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Happy Hollidays: 40th anniversary of the Holliday junction
Author: Y. Liu
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"� 2004 Nature Publishing Group PERSPECTIVES Institut Curie: http://www.curie.fr/ Institute of Physics: http://www.iop.org/ Max-Planck-Gesellschaft: http://www.mpg.de Max-Planck-Institut f�r Dynamik komplexer technischer Systeme: http://www.de.mpi-magdeburg.mpg.de Manchester Interdisciplinary Biocentre: http://www.mib.ac.uk/ Medical Research Council: http://www.mrc.ac.uk/ Nanobiotechnology Center: http://www.nbtc.cornell.edu/ Nano-Science Center, University of Copenhagen: http://www.nano.ku.dk/ Office of Science and Technology (UK): http://www.ost.gov.uk/index_v4.htm Research Councils UK: http://www.rcuk.ac.uk/ Royal Society of Chemistry: http://www.rsc.org/ Royal Academy of Engineering: http://www.raeng.org.uk/ Systeme des Lebens ? Systembiologie: http://www.systembiologie.de/ The Royal Society: http://www.royalsoc.ac.uk/ The Wellcome Trust: http://www.wellcome.ac.uk/ Access to this links box is available online. John McCarthy is at the Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M60 1QD, UK. e-mail: john.mccarthy@umist.ac.uk doi:10.1038/nrm1501 1. von Goethe, J. W. Faust, eine Trag�die. (C. H. Beck, M�nchen, 1989). 2. Schreiber, S. L. The small-molecule approach to biology. Chem. Eng. News 80, 51?61 (2003). 3. Henry, C. M. Systems biology. Chem. Eng. News 81, 45?55 (2003). 4. Kitano, H. Computational systems biology. Nature 420, 206?210 (2002). 5. Barab�si, A.-L. & Bonabeau, E. Scale-free networks. Sci. Am. 288, 50?59 (2003). 6. Oltvai, Z. N. & Barab�si, A.-L. Life?s complexity pyramid. Science 298, 763?764 (2002). 7. Schnitzer, M. J., Visscher, K. & Block, S. M. Force production by single kinesin motors. Nature Cell Biol. 2, 718?723 (2000). 8. Rief, M. et al.Myosin V stepping kinetics: a molecular model for processivity. Proc. Natl Acad. Sci. USA 97, 9482?9486 (2000). 9. Bustamante, C., Bryant, Z. & Smith, S. B. 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All systems go. Nature 427, 568?569 (2004). 18. Chien, K. & Chien, L. The new silk road. Nature 428, 208?209 (2004). 19. Hooke, R. Micrographia: or Some Physiological Descriptions of Minute Bodies Made by Magnifying Glasses With Observations and Inquiries Thereupon. (Royal Society Press, London, 1665). 20. Chapman, A. England?s Leonardo: Robert Hooke (1635?1703) and the art of experiment in Restoration England. Proc. R. Inst. G. Br. 67, 239?275 (1996). 21. Soong, R. K. et al. Powering an inorganic nanodevice with a biomolecular motor. Science 290, 1555?1558 (2000). 22. Yurke, B., Turberfield, A. J., Mills, A. P. Jr, Simmel, F. C. & Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605?608 (2000). 23. Shih, W. M., Quispe, J. D. & Joyce, G. F. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature 427, 618?621 (2004). 24. Ishii, D. et al. Chaperonin-mediated stabilization and ATP-triggered release of semiconductor nanoparticles. Nature 423, 628?632 (2003). 25. Mao, C., LaBean, T. H., Reif, J. H. & Seeman, N. C. Logical computation using algorithmic self-assembly of DNA triple crossover molecules. Nature 407, 493?496 (2000). 26. Benenson, Y., Adar, R., Paz-Elizur, T., Livneh, Z. & Shapiro, E. DNA molecule provides a computing machine with both data and fuel. Proc. Natl Acad. Sci. USA 100, 2191?2196 (2003). Acknowledgements The author is grateful to the Biotechnology and Biological Sciences Research Council, the Engineering and Physical Sciences Research Council, the Medical Research Council and the Wellcome Trust for the support of his research, the Royal Society and the Wolfson Foundation for a Research Merit award, and the Wellcome Trust and the Wolfson Foundation for the generous support of the Manchester Interdisciplinary Biocentre (MIB). He would also like to thank the Royal Society and the Royal Society of Chemistry for their ongoing support of interdisciplinary research. The author is affiliated to the University of Manchester Institute of Science and Technology (UMIST), which merged with the University of Manchester in October 2004. Competing interests statement The author declares no competing financial interests. Online links FURTHER INFORMATION Biotechnology and Biological Sciences Research Council: http://www.bbsrc.ac.uk/ Engineering and Physical Sciences Research Council: http://www.epsrc.ac.uk/ European Science Foundation: http://www.esf.org/ Foresight Institute ? preparing for nanotechnology: http://www.foresight.org/Nanomedicine/ Industrial Technology Research Institute, Taiwan: http://www.itri.org.tw/ NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 937 Happy Hollidays: 40th anniversary of the Holliday junction Yilun Liu and Stephen C. West TIMELINE Abstract | In 1964, the geneticist Robin Holliday proposed a mechanism of DNA- strand exchange that attempted to explain gene-conversion events that occur during meiosis in fungi. His proposal marked the birthday of the now famous cross-stranded DNA structure, or Holliday junction. To understand the importance of the Holliday model we must look back in the history of science beyond the last 40 years, to a time when theories of heredity were being proposed by Gregor Johann Mendel. Gregor Mendel, an Augustinian monk who taught natural science, was a man who paid attention to detail. In 1866 (TIMELINE),on the basis of his studies with pea plants, Mendel published a series of observations describing how characters or traits (now known as genes) are passed from parents to their off- spring. One important conclusion from his study was that hereditary factors do not com- bine, but are passed intact to the offspring, and that each member of the parental genera- tion transmits only half of its hereditary fac- tors to each offspring (with some factors being dominant over others). His work became the foundation for modern genetics; we now interpret it as showing that a parental cell with a pair of heterozygous (that is, differ- ent) alleles will produce gametes with a 2:2 ratio, such that each allele is represented equally in the haploid gametes (FIG. 1).However, although Mendel?s law of segregation was mostly shown to hold true, subsequent studies indi- cated that this was not always the case. Deviations from the expected 2:2 ratio