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Reviews Bacterial artificial chromosomes and herpesvirus genomics 20 Eggleston, A.K. and West, S.C. (1996) Exchanging partners: recombination in E. coli. Trends Genet. 12, 20–26 21 O’Connor, M. et al. (1989) Construction of large DNA segments in Escherichia coli. Science 244, 1307–1312 22 Kempkes, B. et al. (1995) Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein–Barr virus DNA. J. Virol. 69, 231–238 23 Yang, X.W. et al. (1997) Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15, 859–865 24 Brune, W. et al. (1999) Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis. Nat. Biotechnol. 17, 360–364 25 Zhang, Y. et al. (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 26 Muyrers, J.P. et al. (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555–1557 27 Narayanan, K. et al. (1999) Efficient and precise engineering of a 200 kb beta-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. Gene Ther. 6, 442–447 28 Berg, C.M. et al. (1989) Transposable elements and the genetic engineering of bacteria. In Mobile DNA (Berg, D.E. and Howe, M., eds), pp. 879–925, American Society for Microbiology, Washington, DC, USA 29 Chatterjee, P.K. and Coren, J.S. (1997) Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site. Nucleic Acids Res. 25, 2205–2212 30 Chatterjee, P.K. et al. (1999) Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms. Proc. Natl. Acad. Sci. U. S. A. 96, 13276–13281 31 Luckow, V.A. et al. (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposonmediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566–4579 32 Fink, D.J. and Glorioso, J.C. (1997) Engineering herpes simplex virus vectors for gene transfer to neurons. Nat. Med. 3, 357–359 33 Sclimenti, C.R. and Calos, M.P. (1998) Epstein–Barr virus vectors for gene expression and transfer. Curr. Opin. Biotechnol. 9, 476–479 34 Messerle, M. et al. Cytomegalovirus BACs – a new herpesvirus vector approach. Adv. Virus Res. (in press) 35 Banerjee, S. et al. (1995) Therapeutic gene delivery in human B-lymphoblastoid cells by engineered non-transforming infectious Epstein–Barr virus. Nat. Med. 1, 1303–1308 36 Sun, T.Q. et al. (1996) Human artificial episomal chromosomes for cloning large DNA fragments in human cells. Nat. Genet. 8, 33–41 37 Sun, T.Q. et al. (1996) Engineering a mini-herpesvirus as a general strategy to transduce up to 180 kb of functional selfreplicating human mini-chromosomes. Gene Ther. 3, 1081–1088 38 Wang, S. and Vos, J.M. (1996) A hybrid herpesvirus infectious vector based on Epstein–Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo. J. Virol. 70, 8422–8430 39 Suter, M. et al. (1999) BAC-VAC, a novel generation of (DNA) vaccines: a bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1. Proc. Natl. Acad. Sci. U. S. A. 96, 12697–12702 Partners and pathways repairing a double-strand break Double-strand chromosome breaks can arise in a number of ways, by ionizing radiation, by spontaneous chromosome breaks during DNA replication, or by the programmed action of endonucleases, such as in meiosis. Broken chromosomes can be repaired either by one of several homologous recombination mechanisms, or by a number of nonhomologous repair processes. Many of these pathways compete actively for the repair of a double-strand break. Which of these repair pathways is used appears to be regulated developmentally, genetically and during the cell cycle. roken chromosomes pose a serious threat to cell survival. The presence of an unrepaired double-strand break (DSB) will trigger the DNA-damage response systems of a cell to arrest its progression through the cell cycle and, sometimes, to cause apoptotic cell death. But even if a cell with an unrepaired DSB continues to divide, the broken chromosome fragments will mis-segregate and be degraded, producing aneuploidy. In response to this threat, cells have elaborated an impressive arsenal of DNA-repair pathways. There are two general types of repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). These two processes are in competition with each other and one focus of this review is to examine the way that this competition is regulated. But the cell’s options are far more complex than simply electing to employ HR or NHEJ. There are several types of homologous repair: gene conversion, break-induced replication and single-strand annealing (reviewed in Ref. 1). Similarly, there are also several alternative end-joining mechanisms2,3. Moreover, even once a process such as gene conversion is initiated, there are additional genetically regulated decisions in choosing among alternative homologous templates to carry out repair. How does the cell decide to use a template on a sister B 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(00)02022-9 chromatid, on a homologous chromosome or at an ectopic site? Moreover, how are these choices tied to the cell’s DNA damage-sensing checkpoints? This review