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The replication fork's five degrees of freedom, their failure and genome rearrangements
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The replication fork’s five degrees of freedom, their failure and genome rearrangements T Weinert, S Kaochar, H Jones, A Paek and AJ Clark Genome rearrangements are important in pathology and evolution. The thesis of this review is that the genome is in peril when replication forks stall, and stalled forks are normally rescued by error-free mechanisms. Failure of error-free mechanisms results in large-scale chromosome changes called gross chromosomal rearrangements, GCRs, by the aficionados. In this review we discuss five error-free mechanisms a replication fork may use to overcome blockage, mechanisms that are still poorly understood. We then speculate on how genome rearrangements may occur when such mechanisms fail. Replication fork recovery failure may be an important feature of the oncogenic process. (Feedback to the authors on topics discussed herein is welcome.) Address Department of Molecular & Cell Biology, University of Arizona, Tucson, AZ 85721, USA Corresponding author: Weinert, T ([email protected])
Current Opinion in Cell Biology 2009, 21:778–784 This review comes from a themed issue on Cell division, growth and death Edited by Angelika Amon and Mike Tyers Available online 11th November 2009 0955-0674/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.ceb.2009.10.004
The many forms of genome rearrangements Genome instability is a broad term capturing any form of genetic change, from base pair changes, to small insertions, deletions or inversions, to larger scale changes such as translocations, segmental duplications, and whole chromosome loss or duplication [1]. Each type of rearrangement is relevant for pathology and evolution [2,3,4]. Here we focus on mechanisms by which large-scale rearrangements may arise, drawing largely from studies in S. cerevisiae.
General mechanisms underlying genome rearrangement Genome rearrangements may in principle have a simple etiology; chromosomes may break, and two breaks may be joined when error-free repair fails. How chromosomes break probably involves DNA replication forks, and therein lies considerable uncertainty as our understanding of replication fork biology is far from complete. We Current Opinion in Cell Biology 2009, 21:778–784
imagine the following sequence of events may lead to genome rearrangements: when cells replicate their DNA, replication forks stall. Stalling may be stochastic, or may be induced by unusual DNA sequences (secondary structures; [5]), by sites occupied by protein–DNA complexes [6,7], or by DNA damage [8,9]. Replication fork stalling is probably unavoidable because of the existence of protein–DNA complexes involved in gene expression (e.g., RNA polymerases; [6,7] or in chromosome duplication (e.g., centromeres, origins of replication; [7]). When forks stall, regulatory and repair mechanisms rescue the stalled fork by error-free mechanisms outlined below. When error-free fork recovery fails, the strands in the stalled fork may anneal to other sequences in the genome, thereby leading to genome rearrangements. How this ‘ectopic’ annealing of strands occurs is a matter of speculation; annealing may occur either directly from a stalled fork (something we call faulty template switching) or following a double-strand break (DSB). We discuss the forks five error-free mechanisms of recovery, and speculate on how genome rearrangements arise when these fail.
The five degrees of freedom in replication fork recovery Stalled replication forks recover by mechanisms that remain largely speculative to date, largely because of the lack of defined fork-stalling in vivo or in vitro systems. In vitro systems in bacteria hold much promise (for example, see [11,12]), though it is unclear to what extent the generality of bacterial fork behavior will apply to eukaryotic fork behavior. In eukaryotes, even such simple-sounding terms as ‘stalled fork’ and ‘lesion that stalls a fork’ do not yet have concrete molecular descriptions. Nonetheless, molecular studies of fork behavior provide early and important clues on links between replication forks and genome stability. The stalled fork’s five degrees of freedom are shown in the models in Figure 1 that derives from studies in S. cerevisae, leave out many interesting possibilities, and suggest conclusions that may extend to all eukaryotic cells. (Excellent reviews of replication more detailed than this one abound; [10,11,12].) First, stalled forks may recover without fork remodeling; the offending damage is repaired or blocking protein removed (#1). Many regulators (e.g., Mec1 and Rad53) may enforce this first option, though how they may do so is unclear [10]). The next four options have been termed ‘fork collapse’, generally referring to events that ensue after DNA www.sciencedirect.com
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Figure 1
A replication fork encounters a lesion (in this case DNA damage, triangle), and uses one of 5 options to overcome it. The fork may simple resume replication (#1); may undergo sister-strand annealing and replication to form a hemicatenane that is resolved by a host of proteins (#2); may undergo sister-strand annealing to form a regressed fork (‘chicken foot’) that allows lesion repair (#3); may undergo sister-strand annealing to form a regressed fork that allows replication of the annealed strands, followed by regression or re-invasion, forming a Holliday structure that needs to be resolved (#4); for undergoes strand breakage to form a DSB. DSB may also form from the regressed fork.