were first reported by the German scientist Hans Winkler who, in 1930, introduced the term gene conversion to define the aberrant 3:1 ratio that had been observed in yeast tetrads. That is, during the process of segregation of the gametes, a gene-conversion event takes place that converts one allele to the other, so that the ratio of the alleles in the haploid gametes changes from 2:2 to 3:1. How does gene conversion work? In 1964, Robin Holliday (FIG. 2) from the John Innes ?The structure at the point of strand exchange later became known as a Holliday junction, and is embedded in history as a central intermediate in the process of homologous recombination.? � 2004 Nature Publishing Group 938 | NOVEMBER 2004 | VOLUME 5 www.nature.com/reviews/molcellbio PERSPECTIVES tion are crossed over. By contrast, cutting the other pair of strands results in recombination without crossing over (FIG. 3c, purple arrows). In both cases, however, the strand exchange produces heteroduplex DNA that contains mismatched nucleotides. Holliday postulated that mechanisms must exist to repair these mismatches, a suggestion that was later proven to be true when the enzymes of mis- match repair were discovered. Holliday pro- posed that heteroduplex DNA serves as the substrate for the gene-conversion event and, depending on which strand is used as the template for mismatch repair, the allelic ratio can be maintained as 2:2 (FIG. 3d),or changed to the observed aberrant ratio of 3:1 (FIG. 3e). This model provided a mechanistic basis for ?gene conversion?. Forty years after the Holliday model was proposed, we must now look back to see what biophysical analyses have told us about the structure of the Holliday junction, and how biochemical studies have defined the properties of enzymes that can specifically recognize four-way junctions and promote reactions that, originally, could only be imag- ined by Holliday. We will also reinvestigate the dogma that the Holliday junction is the central intermediate of recombination, and discuss how the Holliday model has evolved and been tailored to fit into the present pic- ture of DNA recombination and DNA- strand-break repair. Existence of the Holliday junction The first physical evidence to support Holliday?s proposal of a cross-stranded DNA intermediate was provided by electron- microscope studies that were carried out in the early 1970s. Work with S13 and �X174 ? switching of strands between DNA mole- cules results in the formation of a cross- stranded structure that physically links the two interacting DNA strands. The structure at the point of strand exchange later became known as a Holliday junction, and is embed- ded in history as a central intermediate in the process of homologous recombination (FIG. 3c).A second, critical aspect of the Holliday model invokes cutting (or ?resolu- tion?) of the crossover so that the two DNA helices can separate. Owing to the symmetry of the junction, it was assumed that there might be two possible orientations of resolu- tion, each with a different outcome. If the breaks are introduced in the strands that are complementary to the initiating nicks (FIG. 3c, green arrows), the arms that flank the junc- Institute in the United Kingdom ? who was studying DNA damage and genetic recombi- nation in the smut fungus Ustilago maydis and the budding yeast Saccharomyces cere- visiae at the time ? proposed a model for recombination that provided a molecular basis for both gene conversion and crossing over (that is, how genes linked on the same chromosome could segregate from each other) 1 .The Holliday model suggested that after DNA replication, which generates two copies of each of the two heterozygous alleles, recombination is initiated by the introduction of nicks at the same position in two DNA molecules that have different alleles (FIG. 3a). These breaks in the DNA strands allow single strands to anneal to the complementary sequences in the other duplex (FIG. 3b).The Gregor Mendel studied genetic inheritance and published his laws of heredity. James Watson and Francis Crick published the structure of the DNA double-helix. German scientist Hans Winkler introduced the term ?gene conversion? to explain the observed non-Mendelian ratio in yeast tetrads. Holliday model proposed to explain gene conversion 1 . Holliday junctions visualized by electron microscopy 2 . Holliday junctions made in vitro using purified Eschericia coli RecA recombinase 8 . Proposal of a model for double-strand- break repair 9 . First Holliday-junction resolvase identified in bacteriophage T4 17 . Identification of RuvC as E. coli Holliday-junction resolvase 55,56 . First crystal structure of a Holliday-junction resolvase 25 . Identification of E. coli RuvAB as first branch- migration enzyme 57,58 . Crossovers and non- crossovers are shown to arise at different times and by different mechanisms during meiotic recombination 51 . Synthetic Holliday junctions found to have an anti-parallel configuration 54 . 1850s?1860s 1930 1953 1964 1973 1982 1983 1988 1991 1992 1994 2001 Timeline | History of the Holliday junction Figure 1 | The Mendelian ratio. Based on the laws of segregation, published as part of the theory of heredity by Gregor Mendel in the mid-nineteenth century, a parental cell with a pair of heterozygous alleles (designated A and a), will produce gametes with an A:a = 2:2 ratio (left). However, this is not always the case as the 2:2 rule is violated on rare occasions when the ratio is 3:1 (right). This problem led Robin Holliday to propose his model for recombination to explain gene conversion. AAa a AAa a A aA A aA a A a Meiosis without gene conversion b Meiosis with gene conversion A:a=2:2 A:a=3:1 � 2004 Nature Publishing Group PERSPECTIVES Holliday-junction formation 11 .Whether or not the Holliday junction could survive these ?revo- lutions? will be discussed below. The structure of a Holliday junction The physical existence of Holliday junctions, as indicated by electron microscopy, stimu- lated biophysicists to try to determine its structure. But their goal was not an easy one, as it was extremely difficult to study the struc- ture of a crossover that was just a small part of a larger DNA molecule. The description, by Ned Seeman and colleagues in 1983, of a four-way junction that could be made simply by annealing short synthetic oligonucleotides was therefore a defining moment that led to two Escherichia coli phages that use their host?s enzyme systems for recombination ? revealed the presence of intermediates in which DNA molecules were linked by a ?Holliday junction? 