surveys the impressive recent progress in delineating the different mechanisms of homologous and nonhomologous repair and the way in which they all compete in repairing DSBs. The emphasis will be on what has been learned in the best-studied organism, Saccharomyces cerevisiae, but I also discuss studies in other model eukaryotic systems and in mammalian cells. Homologous recombination mechanisms The three major types of HR all begin in the same way, as the ends of the DSB are resected by 59 to 39 exonucleases or by a helicase coupled to an endonuclease, to produce long, 39-ended single-stranded DNA tails (Fig. 1). James E. Haber [email protected] Single-strand annealing In the simplest process (Fig. 1d), resection exposes complementary regions of homologous sequences originally flanking the DSB, creating a deletion by single-strand annealing (SSA). SSA will occur with as little as 30 bp of homology, although it is much more efficient with 200–400 bp (Ref. 44). TIG June 2000, volume 16, No. 6 Rosentiel Basic Medical Sciences Research Center, MS 029 Brandeis University, 415 South Street, Waltham, MA 02454-9110, USA. 259 Reviews DNA double-stranded break repair FIGURE 1. Alternative outcomes of the homologous recombinational repair of a DSB 5′ to 3′ resection Strand invasion New DNA synthesis (a) Replication fork capture (c) Continued DNA synthesis Holliday junction resolution Break-induced replication No crossing-over (b) Crossing-over Conservative inheritance of DNA strands (d) Continued 5′ to 3′ resection Strand annealing No crossing-over Deletion trends in Genetics After a double-strand break (DSB) is created, the ends are resected and one of the 39 single-stranded ends can invade an intact template. Strand invasion requires the participation of the Rad51p strand exchange protein and a number of associated proteins, including Rad52p, Rad54p and Tid1p, discussed in this review. During meiosis, these recombination proteins are joined by a second strand-exchange protein, Dmc1p. Strand invasion is believed to establish a modified replication fork, in which both leading and lagging-strand DNA synthesis occurs43. As new DNA synthesis proceeds, branch migration displaces the two newly synthesized strands. (a) If the replication fork encounters the other end of the DSB, an intermediate containing two Holliday junctions can be formed, allowing gene conversions to be resolved both with and without crossing-over. (b) If the strands are completely displaced or if the leading strand pairs with the second end of the DSB, a simple synthesisdependent strand annealing (SDSA) will occur, producing gene conversions without crossing-over. (c) If the second end of the DSB fails to engage, replication can proceed all the way to the end of the chromosome (or until it encounters a converging replication fork). This process is known as break-induced replication (BIR). (d) If resection proceeds far enough to expose complementary strands of homologous sequences flanking a DSB (shown as black boxes), repair can occur by single-strand annealing (SSA), leading to a deletion of all intervening sequences. Gene conversion SSA is in competition with gene conversion (Fig. 1a), during which the two resected ends of the DSB invade and copy sequences from a homologous template located on a sister chromatid, a homologous chromosome, or at an ectopic location. The molecular mechanism by which gene conversion occurs is not precisely known; recent evidence has prompted several suggested revisions of the DSB gaprepair mechanism proposed by Szostak et al.4 (these vari260 TIG June 2000, volume 16, No. 6 ations are reviewed in Ref. 1). The mechanism shown in Fig. 1 is termed synthesis-dependent strand annealing (SDSA). It would seem that gene conversion is the most conservative and non-mutagenic process of DNA repair, but surprisingly, in S. cerevisiae, SSA out-competes gene conversion in mitotic cells even when the donor and recipient are intrachromosomal5,6, apparently because the resection of DNA ends is not restrained. It is possible that one reason Reviews DNA double-stranded break repair why SSA is less evident in meiotic cells is that 59 to 39 resection is down-regulated or impaired. Break-induced replication Under certain circumstances only one end of a DSB might be able to engage in homologous recombination, for example in haploid or hemizygous chromosomes of G1 diploids, where there is no homologous chromosome. Sequences near the centromere-proximal side of the DSB might be able to find homologous sequences elsewhere in the genome and create a nonreciprocal translocation by a process known as recombination-dependent DNA replication or break-induced replication (BIR; Fig. 1c)7. This same circumstance appears to occur when chromosomes lack telomerase, the enzyme that normally maintains the short, repeated sequences at telomeres that protect ends from fusions and other types of recombination8. Even when there