polymerases dissociate from the fork [13]). The second, third, and fourth options posit that a stalled fork undergoes a sister-strand annealing reaction, called a template switch or copy choice switch. The idea is that template switching allows either efficient repair/removal, or replication past a lesion. One form of template switching (#2) involves a ‘behind the fork’ event in which sister-strand anneal, forming a hemicatenane (a shorter one of which may form at the initiation of replication; [14,15]). A hemicatenane is a topological intertwining of one of two strands of two DNA duplexes (see blue and green strands of option #2). The hemicatenane is then resolved, most probably by the action of the helicase Sgs1 and topoisomerase Top3. Template switching might also occur at the front of the fork (#3 and #4). In these events, forks first regress to pair newly replicated sister strands as shown, forming a so-called ‘chicken foot’ structure. This regressed structure is then resolved either by fork reversal without replication (option #3), or by more complicated mechanisms (option #4) that involve replication and/or www.sciencedirect.com
strand invasion, and possibly resolution of a Holliday structure (see [12,16]). Finally, a fifth fork option involves a double-strand break, formed by cleavage of the exposed single-stranded DNA at the stalled fork or by cleavage of the regressed fork, followed by strand invasion (not shown in Figure 1). Both fork-mediated and DSB-mediated mechanisms may require homologous recombination proteins to facilitate correct pairing [14,16,51]. Genome rearrangements may arise from a failure of correct annealing during the template switch or DSB options.
Some evidence for the forks five degrees of freedom There is reasonable but far from incontrovertible evidence in support of these five degrees of freedom. The research community is presently identifying specific DNA structures and specific proteins associated with those structures. Evidence on DNA structures (formed Current Opinion in Cell Biology 2009, 21:778–784
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in vivo) comes from two-dimensional agarose gels (2-D gels, [17]) or from electron microscopy (EM; see [8,18]). Two-dimensional gels provide information about where forks stall, and in combination with biochemical techniques can identify potential abnormal structures. For example, key structures identified in 2D gels are so-called ‘X-structures’. One X-structure is probably a Holliday structure, because it is resolvable by enzymes known to resolve bonafide Holliday structures (RuvC and T4 endonuclease VII; [14,16]). A second Xstructure has been identified that is not a Holliday structure, because it cannot be resolved by RuvC, and biochemical studies are consistent with it being a hemicatenane ([14]; see figure option #2). Description of specific fork structures remains a major technical challenge. EM studies can provide less ambiguous descriptions of DNA structures than 2-D gels, though EM requires the technical challenge of purifying DNA structures from cells [8,18]. Given the ambiguities of DNA structures, it is not surprising that the association of specific proteins with specific structures also remains incomplete [10,13,19]. Our current understanding of the fate of stalled replication forks in eukaryotic and prokaryotic cells derives largely from in vivo studies (in vitro studies of prokaryotic replication forks prove informative as well; [11,12]). The in vivo studies involve blocks to DNA replication because of sequence-specific fork-stalling protein complexes or DNA damage. Specific protein-induced fork blocks are referred to as replication fork barriers, or RFBs. These studies provide limited evidence bearing on the options in this model. Two studies of RFBs provide evidence that stalled forks may remain intact and simply proceed (option #1). First, in bacteria a repressor-bound complex [20] can block fork progression, and stalled forks do not appear to break or form unusual structures. Fork recovery resumes promptly upon repressor removal (by adding inducer), and in a recombination protein (RecA) independent fashion. (Prolonged fork arrest was also accomplished in bacteria with an ectopic Ter-site placed away from its normal site of replication termination; [21]. Ter-sites contain specific proteins and DNA sequences that coordinate replication termination.) A second study that supports option 1 comes from studies in budding yeast using the rDNA RFB site system. A protein called Fob1 normally binds to specific RFB sites in the rDNA locus, and blocks replication that would oppose transcription of the rDNA genes. The Fob1-RFB system thus provides for co-directional DNA replication and transcription of the rDNA genes, minimizing head-on DNA polymerase and RNA polymerase collisions. When placed outside the rDNA locus, the RFB-Fob1 system also stalls forks and allows the monitoring of fork behavior Current Opinion in Cell Biology 2009, 21:778–784