2?4 .A spectacular image of two plasmid DNA molecules linked by a Holliday junction is shown in FIG. 4a (REF. 5). Soon afterwards, Holliday junctions that were made by the recombination of 2� plasmid DNA were observed in eukaryotic cells 6 . Our understanding of the detailed mecha- nism of recombination and the formation of Holliday junctions was advanced significantly in the 1980s when several of the key recombi- nation proteins from E. coli were purified and characterized. Using appropriately con- structed DNA substrates, it was shown that purified RecA protein could initiate strand- exchange reactions to form Holliday junc- tions in vitro 7,8 .The concept of the Holliday junction was, by this time, well accepted by the scientific community, and was embraced as a de facto intermediate in recombination for at least the next decade. However, further studies of recombination in eukaryotic cells, which were boosted by the emergence of sophisticated molecular-genetic approaches in yeast, indicated that the Holliday model was too symmetrical, and failed to account for data that had been obtained in S. cerevisiae where little evidence of reciprocal heteroduplex DNA could be found. It also became clear that there were alternative path- ways that could lead to the formation of recombinant products. In the 1980s, two mod- els were proposed that, at present, continue to form the basis of our understanding of recombination. In 1983, Jack Szostak, Terry Orr-Weaver, Rodney Rothstein and Frank Stahl proposed a model that was novel in two respects: first, recombination was initiated by a DNA double-strand break (DSB) rather than by nicks; and second, the recombining DNA helices became linked by two Holliday junc- tions 9 .Soon afterwards, work from Sternberg?s laboratory indicated that recombination could occur between repetitive sequences by a mech- anism that became known as single-strand annealing 10 .More recently, other models have gained in popularity, most notably the concept that recombination can occur by synthesis- dependent strand annealing (SDSA) without NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 939 Figure 2 | Robin Holliday. In May 2004, Robin Holliday attended the EMBO workshop ?Recombination Mechanisms? and presented a lecture to celebrate the 40th anniversary of his model. This photograph was taken at the Chateau de Chenonceau, France, by David Roth. Figure 3 | The Holliday model. After DNA replication and before meiotic cell division, nicks are introduced at a defined point on two homologous chromosomes (a). Strand exchanges take place to generate a crossover, or Holliday junction (b and c). Symmetric resolution in the two possible orientations (indicated by purple and green arrows) allows separation of the recombining chromosomes (c). Crossover or non- crossover products are formed, dependent on the orientation of resolution. DNA mismatches present in the heteroduplex DNA might be corrected, leading to gene conversion (d and e). a a a a A A A A a a a a A A a a A A a a A A a a A A a a A A a a a a a a A A a d e b c A A 2:2 3:1 � 2004 Nature Publishing Group 940 | NOVEMBER 2004 | VOLUME 5 www.nature.com/reviews/molcellbio PERSPECTIVES is seen in the absence of metal ions, which indicates that the metal ions neutralize the electrostatic repulsions that are caused by the phosphates in the DNA backbone at the point of the crossover 13 .Interestingly, enzymes that bind Holliday junctions and catalyse the key reactions that are involved in junction processing (branch migration and nucleolytic resolution) have been shown to bind and stabilize the open planar structure. Therefore, it can be argued that the fourfold, symmetric, unstacked struc- ture might be the more physiologically rele- vant of these two structures. Holliday-junction-processing enzymes In the Holliday model, recombination occurs by a two-step process: symmetric nicking initiates crossover formation, and symmetric nicking resolves the crossover. But in 1964, Robin was not really thinking about the enzymes that were involved and how they might promote such reactions. Indeed, it took until 1982, when biochemi- cal evidence provided the first example of an enzyme that could recognize Holliday junctions and promote a specific cleavage reaction, for us to realize that Holliday-junc- tion-specific proteins existed. The relevant protein from bacteriophage T4 is the prod- uct of gene 49. The enzyme, T4 endonucle- ase VII, is a structure-specific endonuclease that is required for the separation of highly branched DNA before its packaging into phage heads 17 .Subsequently, Holliday- junction resolvases were identified in vari- ous species, including bacteriophage-T7 endonuclease I, pox virus A22, E. coli RuvC and RusA, archaeal Hjc and Hje, and Saccharomyces cerevisiae mitochondrial Cce1 (or Yd c 2 in Schizosaccharomyces pombe) 18 .All of these enzymes recognize Holliday junc- tions and resolve them by the introduction of symmetrically positioned nicks in strands with the same polarity, thereby forming nicked duplex products that can be repaired in a simple nick-ligation reaction. Mammalian resolvases. It has been more dif- ficult to identify Holliday-junction resolvases in eukaryotes, although mammalian activi- ties that fit the resolvase paradigm were first observed in 1990 (REF. 19).The issue has also been complicated by the presence of Mus81, a flap/fork endonuclease from yeast and humans that has a weak Holliday-junction cleavage activity in vitro 20,21 .Because Mus81 is required for the formation of crossover prod- ucts that result from homologous recombi- nation during meiosis in S. pombe, it was suggested that Mus81 could be the eukaryotic In the anti-parallel orientation, the exchanging strands do not cross with each other. The torsional angle of the sugar- phosphate backbone of the DNA at the point of exchange adapts a gauche confor- mation, instead of the trans conformation that is found in normal duplex DNA. This allows it to bend into a U shape and pair with the complementary strand that is run- ning in the