is homology on both sides of a DSB, BIR (using only one of the ends to initiate recombination) appears to be in competition with gene conversion9. This mechanism also accounts for extensive DNA replication found in many gene targeting events10. Nonhomologous recombination DSB ends can be repaired by several nonhomologous repair mechanisms in which the DNA ends are joined with little or no base-pairing at the junction2,3. End joining in yeast and in mammals requires the same core set of proteins: the DNA end-binding proteins Ku70p and Ku80p, as well as DNA ligase IV and its associated Xrcc4 protein. Vertebrate cells also require DNA-PKcs, for which no homologue has been demonstrated in fungi. In budding yeast there is also a requirement for Rad50p, Mre11p and Xrs2p, three proteins that have endo- and exonuclease activity (reviewed in Ref. 11) although there is also a Mre11-independent pathway of chromosome rearrangements12; but in fission yeast13 and vertebrate cells14 the absence of Mre11p has little effect. When the ends of a DSB are created by a nuclease that leaves 4-bp overhanging, complementary ends, yeast cells readily promote their religation by NHEJ, effectively competing with homologous recombinational alternatives15 (Fig. 2). However, when the ends of DNA are not complementary, NHEJ in budding yeast is much less efficient, succeeding in only ~2 in 1000 cells. This is one of the distinctive differences between yeast and mammalian cells, which can efficiently join ends of all types by NHEJ (Ref. 3). The basis of this important difference is not known, but it seems to account in part for why mammalian gene targeting is much more likely to result in mistargeted insertions. There is also likely to be a Ku- and Rad52-independent end-joining process as well, as some deletions are recovered after DSBs are created in yku70D and rad52D cells. No genes have yet been identified to define this pathway. Competition between homologous and nonhomologous recombination: inferences from gene targeting and other types of repair Based on gene-targeting experiments, it appears that mammalian cells are much less adept in accomplishing accurate integration of homologous sequences than is budding yeast. However, several recent observations argue that S. cerevisiae is much more capable of nonhomologous recombination than we had suspected, and that vertebrate FIGURE 2. Nonhomologous end-joining Religation AACA TTGT AACA TTGT A T AACAACA TTGTTGT AACACA TTGTGT Misalignment Fill-in Deletion trends in Genetics Nonhomologous end-joining requires the participation of the DNA end-binding proteins Ku70p and Ku80p and a specialized DNA ligase IV with its associated Xrcc4 protein. In mammalian cells, NHEJ also requires the Ku-associated DNAPKCS, but no homologue of this protein has been found in yeast. In Saccharomyces cerevisiae, most NHEJ processes also require the Mre11p–Rad50p–Xrs2p complex, but their homologues in Schizosaccharomyces pombe or in vertebrate cells do not seem to have a key role. An example is shown in the precise ligation and imprecise end-joining of DNA cleaved by the S. cerevisiae site-specific HO endonuclease. S. cerevisiae efficiently joins sequences with complementary 59 or 39 overhanging ends, but only inefficiently joins unmatched DNA ends. The junctions of deletions and insertions usually have one or a few bp of microhomology. New DNA synthesis is shown in grey. In budding yeast, the insertion pathway is seen only in S and/or G2 cells, whereas the much less efficient deletion pathway (which also does not seem to require Mre11p–Rad50p–Xrs2p) is found at all stages of the cell cycle. Mammalian cells join any DNA ends together efficiently, often creating junctions with 1–5 bp of base pairing (with the removal of the un-paired flaps of the single-stranded ends). cells are far more accomplished in carrying out homologous recombination. In mammalian cells, approximately 30–50% of breaks created by the site-specific I-SceI endonuclease can be repaired by homologous recombination, with the rest repaired by NHEJ (Ref. 16). Similarly, in yeast, when a DSB is made on a chromosome with HO endonuclease, about 30% of the cells simply religate the DSB while the remaining cells engage in a homologous gene conversion to repair the break15. Very little is known in general about the way cells elect homologous or nonhomologous recombination. Such knowledge might provide a way to improve mammalian gene targeting. Two DNA end-binding complexes, Ku70pKu80p and a multimer of Rad52p, might act as ‘gatekeepers’ to control access to NHEJ or HR, respectively17. A remarkable discovery that supports this idea is that the level of the Ku proteins is dramatically lower in mouse cells that are undergoing meiosis18. It is presumed that mouse, like yeast, initiates recombination with hundreds of DSBs created by the Spo11 protein, and it would be highly disadvantageous if these DSBs were repaired by