-like pathways exist in eukaryotic cells was confirmed with the identification of the yeast Rad51 protein as a RecA homolog5,6. The only functional form of the RecA protein had been assumed to be an unusual helical filament, within which DNA is stretched and untwisted so that its normal pitch of ~36 Å is changed to ~95 Å in the complex with the RecA protein7,8. It was shown that the yeast Rad51 protein formed a nearly identical nucleoprotein filament, imposing these same parameters on the DNA (Ref. 9). We also know that the human RAD51 protein induces a very similar structure10. Perhaps the fundamental event in the delineation of the RecA superfamily was the observation that the core of the first helicase, PcrA, to be solved at atomic resolution11 contained two copies of the same nucleotide-binding core first seen in the RecA protein12. This core domain contains a central β sheet with surrounding α helices, with a distinctive topology that had not been observed in any other proteins at the time that the RecA structure
was solved. The core also contains the binding site for ATP or ADP, as well as the catalytic elements used in the hydrolysis of ATP to ADP. Helicases are active not only in DNA replication, where they create a replication fork by opening up double-stranded DNA into two single-stranded templates for DNA polymerases, using energy derived from ATP hydrolysis, but also in many aspects of DNA recombination, repair and transcription13–15. It is puzzling why Escherichia coli has as many as 12 helicases16 and Saccharomyces cerevisiae even more than 134 (Ref. 17). This identification is based purely upon seven conserved sequence motifs that include the Walker A and B phosphate binding loops. This leaves open the possibility that many helicases, based upon such sequence analysis, might actually function in other ways than melting double-stranded DNA. One E. coli helicase, RuvB, is involved in the branch migration that occurs after strand exchange is initiated by the RecA protein18. Electron microscopy studies determined that RuvB exists as a hexameric ring, encircling double-stranded DNA (Ref. 19). This has led to a model for how RuvB functions as a DNA ‘pump’, using the energy of ATP hydrolysis to translocate DNA within the central channel of the ring20,21. In this system, the RuvB ring does not appear to separate doublestranded DNA into two single strands. Rather, the energy derived from ATP hydrolysis is used for translocation of the double-stranded DNA. A similar form of mechanochemical coupling was invoked to explain how the gp4 protein of bacteriophage T7, a replicative helicase, forms a hexameric ring around singlestranded DNA and uses the energy of ATP hydrolysis to translate along the single strand22,23. Because the complementary strand is displaced outside of the ring, this translocation results in strand separation. The comparison between RuvB and gp4 is interesting, because it suggests that the same conserved motor, with only minor differences in detail, can have two entirely different functions. This could provide some insight into the potential diversity of function of many other helicases that might use the energy derived from ATP hydrolysis for very different purposes.
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
The first atomic-level understanding of this helicase motor came with the determination of the structure of PcrA, a helicase from a thermophilic bacterium11. The structure revealed that all of the conserved helicase sequence motifs24,25 resided in the RecA-like domains, suggesting that all helicases would contain this same conserved core. Subsequent structures determined for other helicases have been in agreement with this prediction26–28. The only other protein found to have such a core was the F1-ATPase29, making it highly likely that both F1 and RecA evolved from some common ancestral protein. Our expectation is that individual point mutations can accumulate over time, giving rise to enormous sequence diversity, at the same time that the topology or pattern of connectivity of the b sheets and a helices remains fixed. It is this distinctive topology that defines structural homology and common evolutionary origin. Given that the 134 helicases found in S. cerevisiae (based upon sequence analysis) are all RecA homologs, this means that ~2% of the yeast genes encode proteins in this family. Structural studies suggest that this number could be even larger, as the d9 subunit of the E. coli