opposite direction. This structure, which was observed in vitro,raises interesting questions about its existence in vivo.Within the cell, it is expected that homologous chro- mosomes at meiosis (or sister chromatids undergoing mitotic recombination) will be aligned parallel to each other, at least in a global sense. So if the duplexes at the Holliday junction lie anti-parallel to each other, then, at the local level, one of the two DNA molecules will have to rotate 180� into the anti-parallel orientation. Whether this can occur in vivo, where physical constraints will be imposed by the flanking arms and by proteins that bind to the DNA, remains a puzzle. In addition to the anti-parallel X-structure that is observed in the presence of divalent cations, Holliday junctions are also seen to form an unstacked fourfold symmetric planar structure (FIG. 4d).The square-planar structure significant progress in understanding the three-dimensional structure of the junction 12 . But it was still many years before scientists were able to determine the structure of Holliday?s junction at the atomic level. Initial studies of the Holliday-junction structure were both surprising and contro- versial. Gel electrophoresis and analyses using fluorescent energy transfer (FRET; a technique that measures the distance between the excited states of two fluorescent dyes) revealed that the four-way junction could exist in a variety of structures, none of which resembled the expected structure in which the two linked DNA helices would lie parallel to each other. Robin Holliday was bemused by the physical studies and found it difficult to accept that these structures were truly representative of the Holliday junction as it exists within the cell. What was so controversial was that, in the pres- ence of a divalent metal ion such as Mg 2+ , the junction adopts a twofold symmetric X-shape in which the DNA helices lie anti- parallel to each other 13 (FIG. 4b).But this was no artefact, as the anti-parallel structure of the Holliday junction was later confirmed by three independent X-ray crystallographic structures 14?16 (FIG. 4c). Figure 4 | Structure of the Holliday junction. a | Electron-microscope image of a recombination intermediate. In this image, the Holliday junction was partially denatured to assist its visualization. b | Two possible configurations for the Holliday junction, with the DNA shown in the parallel (left) or anti- parallel configuration (right). c | Three-dimensional view of a Holliday junction, as determined by X-ray crystallography. d | The Holliday junction shown in the anti-parallel stacked-X (left) and open planar (right) configuration. Part a is reproduced with permission from REF. 5 � (1979) Cold Spring Harbor Laboratory Press. Part c is modified with permission from REF. 16 � (2000) the National Academy of Sciences. Parallel Anti-parallel stacked-X Open planar Anti-parallel 5? 5? 5? 5? 5? 5? 5? 5? ab cd � 2004 Nature Publishing Group PERSPECTIVES Analogous branch-migration and resolution activities have been observed in fractionated mammalian extracts 24,37 ,but the proteins that are responsible for branch migration have yet to be identified. Members of the RecQ family of DNA helicases, such as Bloom?s syndrome protein (BLM), and Werner?s syndrome protein (WRN) and RecQ5?,have been shown, at least in vitro,to be capable of catalysing branch migration in addition to unwinding duplex DNA 38?40 . Defects in BLM or WRN lead to inheritable diseases known as Bloom?s syndrome (which Holliday-junction resolvase. However, fur- ther studies showed that the mechanism by which Mus81 cuts Holliday junctions differs from that of all other Holliday-junction resolvases, and that the nuclease had a very potent structure-specific activity on replica- tion-fork or flap structures. It is therefore not a Holliday-junction resolvase in the classic sense 22 .Moreover, in mammalian cell extracts, Holliday-junction-resolution activ- ity can be separated from MUS81 (REF. 23), and the resolvase complex in these cell extracts contains the recombination pro- teins RAD51C and XRCC3 (REF. 24).Further work to identify the nuclease component of the latter complex is underway. Surprisingly, even though Holliday-junc- tion resolvases from different species are functionally conserved, they bear little simi- larity to each other at the level of amino-acid sequence. This makes it particularly difficult to use sequence homology and database analyses to identify related nucleases from higher organisms. The lack of primary sequence conservation indicates that they are unlikely to have evolved from the same ancestral gene, which presents us with a mystery: why are the Holliday-junction resolvases, which function in highly con- served recombination/repair processes, so evolutionarily diverse? And, which features give them the ability to recognize and cleave four-way junctions? Atomic structure of the prokaryotic resolvases. When the crystal structures of various Holliday-junction resolvases were solved, it became clear that most belong to two families: the integrase superfamily (RuvC and Ydc2) or the nuclease superfam- ily (T7 endonuclease I and Hjc) 25?30 (FIG. 5). So, even though the resolvases are not con- served at the amino-acid level, they do resemble each other on the structural level. By contrast, T4 endonuclease VII and E. coli RusA (which itself is phage-derived) seem to have evolved independently. Exactly what the Holliday-junction resolvases have in common remains a difficult question to answer, as the only obvious feature is a cat- alytic domain that contains clusters of basic aspartate and glutamate residues that are required for metal-ion binding. E. coli RuvC is the most well-characterized resolvase and functions as a paradigm for other Holliday-junction resolvases. The mechanism of Holliday-junction resolution can be broken down into a number of experi- mentally separable steps including DNA binding, modification of DNA structure and cleavage 31 .RuvC binds specifically to a Holliday junction as a dimer and unfolds the junction into an open planar configuration (FIG. 6a).In the presence of divalent cations, RuvC introduces symmetrical nicks in strands of the same polarity. Although the protein binds junctions in a structure-specific manner, the cleavage reaction has extra selec- tivity at the sequence level such that the degenerate sequence 5?