[22]. Forks stall briefly (for 30 min) at an ectopic RFB site, the Fob1-stalled forks do not form unusual DNA structures shown in Figure 1, and do not require recombination (Rad52) or regulatory checkpoint proteins (Mec1, Rad53) to resume replication. A conceptually similar fork-stalling system with a very different outcome has been developed in fission yeast [23]. In this system, the Rtf1 protein binds to a single RTS1 DNA sequence, and that protein–DNA complex blocks DNA replication. The Rtf1-RTS1 system normally provides for unidirectional replication through the mating type locus. Lambert et al. manipulated this system to achieve more efficient fork stalling by inserting a pair of RTS1 sequences in inverted orientation such that expression of the Rtf1 protein causes stalling of two converging replication forks. In this setting, forks stall very efficiently (much more so that the Fob1-RFB system in budding yeast). Viability is dependent on homologous recombination, and X-structures are generated suggested that a pairing reaction is involved. This implies that fork recovery may occur via either hemicatenanes or regressed forks or a recombination intermediate. Genome rearrangements occur when forks stall, and it is unclear if failure of a specific option in Figure 1 is involved. One additional site-specific fork-stalling system deserves special mention as it provides evidence for structures that are necessary for options 3 and 4. In bacteriophage T4, Long and Kreuzer describe an elegant system that involves what they call the ‘origin fork’; after T4 infection, bidirection DNA replication occurs from a specific origin from which the two forks emerge at different times; one of the two forks replicates immediately while the other ‘origin fork’ delays and replicates later (an apparent design feature of T4 replication in place for reasons unknown to us; [26]). Long and Kreuzer study the delayed origin fork, can follow its behavior by 2-D gels, and found that certain phage proteins are required for its normal firing. They identified by 2-D gels a specific structure they propose is a regressed fork that accumulates in mutants in specific phage replication proteins. These studies as well as in vitro studies of bacteria DNA replication hold promise for increasing our understanding of eukaryotic stalled forks [11]. Quite a number of studies evaluate how DNA damage induces both fork stalling and genome instability, and those studies suggest options involving template switching (options 3 and 4). Formulation of models is very much a work in progress [18,26,27,28,29,30]. To site but a few observations, Rad5 and Srs2 may be needed to form regressed forks [31,32], Rad53 may both prevent regression and regulate Exo1’s degradation of regressed forks [29,30], and a number of proteins involved in postreplication repair (e.g., Rad18) may be involved in formation and resolution of the hemicatenanes [14,27]. www.sciencedirect.com
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Finally, the extent to which stalled replication forks break (option #5) is unknown. Stalled forks in yeast and bacteria do appear to break (for example, [7,38,39]), though to what extent a DSB pathway is used compared to the other no-DSB options is unclear. In sum, the evidence addressing the forks five degrees of freedom is clearly incomplete, and the model seems based largely on inference. A major technical problem is that neither RFB yeast systems that achieve stalled forms require checkpoint proteins (Mec1 and Rad53 in S. cerevisiae) for their stability. Development of an RFB system that requires Mec1 and Rad53 for stability, as do DNA damage-induced forks [24,25], would clearly be of value. Other features implicit in this model, DNA polymerase coupling and template switching, are discussed next, after which we conclude with discussion of genome instability.
DNA polymerase uncoupling The coupling between leading and lagging strand DNA polymerases is implicit in some aspects of the models shown; for example, damage that stalls the DNA polymerase on one strand only forms a stalled fork if the other DNA polymerase remains coupled to the stalled one. It seems reasonable to speculate that replication polymerases retain some coupling to minimize the amount of single-stranded DNA were leading or lagging DNA polymerases to stall. There is some evidence, for example, that some replication proteins do couple some proteins to a stalled fork (Mrc1/Tof1 in yeast, DnaB helicase in bacteria; [19,33]). This coupling seems to odds, however, with elegant experiments in bacteria and SV40 that demonstrate that leading and lagging strand synthesis, which is normally coupled, can uncouple when the leading strand or lagging strand is blocked by damage [8,9]. We do not know the answer to this conondrum; it may be as simple as suggesting that experiments that use DNA damage generate lesions in close proximity on both strands.