clamp loader complex is also a RecA homolog30. The clamp loader complex is involved in positioning a clamp, or processivity factor, around the DNA that keeps the polymerase topologically tethered to its template during replication. However, the sequence of the d9 subunit has diverged so far from RecA that it no longer contains a recognizable ATP-binding motif, and does not bind to ATP. This suggests that other proteins involved in DNA replication, recombination, repair and transcription are also part of this superfamily, but no longer have identifiable sequence homology. The connection between RecA, Rad51 and helicases has been strengthened by the observation that RecA assembles into hexameric rings31, which are similar to the rings formed by helicases such as T7 gp4 (Refs 22,32), SV40 large T (Ref. 33), E. coli DnaB (Refs 34,35), E. coli RuvB (Ref. 19), phage SPP1 g40p (Ref. 36) and papilloma virus E1 (Ref. 37). Strikingly, the RecA ring can exist in two states, a symmetrical hexamer in which every subunit is equivalent, and a trimer of dimers. Within the trimer of dimers, there are two different subunit conformations, and the dimers are arranged so that the ring has a three-fold symmetry, rather than the six-fold symmetry of the symmetrical hexamer. A very similar polymorphism has been shown for DnaB (Ref. 35), SPP1
PII: S0968-0004(00)01570-X
183
COMMENT g40p (Ref. 36) and papilloma E1 (Ref. 37). The RecA ring structure appears to be conserved – both yeast (X. Yu, T. Ogawa and E. Egelman, unpublished) and human (X. Yu, S. West and E. Egelman, unpublished) Rad51 proteins also forms rings, but they are octameric, rather than hexameric. The conservation of these rings from bacteria to humans suggests that they are functional. The possibility that they are intermediates in the assembly of filaments can be excluded, as the subunit– subunit contacts within the rings are very different from those in the filaments31,38. Unfortunately, no mutants of RecA or Rad51 have yet been isolated that only form the ring or only form the filament, which could provide a clue to the function of the ring structure. It thus appears that RecA and Rad51 might exist in two functional forms, helical filaments and rings. The expectation was that Dmc1, a meiotic homolog of RecA (Ref. 39), would also exist as a helical filament. However, the only form of Dmc1 seen in vitro, under conditions where DNA-binding, DNA-activated ATPase and strand exchange have been assayed40,41, is an octameric ring42. Although we cannot exclude the possibility that within the cell other cofactors are present which allow Dmc1 to form helical filaments, it does suggest that in vivo RecA and Rad51 could function as both filaments and rings, whereas Dmc1 only functions as a ring.
In summary Structural studies, employing X-ray crystallography to determine the atomic structure of protein subunits, and electron microscopy to illuminate the higherorder structures of filaments and rings, have been instrumental in characterizing a polymorphic superfamily of proteins that all contain the same nucleotidebinding core. At least 2% of the genes in yeast encode for members of this superfamily, and in humans the percentage might be even higher. The RecA protein, whose structure was the first to be solved in this superfamily, is integrally involved in recombination, repair and replicative bypass of lesions43–45 in bacteria. It has been studied for many years as a model for how homologous recombination is catalysed by proteins. But RecA is not an essential gene in bacteria, except under conditions where DNA is subject to massive amounts of damage. We now know that the eukaryotic homolog of RecA, Rad51, might play an even more important role in humans, as it is probably an essential gene in higher eukaryotes46,47.
184
TIBS 25 – APRIL 2000 Similarly, helicases, whose mutations in humans can lead to premature aging, as in Werner’s Syndrome48, or predisposition to cancer, as in Bloom’s Syndrome49, are now known to be homologs of the RecA protein. We are at a very early stage in understanding what mechanisms all of these proteins employ in common, but the functional divergence could be very large. What is clear is that in the course of evolution, cells have created a vast repertoire of structures acting in replication, recombination, transcription and repair, functioning as monomers, dimers, hexamers, octamers and long helical filaments, that are all built around the same highly conserved core.