-(A/T)TT?(G/C)-3? is the preferred cleavage site. Junction migration. The concept that junc- tions might ?slide? along DNA to extend the length of the heteroduplex was also pro- posed in Holliday?s 1974 paper 32 .We now refer to this reaction as branch migration, and from studies of bacterial proteins we have a good understanding of how it takes place 31 .In E. coli,Holliday junctions are branch migrated by the products of the DNA-damage-inducible ruvA and ruvB genes. RuvA protein, a tetramer, binds the junction in the unfolded open-square con- figuration, with the four DNA arms lying in positively charged grooves on its surface (FIG. 6b).The unfolded configuration favours branch migration, as do four acidic amino acids on the surface of the RuvA tetramer, which function as guides to facilitate the transient opening of base pairs as strands pass from one helical axis to another 33,34 .The motor of branch migration is RuvB, a hexa- meric ring protein, which associates with RuvA to form the tripartite structure that is shown in FIG. 6b (REF. 35,36).The rings are positioned in the opposite orientation rela- tive to the Holliday junction, and therefore exert equal and opposite forces after ATP hydrolysis in a reaction that results in the passage of DNA helices through the protein complex. In vivo, it is thought that the RuvA?RuvB complex functions together with RuvC resolvase as part of a ?resolvasome? complex. NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 941 Figure 5 | Structural comparison of the Holliday-junction resolvases. Comparison of the crystallographic structures of various Holliday-junction resolvases indicate that Escherichia coli RuvC and Schizosaccharomyces pombe Ydc2 belong to the integrase superfamily, whereas T7 endonuclease I (T7 endo I) and Sulfolobus solfataricus Hjc are members of the nuclease superfamily. The origins of T4 endonuclease VII and RusA are unknown. The structures were obtained from the National Center for Biotechnology Information (NCBI) structure database (see the online links box) and illustrated using CN3D software. E. coli RuvC S. pombe Ydc2 a Integrase superfamily T7 endo I S. solfataricus Hjc b Nuclease superfamily T4 endo VII E. coli RusA c Unrelated ??since the Holliday model was first proposed, we have seen its re-evaluation and continued modification ? Perhaps the most significant change lies in the complexity of the process, in which alternative events can occur.? � 2004 Nature Publishing Group 942 | NOVEMBER 2004 | VOLUME 5 www.nature.com/reviews/molcellbio PERSPECTIVES to the dynamic picture that represents our present understanding of how recombi- nants form in eukaryotic cells (FIG. 7). Perhaps the most significant change lies in the complexity of the process, in which alternative events can occur. Most recombi- nation events are thought to result from the formation of a DSB: in meiotic recombina- tion DNA-strand breaks are a consequence of DSBs that are introduced by a topoiso- merase-like protein known as SPO11 (REF. 44), whereas in a mitotic cell they might be radi- ation-induced breaks or DSBs that arise from stalled and broken replication forks 45,46 .The ends of the DNA are resected to produce single-stranded DNA that recruits recombination proteins such as replication protein A (RPA), RAD52 and RAD51.The assembly of a RAD51 nucleo- protein filament leads to interactions with homologous duplex DNA and strand inva- sion. This process is known as single-end invasion (SEI), and RAD54 is thought to stabilize this recombination intermediate to allow the subsequent events to take place. In some recombination pathways, SEI is followed by the annealing of the second DNA end in a reaction that might involve the single- strand-annealing activity of RAD52 (FIG. 7). This intermediate can proceed to form dou- ble Holliday junctions, and any remaining gaps might be filled by new DNA synthesis. The resulting Holliday junctions might then serve as the substrate for a classic Holliday- junction-resolution reaction ? which involves RAD51C, XRCC3 and other as-yet- unidentified factors 24 ? or be dissociated by the combined actions of BLM and topoiso- merase III? (REF. 42). Recombinants can also form by path- ways that do not involve Holliday junctions (FIG. 7).For example, the formation of dou- ble Holliday junctions can be prevented by the MUS81 complex, which cleaves strand- invasion intermediates (FIG. 7,green arrows) before they can mature into Holliday junc- tions 47?49 .Similarly, DSBs can be repaired by SDSA, a pathway that is dependent on the SRS2 helicase 43,50 .In SDSA, nucleopro- tein filaments of the RAD51 recombinase Holliday junctions 42 .As Holliday-junction ?dissolution? by BLM and topoisomerase III? gives rise to non-crossover products, the disruption of this pathway in Bloom?s syn- drome cells provides a satisfying explana- tion for the elevated level of crossovers that are observed in the mutant. The S. cere- visiae homologues of BLM and topoiso- merase III? ? Sgs1 and Top 3 ,respectively ?promote similar reactions in yeast 43 ,so in some situations (that is, recombination at blocked replication forks), this system might provide an alternative pathway to process double Holliday junctions (FIG. 7). Crossover versus non-crossover In the 40 years since the Holliday model was first proposed, we have seen its re-evaluation and continued modification, which has led afflicts more than 1 in a 100 Ashkenazi Jews) and Werner?s syndrome (which is character- ized by premature ageing). On a cellular level, mutations in RecQ-family proteins give rise to genomic instability and a sensi- tivity to DNA-damaging agents 41 .However, RecQ-family proteins, BLM in particular, are unlikely to be branch-migration motors equivalent to RuvB, because mutations in the BLM gene lead to a phenotypic increase in the frequency of sister-chromatid exchanges (that is, crossovers) as a result of homologous recombination at stalled repli- cation forks. In this respect, BLM could be regarded as an anti-recombinase. New insight into this phenomenon was recently gained when it was shown that BLM and topoisomerase III? function together to dissociate structures that contain double Figure 6 | Three-dimensional structure of RuvC? and