pairing and replication, the replication complex appears to dissociate from the first chromosome and resume replication after it pairs with allelic sequences on the second homologous chromosome. In a separate study, Schmidt et al. provided evidence for what we believe may be a similar template-switch reaction, this time involving non-allelic sequences and forming genome rearrangements [36]. The simplest model of such template-switch events envisions the 30 end of the polymer that DNA polymerase is extending dissociating from one sequence and reannealing to another, presumably in a single cell cycle. Molecular details that underlie such a template switch are unknown, and are difficult to study given the rarity of events. One feature that can be analyzed is the role of sequence homology in template switching. Several studies suggest to us that extensive sequence homology may not be required. For example, a human genome disorder that arises from multiple complex rearrangements was proposed to involve switching between short sequence homologies (<10 bp; [3]). In yeast Rattray et al. provide evidence that single-strand annealing and polymerase extension in vivo may also involve only short sequence homologies [37]. Finally, we find intrachromosomal rearrangements between repeat sequences that may involve a template-switch reaction, and appear to involve only limited sequence homology (Admire et al., 2006; unpublished results).
The role of DSBs versus template switch in fork recovery Most models of genome instability do favor a DSB intermediate, as opposed to a template-switch mechanism. What difference would it make whether a DSB or a template-switch-based mechanism were involved in fork recovery and in genome rearrangements? One idea is that a template-switch mechanism, without a DSB intermediate, might favor the correct pairing to nearby sister strands simply by greater geometrical constraint than that afforded were a DSB intermediate involved.
Template switching One major feature of replication fork recovery proposes template switches (Figure 1), and some genome rearrangements come about by a faulty template switch. There is now substantial evidence for template-switchlike events in bacteria [34] and in budding yeast [35,36], though molecular details remain obscure. In yeast, Smith et al. [35] showed that template switching occurs during repair of a DSB. They introduced a chromosome fragment with a DSB into a cell that contains two chromosomes with homology to the introduced chromosome fragment. The DSB of the chromosome fragment pairs with one of the homologous chromosomes to begin ‘repair’ of the DSB. Repair involves formation of a replication fork in a process referred to as break-induced replication (BIR). Smith et al. found that after the initial www.sciencedirect.com
The role of sequence homology and location in genome rearrangements Whatever the mechanism, rearrangements in budding yeast occur frequently between two sequences sharing sequence homology, ranging from several bases to several hundred bases [40,41,53]. A recent study in yeast suggests quite reasonably that the genetic requirements for rearrangements involving extensive sequence homology differ from those involved in short or no sequence homology [53]. Rearrangements in mammalian cells may also involve two sequences having only short homologies [42], though rearrangements arising from fusions with longer stretches occur experimentally [43] and probably in genomic disorders (e.g., involving copy number variability; [4]). Current Opinion in Cell Biology 2009, 21:778–784
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Co-localization of two sequences that fuse is also probably a relevant variable. One study reported a higher frequency of co-localization of RET and ELE1 genes in thyroid cell as opposed to epithelial cells, and fusion of these two sequences is commonly found in thyroid cancer [44]. Specific sequences in yeast also co-localize and preferentially recombine [45,46], and in bacteria sequences that are closer also recombine more preferentially [47]. How might two sequences co-localize? Replication factories [48] may bring together two sequences that are not directly adjacent.