References 1 West, S.C. (1995) Formation, translocation and resolution of Holliday junctions during homologous genetic recombination. Phil. Trans. Roy. Soc. B 347, 21–25 2 Kowalczykowski, S.C. et al. (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol. Rev. 58, 401–465 3 Roca, A.I. and Cox, M.M. (1997) RecA protein: structure, function, and role in recombinational DNA repair. Prog. Nucleic Acids Res. Mol. Biol. 56, 129–223 4 Radding, C.M. (1991) Helical interactions in homologous pairing and strand exchange driven by RecA protein. J. Biol. Chem. 266, 5355–5358 5 Shinohara, A. et al. (1992) Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69, 457–470 6 Aboussekhra, A. et al. (1992) Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol. Cell Biol. 12, 3224–3234 7 Stasiak, A. and DiCapua, E. (1982) The helicity of DNA in complexes with RecA protein. Nature 229, 185–186 8 Stasiak, A. et al. (1981) Elongation of duplex DNA by RecA protein. J. Mol. Biol. 151, 557–564 9 Ogawa, T. et al. (1993) Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 259, 1896–1899 10 Benson, F.E. et al. (1994) Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 13, 5764–5771 11 Subramanya, H.S. et al. (1996) Crystal structure of a DExx box DNA helicase. Nature 384, 379–383 12 Story, R.M. et al. (1992) The structure of the E. coli recA protein monomer and polymer. Nature 355, 318–325 13 Lohman, T.M. (1993) Helicase-catalysed DNA unwinding. J. Biol. Chem. 268, 2269–2272 14 Matson, S.W. et al. (1994) DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. BioEssays 16, 13–22 15 Mendonca, V.M. et al. (1995) DNA helicases in recombination and repair: construction of a DuvrD DhelD DrecQ mutant deficient in recombination and repair. J. Bacteriol. 177, 1326–1335 16 Matson, S.W. (1991) DNA helicases of Escherichia coli. Prog. Nucleic Acids Res. Mol. Biol. 40, 289–326 17 Shiratori, A. et al. (1999) Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis. Yeast 15, 219–253 18 West, S.C. (1996) The RuvABC proteins and Holliday junction processing in Escherichia coli. J. Bacteriol. 178, 1237–1241 19 Stasiak, A. et al. (1994) The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA. Proc. Natl. Acad. Sci. U. S. A. 91, 7618–7622 20 Yu, X. et al. (1997) Structure and subunit composition of the RuvAB–Holliday junction complex. J. Mol. Biol. 266, 217–222 21 Parsons, C.A. et al. (1995) Structure of a multisubunit complex that promotes DNA branch migration. Nature 374, 375–378 22 Yu, X. et al. (1996) DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase. Nat. Struct. Biol. 3, 740–743
23 Hacker, K.J. and Johnson, K.A. (1997) A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding. Biochemistry 36, 14080–14087 24 Gorbalenya, A.E. and Koonin, E.V. (1993) Helicases: amino acid sequence comparisons and structure–function relationships. Curr. Opin. Struct. Biol. 3, 419–429 25 Gorbalenya, A.E. et al. (1989) Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 17, 4713–4730 26 Korolev, S. et al. (1998) Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases. Protein Sci. 7, 605–610 27 Korolev, S. et al. (1997) Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell 90, 635–647 28 Sawaya, M.R. et al. (1999) Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7. Cell 99, 167–177 29 Abrahams, J.P. et al. (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–628 30 Guenther, B. et al. (1997) Crystal structure of the d9 subunit of the clamp-loader complex of E. coli DNA polymerase III. Cell 91, 335–345 31 Yu, X. and Egelman, E.H. (1997) The RecA hexamer is a structural homologue of ring helicases. Nat. Struct. Biol. 4, 101–104 32 Egelman, E.H. et al. (1995) Bacteriophage T7 helicase/ primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases. Proc. Natl. Acad. Sci. U. S. A. 92, 3869–3873 33 San Martin, M.C. et al. (1997) Six molecules of SV40 large T antigen assemble in a propeller-shaped particle around a channel. J. Mol. Biol. 268, 15–20 34 San Martin, M.C. et al. (1995) A structural model for the Escherichia coli DnaB helicase based on electron microscopy data. J. Struct. Biol. 114, 167–176 35 Yu, X. et al. (1996) The hexameric E. coli DnaB helicase can exist in different quaternary hexameric states. J. Mol. Biol. 259, 7–14 36 Barcena, M. et al. (1998) Polymorphic quaternary organization of the Bacillus subtilis bacteriophage SPP1 replicative helicase (G40 P). J. Mol. Biol. 283, 809–819 37 Fouts, E.T. et al. (1999) Biochemical and electron microscopic image analysis of the hexameric E1 helicase. J. Biol. Chem. 274, 4447–4458 38 Yu, X. et al. (1995) Structural polymorphism of the RecA protein from the thermophilic bacterium Thermus aquaticus. Biophys. J. 69, 2728–2738 39 Bishop, D.K. et al. (1992) DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69, 439–456 40 Li, Z. et al. (1997) Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein. Proc. Natl. Acad. Sci. U. S. A. 94, 11221–11226 41 Masson, J.Y. et al. (1999) The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51. EMBO J. 18, 6552–6560 42 Passy, S.I. et al. (1999) Human Dmc1 protein binds DNA as an octameric ring. Proc. Natl. Acad. Sci. U. S. A. 96, 10684–10688 43 Rajagopalan, M. et al. (1992) Activity of the purified mutagenesis proteins UmuC, UmuD9, and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III. Proc. Natl. Acad. Sci. U. S. A. 89, 10777–10781 44 Tang, M. et al. (1999) UmuD9(2)C is an error-prone DNA polymerase, Escherichia coli pol V. Proc. Natl. Acad. Sci. U. S. A. 96, 8919–8924 45 Rehrauer, W.M. et al. (1998) Modulation of RecA nucleoprotein function by the mutagenic UmuD9C protein complex. J. Biol. Chem. 273, 32384–32387 46 Sharan, S.K. et al. (1997) Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature 386, 804–810 47 Lim, D.S. and Hasty, P. (1996) A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol. Cell Biol. 16, 7133–7143 48 Yu, C-E. et al. (1996) Positional cloning of the Werner’s syndrome gene. Science 272, 258–262 49 Ellis, N.A. et al. (1995) The Bloom’s syndrome gene product is homologous to RecQ helicases. Cell 83, 655–666