RuvAB?Holliday-junction complexes. a | Atomic model of RuvC binding to a Holliday junction. The dimeric RuvC protein introduces nicks at symmetric positions in strands with the same polarity. b | Model of the RuvAB?Holliday-junction complex 53 . Part a is modified from REF. 53 � (1996) American Association for the Advancement of Science. The image in part b is reproduced with permission from the University of Sheffield web site (see online links box). a b ?So, 40 years on, the importance of the Holliday model is evidenced by the fact that it has evolved rather than having been replaced?? � 2004 Nature Publishing Group PERSPECTIVES Holliday junctions, the intermediate is already destined to be resolved in a specific orienta- tion that leads to crossover 51,52 .Whether or not this is also true for organisms other than yeast remains to be answered. So, 40 years on, the importance of the Holliday model is evidenced by the fact that it has evolved rather than having been replaced, even in the face of the genetic revolution that has taken place during this time. The Holliday junction still exists and takes its rightful place in many recombination pathways. We know its atomic structure, and we have discovered pro- tein complexes that move it and cut it. But we are still left with so many mysteries, in particu- lar, what factors control how, and into what, it is resolved. These puzzles cannot be explained by our present knowledge of the structure of the Holliday junction and the enzymes that resolve it. These challenges are for the future, for us to investigate now that the model has been celebrated four decades after it was born. Yilun Liu and Stephen C. West are at Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK. Correspondence to S.C.W. e-mail: stephen.west@cancer.org.uk doi:10.1038/nrm1502 1. Holliday, R. A mechanism for gene conversion in fungi. Genet. Res. Camb. 5, 282?304 (1964). 2. Doniger, J., Warner, R. C. & Tessman, I. Role of circular dimer DNA in the primary recombination mechanism of bacteriophage S13. Nature New Biol. 242, 9?12 (1973). 3. Thompson, B. J. et al. Figure-8 configuration of dimers of S13 and �X174 replicative form DNA. J. Mol. Biol. 91, 409?419 (1975). 4. Benbow, R. M., Zuccarelli, A. J. & Sinsheimer, R. L. Recombinant DNA molecules of �X174. Proc. Natl Acad. Sci. USA 72, 235?239 (1975). 5. Potter, H. & Dressler, D. DNA recombination: in vivo and in vitro studies. Cold Spring Harb. Symp. Quant. Biol. XLIII, 969?985 (1979). 6. Bell, L. & Byers, B. Occurrence of crossed strand- exchange forms in yeast during meiosis. Proc. Natl Acad. Sci. USA 76, 3445?3449 (1979). 7. Cunningham, R. P., DasGupta, C., Shibata, T. & Radding, C. M. Homologous pairing in genetic recombination: RecA protein makes joint molecules of gapped circular DNA and closed circular DNA. Cell 20, 223?235 (1980). 8. West, S. C., Countryman, J. K. & Howard-Flanders, P. Enzymatic formation of biparental figure-8 molecules from plasmid DNA and their resolution in Escherichia coli. Cell 32, 817?829 (1983). 9. Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J. & Stahl, F. W. The double-strand break repair model for recombination. Cell 33, 25?35 (1983). 10. Lin, F. L., Sperle, K. & Sternberg, N. Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process. Mol. Cell. Biol. 4, 1020?1034 (1984). 11. Nassif, N., Penney, J., Pal, S., Engels, W. R. & Gloor, G. B. Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol. Cell. Biol. 14, 1613?1625 (1994). 12. Kallenbach, N. R., Ma, R. I. & Seeman, N. C. An immobile nucleic acid junction constructed from oligonucleotides. Nature 305, 829?831 (1983). 13. Lilley, D. M. J. Structures of helical junctions in nucleic acids. Q. Rev. Biophys. 33, 109?159 (2000). 14. Nowakowski, J., Shim, P. J., Prasad, G. S., Stout, C. D. & Joyce, G. F. Crystal structure of an 82-nucleotide RNA?DNA complex formed by the 10-23 DNA enzyme. Nature Struct. Biol. 6, 151?156 (1999). 15. Ortiz-Lombardia, M. et al. Crystal structure of a DNA Holliday junction. Nature Struct. Biol. 6, 913?917 (1999). promote SEI, and the reaction is followed by DNA synthesis to fill the gap. At this stage, SRS2 helicase is thought to dissociate heteroduplex intermediates that are formed within the RAD51 filament, so that the invading strand that has a newly synthe- sized 3? end is available to reanneal with the second end of the same DNA molecule at the break site. DNA synthesis can then take place to restore the integrity of the DNA. Although all these recombination pathways can result in gene conversion, there is a very significant difference between them in that some pathways commit to the formation of either a crossover or a non-crossover prod- uct. For example, BLM/topoisomerase-III?- dependent Holliday-junction dissolution and SDSA events generally produce non- crossover products, whereas MUS81- dependent reactions primarily yield the crossovers. In the Holliday model it was suggested that a junction could be resolved in one of two possible orientations to yield either the crossover or non-crossover product. However, this concept is not necessarily true as studies of yeast meiosis indicate that non- crossovers appear earlier than crossovers. Moreover, mutations in Ndt80 ? a yeast meiosis-specific transcription factor that is important for the completion of prophase in meiosis I ? cause meiotic arrest with unre- solved Holliday-junction intermediates. Surprisingly, the mutant cells show a signifi- cant reduced frequency of crossover products, whereas the non-crossover products remain at a normal level. This finding indicates that, at least in yeast meiosis, crossover and non- crossover products are formed by different mechanisms, and, more importantly, that once the DSB-repair reaction has committed to a resolution pathway that involves double NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 943 Figure 7 | Homologous recombination, the big picture. Summary of our current understanding of recombination pathways that are initiated by a DNA double-strand break (DSB) and which lead to gene conversion with or without crossover. First, the ends of the DSB are resected to produce single- stranded DNA that recruits the recombination protein RAD51. The assembly of a RAD51 nucleoprotein filament leads to interactions with homologous duplex DNA and strand invasion. This