Studies of genome instability in budding yeast Genome instability systems have been described in budding yeast [1,5,23,36,37,41,49,50,53]. These systems usually possess specific DNA sequence features causing instability (e.g., instability of palindromes; [5], inverted repeats; [49,50]). How instability occurs relevant to the models shown in the figure is often not obvious from these studies. We are left wondering exactly how defects in replication fork recovery are converted into rearranged genomes. We conclude with discussion of two studies that may address key issues relevant to the model in the figure. One study by Pannunzio, Bailis and colleagues provides, in our view, a suggestion that DSBs may not be intermediates in some genome rearrangements [52]. The authors divided the HIS3 gene into two DNA fragments that were inserted at two sites on different chromosomes. The HIS3 gene fragments share 300 bases of sequence overlap, allowing their recombination to form an intact HIS3 gene. Engineered adjacent to each sequence was a DNA sequence that can be cleaved by the HO endonuclease (a site-specific endonuclease that is normally involved in a gene conversion involved in mating type switching). They then isolated His+ cells, either formed spontaneously or after formation of a DSB at the HO site. The His+ cells contained translocations that fused the HIS3 fragments, as expected. Induction of the DSB increased the frequency of His+ translocations dramatically, as expected. What was striking to us is that the genetics of the spontaneous His+ and DSB-mediated His+ events were completely different (the spontaneous events had no requirement for Mre11, Rad1, Rad59 nor Ku70, while the DSB-mediated events required these proteins. Both events did require RAD52, a key strand annealing protein). That the genetics of spontaneous and DSB-induced events differed so dramatically suggests to us that spontaneous events are not proceeding by a DSBmediated mechanism; perhaps they proceed by a forkmediated template-switch process instead. We finish by citing another study by Payen, Fischer and colleagues that is of interest for a several reasons. They developed a genome instability assay that detects segmental duplications [41]. Their system uses the fact that Current Opinion in Cell Biology 2009, 21:778–784
two genes encode identically acting ribosomal proteins, deletion of one results in very poor growth, and subsequent duplication of the existing gene restores growth. They selected for growth of a strain deleted for one of the two genes, and found that many survivors contained segmental duplications containing the remaining gene. The segmental duplications involve either intra or intermolecular events, and duplications of different sizes. (The authors favor the view that segmental duplications occur via DSBs, though fork-mediated mechanisms seem possible as well.) Interestingly, the Rad52 strand annealing protein is required when duplications involve two DNA sequences that lie far apart (on the same chromosome or interchromosomally), but Rad52 was not required for the fusion of sequences that were closer together on the same chromosome. (Rad52-dependent fusions involved longer sequence homologies while Rad52-independent fusions involved shorter sequence homologies.) This suggests to us that, in this system, rearrangements involving nearby sequences may occur by a fundamentally different mechanism (faulty template switch?) than sequences that lie further apart.
Conclusion Studies of replication fork biology and genome instability are progressing at a rapid pace. Understanding how normal replication fork recovery mechanisms rescue a stalled fork is key to understanding how genome rearrangements arise. Either DSB or fork-mediated (template switch) events may drive genome rearrangements. Finally, the mechanisms underlying replication fork biology and genome instability may be crucial to the oncogenic process [54–56].
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36. Schmidt KH, Wu J, Kolodner RD: Control of translocations between highly diverged genes by Sgs1, the Saccharomyces cerevisiae homolog of the Bloom’s syndrome protein. Mol Cell Biol 2006, 26(14):5406-5420. 37. Rattray AJ, Shafer BK, Neelam B, Strathern JN: A mechanism of palindromic gene amplification in Saccharomyces cerevisiae. Genes Dev 2005, 19(11):1390-1399. A genomic instability system induced by a DSB that generates palindromes by the single-strand DNA ‘looping’ back and pairing with small regions of homology. The mechanism was deduced from sequencing the rearrangements formed. 38. Weitao T, Budd M, Hoopes LL, Campbell JL: Dna2 helicase/ nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae. J Biol Chem 2003, 278(25):22513-22522. 39. Seigneur M, Bidnenko V, Ehrlich SD, Michel B: RuvAB acts at arrested replication forks. Cell 1998, 95(3):419-430. 40. Putnam CD, Pennaneach V, Kolodner RD: Saccharomyces cerevisiae as a model system to define the chromosomal instability phenotype. Mol Cell Biol 2005, 25(16): 7226-7238. 41. Payen C, Koszul R, Dujon B, Fischer G: Segmental duplications arise from Pol32-dependent repair of broken forks through two alternative replication-based mechanisms. PLoS Genet 2008, 4(9):pe1000175. A study of segmental duplication in budding yeast. The system is described briefly in this review. It is a lovely study. 42. Elliott B, Jasin M: Double-strand breaks and translocations in cancer. Cell Mol Life Sci 2002, 59(2):373-385. 43. Elliott B, Richardson C, Jasin M: Chromosomal translocation mechanisms at intronic alu elements in mammalian cells. Mol Cell 2005, 17(6):885-894. 44. Nikiforova MN, Stringer JR, Blough R, Medvedovic M, Fagin JA, Nikiforov YE: Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells. Science 2000, 290(5489):138-141. 45. Lichten M, Haber JE: Position effects in ectopic and allelic mitotic recombination in Saccharomyces cerevisiae. Genetics 1989, 123(2):261-268. 46. Burgess SM, Kleckner N: Collisions between yeast chromosomal loci in vivo are governed by three layers of organization. Genes Dev 1999, 13(14):1871-1883. 47. Bi X, Liu LF: DNA rearrangement mediated by inverted repeats. Proc Natl Acad Sci U S A 1996, 93(2):819-823.