EDWARD EGELMAN Dept of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, Virginia, USA. Email: [email protected]
TIBS 25 – APRIL 2000
A ubiquitous structural core Over the last few years, the existence of a huge superfamily of structurally homologous proteins that are active in DNA recombination, replication, repair and transcription has been revealed. The underlying structural homology, with divergence of both sequence and function, suggests that all of these proteins (both eukaryotic and prokaryotic) are derived from some common prokaryotic ancestor. The extent of the functional divergence, however, suggests that the common ancestry might be more important in understanding the evolution of these proteins rather than in deducing their current functions. The bacterial RecA protein had been intensively studied for many years as the key catalyst in prokaryotic homologous recombination1–4. Remarkably, this single protein was capable of promoting strand exchange reactions in vitro involving: (i) the formation of joint molecules between two DNA substrates sharing common sequences, and (ii) the exchange of strands between these molecules. Within the bacterial cell, RecA plays a pivotal role in both recombination and repair, establishing that recombination occurs in many circumstances as a tool for the repair of DNA and the maintenance of genome stability. The possibility, long surmised, that RecA-like pathways exist in eukaryotic cells was confirmed with the identification of the yeast Rad51 protein as a RecA homolog5,6. The only functional form of the RecA protein had been assumed to be an unusual helical filament, within which DNA is stretched and untwisted so that its normal pitch of ~36 Å is changed to ~95 Å in the complex with the RecA protein7,8. It was shown that the yeast Rad51 protein formed a nearly identical nucleoprotein filament, imposing these same parameters on the DNA (Ref. 9). We also know that the human RAD51 protein induces a very similar structure10. Perhaps the fundamental event in the delineation of the RecA superfamily was the observation that the core of the first helicase, PcrA, to be solved at atomic resolution11 contained two copies of the same nucleotide-binding core first seen in the RecA protein12. This core domain contains a central β sheet with surrounding α helices, with a distinctive topology that had not been observed in any other proteins at the time that the RecA structure
was solved. The core also contains the binding site for ATP or ADP, as well as the catalytic elements used in the hydrolysis of ATP to ADP. Helicases are active not only in DNA replication, where they create a replication fork by opening up double-stranded DNA into two single-stranded templates for DNA polymerases, using energy derived from ATP hydrolysis, but also in many aspects of DNA recombination, repair and transcription13–15. It is puzzling why Escherichia coli has as many as 12 helicases16 and Saccharomyces cerevisiae even more than 134 (Ref. 17). This identification is based purely upon seven conserved sequence motifs that include the Walker A and B phosphate binding loops. This leaves open the possibility that many helicases, based upon such sequence analysis, might actually function in other ways than melting double-stranded DNA. One E. coli helicase, RuvB, is involved in the branch migration that occurs after strand exchange is initiated by the RecA protein18. Electron microscopy studies determined that RuvB exists as a hexameric ring, encircling double-stranded DNA (Ref. 19). This has led to a model for how RuvB functions as a DNA ‘pump’, using the energy of ATP hydrolysis to translocate DNA within the central channel of the ring20,21. In this system, the RuvB ring does not appear to separate doublestranded DNA into two single strands. Rather, the energy derived from ATP hydrolysis is used for translocation of the double-stranded DNA. A similar form of mechanochemical coupling was invoked to explain how the gp4 protein of bacteriophage T7, a replicative helicase, forms a hexameric ring around singlestranded DNA and uses the energy of ATP hydrolysis to translate along the single strand22,23. Because the complementary strand is displaced outside of the ring, this translocation results in strand separation. The comparison between RuvB and gp4 is interesting, because it suggests that the same conserved motor, with only minor differences in detail, can have two entirely different functions. This could provide some insight into the potential diversity of function of many other helicases that might use the energy derived from ATP hydrolysis for very different purposes.