process is known as single-end invasion (SEI) and the intermediate structures might be stabilized by the RAD54 protein. In some pathways for recombination (centre), SEI is followed by capture of the second DNA end in reactions that are likely to involve RAD52. This intermediate can proceed to form double Holliday junctions, and any remaining gaps might be filled by new DNA synthesis. The resulting Holliday junctions might then serve as the substrate for a classic Holliday-junction-resolution reaction, involving RAD51C, XRCC3 and other as-yet-unidentified factors, or be dissociated by the combined actions of BLM (Bloom?s syndrome protein) and topoisomerase III? (Topo III). The BLM?Topo-III reaction primarily leads to the formation of non-crossover products, as mutations in BLM cause an increase in crossover formation. Recombinants can also form by a MUS81-dependent pathway that does not involve Holliday-junction formation (right). Similarly, DSBs can be repaired by synthesis-dependent strand annealing (SDSA), a pathway that is dependent on the SRS2 helicase (left). RAD51 RAD54 RAD52 Double Holliday junctions (SRS2) SDSA Non-crossover Non-crossover BLM?Topo III Dissolution Crossover Crossover Resolution RAD51C?XRCC3 MUS81-dependent MUS81? EME1 � 2004 Nature Publishing Group 944 | NOVEMBER 2004 | VOLUME 5 www.nature.com/reviews/molcellbio PERSPECTIVES 53. Rafferty, J. B. et al. Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction. Science 274, 415?421 (1996). 54. Duckett, D. R. et al. The structure of the Holliday junction and its resolution. Cell 55, 79?89 (1988). 55. Dunderdale, H. J. et al. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature 354, 506?510 (1991). 56. Iwasaki, H., Takahagi, M., Shiba, T., Nakata, A. & Shinagawa, H. Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J. 10, 4381?4389 (1991). 57. Tsaneva, I. R., M�ller, B. & West, S. C. ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coli. Cell 69, 1171?1180 (1992). 58. Iwasaki, H., Takahagi, M., Nakata, A. & Shinagawa, H. Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration. Genes Dev. 6, 2214?2220 (1992). Acknowledgements Work in the West laboratory is supported by Cancer Research UK, and by the Breast Cancer Campaign. Y.L. is a recipient of a post-doctoral fellowship from the American Cancer Society. Competing interests statement The authors declare no competing financial interests. Online links DATABASES The following terms in this article are linked online to: Entrez: http://www.ncbi.nlm.nih.gov/entrez archaeal Hjc | archaeal Hje | RecA | RusA | ruvA | ruvB | RuvC | T4 endonuclease VII Saccharomyces genome database: http://db.yeastgenome.org/ Cce1 | Mus81 | Ndt80 | Sgs1 | Top3 S. pombe gene database: http://www.genedb.org/genedb/pombe/index.jsp Ydc2 SwissProt: http://us.expasy.org/sprot/ BLM | RAD51 | RAD51C | RAD52 | SPO11 | topoisomerase III? | WRN | XRCC3 FURTHER INFORMATION Bill Engels?s laboratory (movies of Holliday junctions and mechanisms of recombination): http://engels.genetics.wisc.edu/Holliday/index.html Human DNA-repair genes: http://www.cgal.icnet.uk/DNA_Repair_Genes.html NCBI structure database: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Structure&i tool=toolbar Steve West?s laboratory: http://science- edit.cancerresearchuk.org/sci/genrecombi/?version=1 University of Sheffield: model for the mechanism of RuvAB-mediated branch migration: http://www.shef.ac.uk/mbb/ruva/abcomplex.html Access to this links box is available online. 35. Parsons, C. A., Stasiak, A., Bennett, R. J. & West, S. C. Structure of a multisubunit complex that promotes DNA branch migration. Nature 374, 375?378 (1995). 36. Yamada, K. et al. Crystal structure of the RuvA?RuvB complex: a structural basis for the Holliday junction migrating motor machinery. Mol. Cell 10, 671?681 (2002). 37. Constantinou, A., Davies, A. A. & West, S. C. Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells. Cell 104, 259?268 (2001). 38. Constantinou, A. et al. Werner?s syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest. EMBO Rep. 1, 80?84 (2000). 39. Karow, J. K., Constantinou, A., Li, J.-L., West, S. C. & Hickson, I. D. The Bloom?s syndrome gene product promotes branch migration of Holliday junctions. Proc. Natl Acad. Sci. USA 97, 6504?6508 (2000). 40. Garcia, P. L., Liu, Y., Jiricny, J., West, S. C. & Janscak, P. Human RecQ5?, a protein with DNA helicase and strand- annealing activities in a single polypeptide. EMBO J. 23, 2882?2891 (2004). 41. Hickson, I. D. RecQ helicase: caretakers of the genome. Nature Rev. Mol. Cell Biol. 3, 169?178 (2003). 42. Wu, L. & Hickson, I. D. The Bloom?s syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870?874 (2003). 43. Ira, G., Malkova, A., Liberi, G., Foiani, M. & Haber, J. E. Srs2 and Sgs1?Top3 suppress crossovers during double-strand break repair in yeast. Cell 115, 401?411 (2003). 44. Keeney, S., Giroux, C. N. & Kleckner, N. Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375?384 (1997). 45. Cox, M. M. et al. The importance of repairing stalled replication forks. Nature 404, 37?41 (2000). 46. Haber, J. E. DNA recombination: the replication connection. Trends Biochem. Sci. 24, 271?275 (1999). 47. Osman, F., Dixon, J., Doe, C. L. & Whitby, M. C. Generating crossovers by resolution of nicked Holliday junctions: a role of Mus81?Eme1 in meiosis. Mol. Cell 12, 761?774 (2003). 48. Gaillard, P.-H. L., Noguchi, E., Shanahan, P. & Russell, P. The endogenous Mus81?Eme1 complex resolves Holliday junctions by a nick and counternick mechanism. Mol. Cell 12, 747?759 (2003). 49. Heyer, W. D. Recombination: Holliday junction resolution and crossover formation. Curr. Biol. 14, R56?R58 (2004). 50. Fabre, F., Chan, A., Heyer, W. D. & Gangloff, S. Alternate pathways involving Sgs1/Top3, Mus81/Mus81, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc. Natl Acad. Sci. USA 99, 16887?16892 (2002). 51. Allers, T. & Lichten, M. Differential timing and control of noncrossover and crossover recombination during meiosis. Cell 106, 47?57 (2001). 52. Borner, G. V., Kleckner, N. & Hunter, N. Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis. Cell 117, 29?45 (2004). 16. Eichman, B. F., Vargason, J. M., Mooers, B. H. M. & Ho, P. S. The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions. Proc. Natl Acad. Sci. USA 97, 3971?3976 (2000). 