imprecise NHEJ. Yeast has provided additional insights into choice between HR and NHEJ. The largest effects are associated with expression of yeast’s mating-type locus. Haploid cells express either MATa or MATa alleles, whereas TIG June 2000, volume 16, No. 6 261 Reviews DNA double-stranded break repair FIGURE 3. Alternative homologous partners with which homologous recombination can occur Allelic Sister Ectopic trends in Genetics In meiosis, sister-chromatid recombination is apparently suppressed by the presence of protein structures (axial elements of the synaptonemal complex) and by the action of a meiosis-specific recombination complex (Dmc1p–Tid1p) that direct recombination to non-sister homologous sequences. Partner choice is also changed by mutations in genes that are involved in the DNA-damage checkpoint pathway, leading to an increased use of ectopic and sister sequences over allelic sites. diploids are normally MATa/MATa. MATa/MATa diploids are more radiation-resistant and are significantly more adept in repairing an HO-induced DSB than diploids expressing only one MAT allele15,19. NHEJ is also under mating-type control15,20; both haploid and diploid cells expressing one MAT allele are much more proficient in NHEJ than cells expressing both MATa and MATa. These differences make sense: haploid strains in G1 can only repair DSBs by NHEJ, whereas diploids always have a homologous partner with which to repair a DSB and hence might be expected to down-regulate NHEJ. The search is on for the genes controlled by mating type that carry out this regulation. Another key question is whether a cell irrevocably commits the DNA ends towards one repair pathway. For example, when a haploid G1 cell suffers a DSB, the ends do not have homologous sequences with which to repair the damage, and hence repair can only occur by NHEJ. But as DNA ends are resected, flanking homologous sequences can become exposed, allowing SSA to occur, sometimes as long as several hours later5. By that time, has the cell given up trying to complete NHEJ, or are all solutions being tried out at the same time? Choosing a template for gene conversion in mitotic cells: a sister chromatid or a homologue? As noted above, gene conversion, BIR and SSA all compete with each other; but even if we consider only the process of gene conversion, the cell still confronts important choices. In a diploid cell, after DNA replication, a DSB can be repaired either from a sister chromatid or from a homologous sequence located either in an allelic or ectopic location (Fig. 3). There is increasing evidence that 262 TIG June 2000, volume 16, No. 6 the choice between inter-homologue recombination and sister-chromatid exchange is strongly regulated. The selection of a partner for HR is particularly crucial during meiosis. Recombination is initiated by DSBs, but if repair occurs between sister chromatids, there will be no inter-homologue crossing-over, which is essential for proper chromosome segregation. Thus, meiotic cells have evolved mechanisms to ensure that most recombination events will occur between non-sister chromatids21, apparently the opposite of what occurs in mitotic cells, where most DSBs are likely to occur during replication and the most conservative choice of a partner is the sister chromatid22. There seem to be two major components involved in this process, the induction of a meiosis-specific recombination protein and the meiosis-specific formation of proteinaceous axial elements between sister chromatids (reviewed in Ref. 23). The new recombination protein, Dmc1p, is found in all meiotic cells, from yeast to humans, in addition to its homologue, the Rad51p strand-exchange protein, which is also present in mitotic cells. Two-dimensional gel analysis shows that a dmc1 mutant strain fails to produce the joint molecule (JM) intermediates either formed between homologous sister- or between non-sister chromatids, whereas the absence of Rad51p particularly reduces interhomologue exchanges21. Interestingly, the defects caused by dmc1 are partially suppressed by a red1 deletion that prevents normal axial element formation21. red1 dmc1 double mutants can form JMs with normal kinetics, but they are almost exclusively derived from sister-chromatid exchanges, apparently by allowing Rad51p to work on substrates from which it is somehow normally excluded. Recently, Thompson et al.24 showed that red1, mek1 and hop1 mutations that prevent normal axial element formation all increase sister-chromatid recombination. Another pair of proteins involved in this decision are Rad54p and its homologue Tid1p (Rdh54p). Tid1p interacts strongly with Dmc1p and less so with Rad51p; conversely, Rad54p strongly interacts with Rad51p but apparently not with Dmc1p (Ref. 25). These proteins also play important roles in mitotic cells. Rad54p seems to be critical in sister-chromatid repair, whereas the absence of Tid1p has no effect26. Conversely, the