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
The first atomic-level understanding of this helicase motor came with the determination of the structure of PcrA, a helicase from a thermophilic bacterium11. The structure revealed that all of the conserved helicase sequence motifs24,25 resided in the RecA-like domains, suggesting that all helicases would contain this same conserved core. Subsequent structures determined for other helicases have been in agreement with this prediction26–28. The only other protein found to have such a core was the F1-ATPase29, making it highly likely that both F1 and RecA evolved from some common ancestral protein. Our expectation is that individual point mutations can accumulate over time, giving rise to enormous sequence diversity, at the same time that the topology or pattern of connectivity of the b sheets and a helices remains fixed. It is this distinctive topology that defines structural homology and common evolutionary origin. Given that the 134 helicases found in S. cerevisiae (based upon sequence analysis) are all RecA homologs, this means that ~2% of the yeast genes encode proteins in this family. Structural studies suggest that this number could be even larger, as the d9 subunit of the E. coli clamp loader complex is also a RecA homolog30. The clamp loader complex is involved in positioning a clamp, or processivity factor, around the DNA that keeps the polymerase topologically tethered to its template during replication. However, the sequence of the d9 subunit has diverged so far from RecA that it no longer contains a recognizable ATP-binding motif, and does not bind to ATP. This suggests that other proteins involved in DNA replication, recombination, repair and transcription are also part of this superfamily, but no longer have identifiable sequence homology. The connection between RecA, Rad51 and helicases has been strengthened by the observation that RecA assembles into hexameric rings31, which are similar to the rings formed by helicases such as T7 gp4 (Refs 22,32), SV40 large T (Ref. 33), E. coli DnaB (Refs 34,35), E. coli RuvB (Ref. 19), phage SPP1 g40p (Ref. 36) and papilloma virus E1 (Ref. 37). Strikingly, the RecA ring can exist in two states, a symmetrical hexamer in which every subunit is equivalent, and a trimer of dimers. Within the trimer of dimers, there are two different subunit conformations, and the dimers are arranged so that the ring has a three-fold symmetry, rather than the six-fold symmetry of the symmetrical hexamer. A very similar polymorphism has been shown for DnaB (Ref. 35), SPP1
PII: S0968-0004(00)01570-X
183
COMMENT g40p (Ref. 36) and papilloma E1 (Ref. 37). The RecA ring structure appears to be conserved – both yeast (X. Yu, T. Ogawa and E. Egelman, unpublished) and human (X. Yu, S. West and E. Egelman, unpublished) Rad51 proteins also forms rings, but they are octameric, rather than hexameric. The conservation of these rings from bacteria to humans suggests that they are functional. The possibility that they are intermediates in the assembly of filaments can be excluded, as the subunit– subunit contacts within the rings are very different from those in the filaments31,38. Unfortunately, no mutants of RecA or Rad51 have yet been isolated that only form the ring or only form the filament, which could provide a clue to the function of the ring structure. It thus appears that RecA and Rad51 might exist in two functional forms, helical filaments and rings. The expectation was that Dmc1, a meiotic homolog of RecA (Ref. 39), would also exist as a helical filament. However, the only form of Dmc1 seen in vitro, under conditions where DNA-binding, DNA-activated ATPase and strand exchange have been assayed40,41, is an octameric ring42. Although we cannot exclude the possibility that within the cell other cofactors are present which allow Dmc1 to form helical filaments, it does suggest that in vivo RecA and Rad51 could function as both filaments and rings, whereas Dmc1 only functions as a ring.