17. Mizuuchi, K., Kemper, B., Hays, J. & Weisberg, R. A. T4 endonuclease VII cleaves Holliday structures. Cell 29, 357?365 (1982). 18. Sharples, G. J. The X philes: structure-specific endonucleases that resolve Holliday junctions. Mol. Microbiol. 39, 823?834 (2001). 19. Elborough, K. M. & West, S. C. Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus. EMBO J. 9, 2931?2936 (1990). 20. Boddy, M. N. et al. Mus81?Eme1 are essential components of a Holliday junction resolvase. Cell 107, 537?548 (2001). 21. Chen, X. B. et al. Human Mus81-associated endonuclease cleaves Holliday junctions in vitro. Mol. Cell 8, 1117?1127 (2001). 22. Hollingsworth, N. M. & Brill, S. J. The Mus81 solution to resolution: generating meiotic crossovers without Holliday junctions. Genes Dev. 18, 117?125 (2004). 23. Constantinou, A., Chen, X.-B., McGowan, C. H. & West, S. C. Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities. EMBO J. 21, 5577?5585 (2002). 24. Liu, Y., Masson, J.-Y., Shah, R., O?Regan, P. & West, S. C. RAD51C is required for Holliday junction processing in mammalian cells. Science 303, 243?246 (2004). 25. Ariyoshi, M. et al. Atomic structure of the RuvC resolvase: a Holliday junction-specific endonuclease from E. coli. Cell 78, 1063?1072 (1994). 26. Ceschini, S. et al. Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2. EMBO J. 20, 6601?6611 (2001). 27. Raaijmakers, H. et al. X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture. EMBO J. 18, 1447?1458 (1999). 28. Hadden, J. M., Convery, M. A., Declais, A. C., Lilley, D. M. J. & Phillips, S. E. V. Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I. Nature Struct. Biol. 8, 62?67 (2001). 29. Bond, C. S., Kvaratskhelia, M., Richard, D., White, M. F. & Hunter, W. N. Structure of Hjc, a Holliday junction resolvase, from Sulfolobus solfataricus. Proc. Natl Acad. Sci. USA 98, 5509?5514 (2001). 30. Rafferty, J. B. et al. The structure of Escherichia coli RusA endonuclease reveals a new Holliday junction DNA binding fold. Structure 11, 1557?1567 (2003). 31. West, S. C. Processing of recombination intermediates by the RuvABC proteins. Annu. Rev. Genet. 31, 213?244 (1997). 32. Holliday, R. Molecular aspects of genetic exchange and gene conversion. Genetics 78, 273?287 (1974). 33. Hargreaves, D. et al. Crystal structure of E. coli RuvA with bound DNA Holliday junction at 6� resolution. Nature Struct. Biol. 5, 441?446 (1998). 34. Ariyoshi, M., Nishino, T., Iwasaki, H., Shinagawa, H. & Morikawa, K. Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer. Proc. Natl Acad. Sci. USA 97, 8257?8262 (2000). � 2004 Nature Publishing Group PERSPECTIVES NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 945 MCCARTHY ONLINE ONLY Author biography John McCarthy?s first degree at Oxford University, UK, was followed by a Ph.D. on the biochemistry and biophysics of electron- transport-coupled ATP synthesis. As an EMBO research fellow in Germany, he then elucidated novel post-transcriptional control mechanisms that underlie the synthesis and assembly of the Escherichia coli (H + )ATPase. His research subsequently re-focused on the mechanisms of eukaryotic post-transcrip- tional control, and he became Head of a Research and Development Department at the Federal Biotechnology Institute, Braunschweig, Germany, and a ?habilitated? university teacher. He was appointed full pro- fessor at the University of Manchester Institute of Science and Technology (UMIST), UK, in 1996, and chaired the Department of Biomolecular Sciences from 1998 to 2000. At present, he is Director of the Manchester Interdisciplinary Biocentre and a Wolfson?Royal-Society Research Fellow. Recent work includes the application of bio- physical methods, including single-molecule techniques, to the study of the yeast ribo- some. � 2004 Nature Publishing Group 946 | NOVEMBER 2004 | VOLUME 5 www.nature.com/reviews/molcellbio WEST ONLINE bin/locus.pl?locus=cce1 Mus81 http://db.yeastgenome.org/cgi- bin/locus.pl?locus=Mus81 Ndt80 http://db.yeastgenome.org/cgi- bin/locus.pl?locus=ndt80 Sgs1 http://db.yeastgenome.org/cgi- bin/locus.pl?locus=Sgs1 Top 3 http://db.yeastgenome.org/cgi- bin/locus.pl?locus=top3 S. pombe gene database: http://www.genedb.org/genedb/pombe/index .jsp Yd c 2 http://www.genedb.org/genedb/Search?name =ydc2&organism=pombe&desc=yes&wild- card=yes SwissProt: http://us.expasy.org/sprot/ BLM http://us.expasy.org/cgi- bin/niceprot.pl?P54132 RAD51 http://us.expasy.org/cgi- bin/niceprot.pl?Q06609 RAD51C http://us.expasy.org/cgi- bin/niceprot.pl?O43502 RAD52 http://us.expasy.org/cgi- bin/niceprot.pl?P43351 SPO11 http://us.expasy.org/cgi- bin/niceprot.pl?Q9Y5K1 topoisomerase III? http://us.expasy.org/cgi- bin/niceprot.pl?Q13472 WRN http://us.expasy.org/cgi- bin/niceprot.pl?Q14191 XRCC3 http://us.expasy.org/cgi- bin/niceprot.pl?O43542 Biographies Stephen C. West received his Ph.D. from Newcastle University, UK, and carried out his postdoctoral studies with Paul Howard- Flanders at Yale University, USA. He is now a Principal Scientist with Cancer Research UK and his laboratory is based at Clare Hall in Hertfordshire, UK. His interests include the mechanisms of genetic recombination and DNA repair, and how these processes are important for genome stability. Yilun Liu received her Bachelor?s degree in biology from the Massachusetts Institute of Te chnology, Cambridge, USA, and her Ph.D. degree from Yale University, USA. At present, she is a post-doctoral fellow with Steve West. West Links archaeal Hjc http://www.ncbi.nlm.nih.gov/entrez/viewer.fc gi?db=protein&val=14278692 archaeal Hje http://www.ncbi.nlm.nih.gov/entrez/viewer.fc gi?db=protein&val=13814368 RecA http://www.ncbi.nlm.nih.gov/entrez/query.fc gi?dopt=GenPept&cmd=Retrieve&db=pro- tein&list_uids=26249094 RusA http://www.ncbi.nlm.nih.gov/entrez/viewer.fc gi?db=protein&val=26247426 ruvA http://www.ncbi.nlm.nih.gov/entrez/query.fc gi?db=gene&cmd=Retrieve&dopt=Graphics &list_uids=1036829 ruvB http://www.ncbi.nlm.nih.gov/entrez/query.fc gi?db=gene&cmd=Retrieve&dopt=Graphics &list_uids=1036828 RuvC http://www.ncbi.nlm.nih.gov/entrez/viewer.fc gi?db=protein&val=26248129 T4 endonuclease VII http://www.ncbi.nlm.nih.gov/entrez/viewer.fc gi?db=protein&val=119331 Saccharomyces genome database: http://db.yeastgenome.org/ Cce1 http://db.yeastgenome.org/cgi- "
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