absence of Tid1p reduces interhomologue mitotic recombination but does not affect intrachromosomal or inter-sister recombination27,28. Taken together, these data argue that normally Dmc1p and Tid1p direct DSB repair towards non-sisters, whereas Red1p prevents sister-chromatid exchange that would be mediated by Rad51p and Rad54p. The absence both of Red1p and of Dmc1p permits DSB repair from sisters (presumably carried out by Rad51p and Rad54p) and improves spore viability. Allelic and ectopic partners in mitosis In some instances, the cell must make a further decision, whether to recombine with an allelic partner or with homologous sequences located at ectopic sites. Some sequences are found in multiple copies, dispersed throughout the genome and crossovers between such non-allelic sites can lead to chromosome rearrangements. One way to restrict ectopic interactions would be to make the lengths of homology needed for a successful encounter greater than the size of the dispersed elements. This might be the case for small sequences such as Alu sequences in humans or delta sequences in yeast, but recombination between larger transposable elements do not appear to be limited Reviews DNA double-stranded break repair by this constraint29,30. Recently Inbar and Kupiec31 presented evidence that yeast will forgo recombining with a homologous partner that shares a few hundred bp of homology located at the very ends of a DSB in favor of recombining with a second, competing locus that shares greater homology, but further away from the DSB ends. This could explain how recombination between short, dispersed sequences might be avoided and why allelic events (where the homology stretches out to the ends of the chromosome) would be favored over ectopic ones. Burgess and colleagues32,33 have used fluorescent in situ hybridization and site-specific recombination to examine allelic and ectopic recombination in mitotic yeast cells. Their results reveal several layers of interaction. First, homologous chromosomes show frequent, but transient pairing at most times in the cell cycle. Second, because centromeres cluster, two sequences equally distant from their respective centromeres are more likely to recombine than those at different distances. Finally, for three of the four sites analysed there was a fourfold advantage to being in allelic, rather than ectopic, positions. Studies of HR initiated by endogenous nucleases have not found much constraint on the ability of broken chromosomes to search the entire genome for homologous partners. When cells were given a choice between repairing simultaneous DSBs on two different chromosomes, either by a pair of intrachromosomal deletions or by two interchromosomal translocations, the two outcomes occurred at equal frequencies34. Even though mammalian chromosomes are much larger and homology searches must be more difficult, quite similar results have been found in mouse cells. Expressing the site-specific I-SceI endonuclease to create DSBs in mouse cells, Jasin and her colleagues have shown that ectopic recombination is, at best , eightfold less frequent than allelic events35. It is possible that in these cases, the presence of DSB damage induces a checkpoint gene-mediated response that causes the disruption of the normal localization of sequences and promotes a search of the entire genome for partners. In meiosis, allelic recombination is about five times more frequent than ectopic events36. Moreover, when the ectopic events are between different locations on two homologous chromosomes, the advantage of allelic over ectopic recombination increases as the two sequences are moved further apart, leading to the suggestion that homologous chromosomes are already loosely aligned before the initiation of recombination that will cause their true synapsis. Homologues are associated even in cells that cannot recombine37. The advantage of allelic over ectopic events can also be explained if the recombination event under study occurs later than another recombination event on the same chromosome that would bring the rest of the sequences closer together. Efficient meiotic ectopic recombination is not simply a feature of fungi, whose small chromosomes might not be as organized as those in mammals. For example, in meiosis of male mice, Schimenti and his colleagues30 have shown that ectopic recombination is surprisingly robust. One set of DNA sequences seems to be particularly restricted in ectopic interactions, both in meiosis and mitosis. Sequences near telomeres can recombine freely with homologous sequences at other chromosome ends38,39, but these subtelomeric regions are somehow insulated from recombining with similar homologous regions that are located away from telomeres36. It is important to know if ectopic meiotic recombination is carried out by the same machinery that performs allelic events. Recent results from Grushcow et al.40 argue that