In summary Structural studies, employing X-ray crystallography to determine the atomic structure of protein subunits, and electron microscopy to illuminate the higherorder structures of filaments and rings, have been instrumental in characterizing a polymorphic superfamily of proteins that all contain the same nucleotidebinding core. At least 2% of the genes in yeast encode for members of this superfamily, and in humans the percentage might be even higher. The RecA protein, whose structure was the first to be solved in this superfamily, is integrally involved in recombination, repair and replicative bypass of lesions43–45 in bacteria. It has been studied for many years as a model for how homologous recombination is catalysed by proteins. But RecA is not an essential gene in bacteria, except under conditions where DNA is subject to massive amounts of damage. We now know that the eukaryotic homolog of RecA, Rad51, might play an even more important role in humans, as it is probably an essential gene in higher eukaryotes46,47.
184
TIBS 25 – APRIL 2000 Similarly, helicases, whose mutations in humans can lead to premature aging, as in Werner’s Syndrome48, or predisposition to cancer, as in Bloom’s Syndrome49, are now known to be homologs of the RecA protein. We are at a very early stage in understanding what mechanisms all of these proteins employ in common, but the functional divergence could be very large. What is clear is that in the course of evolution, cells have created a vast repertoire of structures acting in replication, recombination, transcription and repair, functioning as monomers, dimers, hexamers, octamers and long helical filaments, that are all built around the same highly conserved core.
References 1 West, S.C. (1995) Formation, translocation and resolution of Holliday junctions during homologous genetic recombination. Phil. Trans. Roy. Soc. B 347, 21–25 2 Kowalczykowski, S.C. et al. (1994) Biochemistry of homologous recombination in Escherichia coli. Microbiol. Rev. 58, 401–465 3 Roca, A.I. and Cox, M.M. (1997) RecA protein: structure, function, and role in recombinational DNA repair. Prog. Nucleic Acids Res. Mol. Biol. 56, 129–223 4 Radding, C.M. (1991) Helical interactions in homologous pairing and strand exchange driven by RecA protein. J. Biol. Chem. 266, 5355–5358 5 Shinohara, A. et al. (1992) Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69, 457–470 6 Aboussekhra, A. et al. (1992) Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol. Cell Biol. 12, 3224–3234 7 Stasiak, A. and DiCapua, E. (1982) The helicity of DNA in complexes with RecA protein. Nature 229, 185–186 8 Stasiak, A. et al. (1981) Elongation of duplex DNA by RecA protein. J. Mol. Biol. 151, 557–564 9 Ogawa, T. et al. (1993) Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 259, 1896–1899 10 Benson, F.E. et al. (1994) Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 13, 5764–5771 11 Subramanya, H.S. et al. (1996) Crystal structure of a DExx box DNA helicase. Nature 384, 379–383 12 Story, R.M. et al. (1992) The structure of the E. coli recA protein monomer and polymer. Nature 355, 318–325 13 Lohman, T.M. (1993) Helicase-catalysed DNA unwinding. J. Biol. Chem. 268, 2269–2272 14 Matson, S.W. et al. (1994) DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. BioEssays 16, 13–22 15 Mendonca, V.M. et al. (1995) DNA helicases in recombination and repair: construction of a DuvrD DhelD DrecQ mutant deficient in recombination and repair. J. Bacteriol. 177, 1326–1335 16 Matson, S.W. (1991) DNA helicases of Escherichia coli. Prog. Nucleic Acids Res. Mol. Biol. 40, 289–326 17 Shiratori, A. et al. (1999) Systematic identification, classification, and characterization of the open reading frames which encode novel helicase-related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis. Yeast 15, 219–253 18 West, S.C. (1996) The RuvABC proteins and Holliday junction processing in Escherichia coli. J. Bacteriol. 178, 1237–1241 19 Stasiak, A. et al. (1994) The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA. Proc. Natl. Acad. Sci. U. S. A. 91, 7618–7622 20 Yu, X. et al. (1997) Structure and subunit composition of the RuvAB–Holliday junction complex. J. Mol. Biol. 266, 217–222 21 Parsons, C.A. et al. (1995) Structure of a multisubunit complex that promotes DNA branch migration. Nature 374, 375–378 22 Yu, X. et al. (1996) DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase. Nat. Struct. Biol. 3, 740–743
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EDWARD EGELMAN Dept of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, Virginia, USA. Email: [email protected]