meiotic, ectopic and allelic recombination might, indeed, use somewhat different recombination enzymes. A dmc1 mutant enhances the frequency with which ectopic sites are used, suggesting that Dmc1p (and Tid1p, perhaps) are especially focused on interallelic events. A similar finding has been made by Thompson and Stahl24, who observed that a dmc1 mutation (as well as mutations in a number of other genes) increases unequal sister-chromatid recombination at the expense of normal allelic recombination. Effect of checkpoint genes on DSB repair in mitosis and meiosis One critical function of the DNA-damage checkpoint response is to give cells enough time to complete recombination. Consequently, deletion of S. cerevisiae checkpoint genes MEC1, RAD17 or RAD24 produces a loss of spore viability. The mutations also suppress the arrest of cells at the pachytene stage of meiosis in dmc1 recombinationdefective mutants41. Recently, Grushcow et al.40 have discovered that checkpoint genes also regulate both the choice of homologous partners and the frequency of crossing-over. There is an increase both in inter- and intra-chromosomal ectopic recombination that is independent of the increase seen with dmc1 mutants, discussed above. Moreover the proportion of ectopic gene convertants undergoing crossovers increased from 65% to 90%. It would be fascinating to know if checkpoint mutations also affect the low level of crossing-over in msh4 or zip1 mutants, as it is not clear that ectopic recombination between short sequences will involve the synaptonemal complex. Another recent study by Thompson and Stahl24 found that mutations in some checkpoint genes also altered the choice between an allelic partner or a sister chromatid during yeast meiosis, including RAD17, RAD24 and MEC3. So it looks like checkpoint genes are important to ensure inter-homologue allelic recombination in competition both with ectopic and with (unequal) sister partners. Checkpoint defects also affect ectopic recombination in mitotic cells. A rad9 deletion increased translocations five- to tenfold, but this was suppressed if cells were arrested in G2/M before induction42, suggesting that cells that are capable of G2/M arrest can repair lesions by sister-chromatid exchange, so that the possibility of translocations is reduced. But Fasullo et al. also note that translocations might arise from repair of the damaged chromosome following mitosis42; indeed, half of selected events carry nonreciprocal translocations, in addition to intact copies of the two chromosomes involved in these rearrangements. This can be explained if a broken chromosome is segregated during mitosis, in the absence of checkpoint arrest, and can then initiate BIR to create a nonreciprocal translocation. Conclusions We are beginning to see how the cell can achieve a balance between competing mechanisms to repair a chromosomal DSB. Insights from these studies may help provide new approaches to ensuring more accurate gene targeting in mammalian cealls, as well as in other organisms, where this has proven difficult. We are still left with many questions about how homology searches are carried out and about the precise mechanisms of any of these pathways, but the rate of information is growing at an ever more rapid pace. TIG June 2000, volume 16, No. 6 263 Reviews DNA double-stranded break repair Acknowledgements The author wishes to thank S. Lovett and members of the Haber lab for generously providing comments. Work from References 1 Pâques, F. and Haber, J.E. (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 2 Moore, J.K. and Haber, J.E. 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(1999) Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases. Cell 96, 415–424 Reference added in proof 44 Sugawara, N. et al. DNA length dependence of the singlestrand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair. Mol. Cell. Biol. (in press) http://tto.trends.com Editor: Adrian Bird Institute for Cell and Molecular Biology at the University of Edinburgh, UK New peer-reviewed articles published recently in Elsevier Trends Journals Technical Tips Online include: • Galassi, L. (2000) A gas-proof temperature-controlled microscope chamber that accepts oil or glycerolimmersion objectives for cytofluorimetry (http://tto.trends.com) t01920 • Wiener, F. and Mai, S. (2000) Banding of mouse chromosomes after spectral karyotyping (http://tto.trends.com) t01884 • Penalva, L.O.F. and Valcárcel, J. (2000) An optimized procedure to mutagenize long (>10kb) plasmids by PCR (http://tto.trends.com) t01918 • Bayani, J. et al. (2000) Sequential G-banding, SKY and FISH provide a refined identification of translocation breakpoints and complex chromosomal rearrangements (http://tto.trends.com) t01864 • Tanaka, K.J. and Nishikata, T. (1999) A non-radioactive gel shift protocol enables recovery of RNA-binding proteins (http://tto.trends.com) t01794 264 TIG June 2000, volume 16, No. 6