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48. Kitamura E, Blow JJ, Tanaka TU: Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories. Cell 2006, 125(7):1297-1308. 49. Admire A, Shanks L, Danzl N, Wang M, Weier U, Stevens W, Hunt E, Weinert T: Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast. Genes Dev 2006, 20(2):159-173. 50. Lemoine FJ, Degtyareva NP, Lobachev K, Petes TD: Chromosomal translocations in yeast induced by low levels of DNA polymerase a model for chromosome fragile sites. Cell 2005, 120(5):587-598. 51. Mimitou EP, Symington LS: Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 2008, 455(7214):770-774. A soon to be classic study of how a DSB is degraded first by Sae2directed mechanism, and then Exo1 and Sgs1. 52. Pannunzio NR, Manthey GM, Bailis AM: RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae. DNA Repair (Amst) 2008, 7(5):788-800. A study of genome instability described in this review. The genetic data suggest to us that genome rearrangements that are spontaneous occur by a different mechanism (fork-mediated?) compared to DSB-mediated events. The author focuses on the role of Rad59 in mediating fusion between short sequences of homology. 53. Putnam CD, Hayes TK, Kolodner RD: Specific pathways prevent duplication-mediated genome rearrangements. Nature 2009, 460(7258):984-989. A study of genome rearrangements using paralogous sequences, defining the events and the genetic requirements. The results from this study were compared to the genetic requirements for rearrangements involving singlecopy sequences; they are different. 54. Halazonetis TD, Gorgoulis VG, Bartek J: An oncogene-induced DNA damage model for cancer development. Science 2008, 319(5868):1352-1355. 55. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV, Kolettas E, Niforou K, Zoumpourlis VC et al.: Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006, 444(7119):633-637. 56. Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, Venere M, Ditullio RA Jr, Kastrinakis NG, Levy B: Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005, 434(7035):907-913.
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The replication fork’s five degrees of freedom, their failure and genome rearrangements T Weinert, S Kaochar, H Jones, A Paek and AJ Clark Genome rearrangements are important in pathology and evolution. The thesis of this review is that the genome is in peril when replication forks stall, and stalled forks are normally rescued by error-free mechanisms. Failure of error-free mechanisms results in large-scale chromosome changes called gross chromosomal rearrangements, GCRs, by the aficionados. In this review we discuss five error-free mechanisms a replication fork may use to overcome blockage, mechanisms that are still poorly understood. We then speculate on how genome rearrangements may occur when such mechanisms fail. Replication fork recovery failure may be an important feature of the oncogenic process. (Feedback to the authors on topics discussed herein is welcome.) Address Department of Molecular & Cell Biology, University of Arizona, Tucson, AZ 85721, USA Corresponding author: Weinert, T ([email protected])
Current Opinion in Cell Biology 2009, 21:778–784 This review comes from a themed issue on Cell division, growth and death Edited by Angelika Amon and Mike Tyers Available online 11th November 2009 0955-0674/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.ceb.2009.10.004
The many forms of genome rearrangements Genome instability is a broad term capturing any form of genetic change, from base pair changes, to small insertions, deletions or inversions, to larger scale changes such as translocations, segmental duplications, and whole chromosome loss or duplication [1]. Each type of rearrangement is relevant for pathology and evolution [2,3,4]. Here we focus on mechanisms by which large-scale rearrangements may arise, drawing largely from studies in S. cerevisiae.
General mechanisms underlying genome rearrangement Genome rearrangements may in principle have a simple etiology; chromosomes may break, and two breaks may be joined when error-free repair fails. How chromosomes break probably involves DNA replication forks, and therein lies considerable uncertainty as our understanding of replication fork biology is far from complete. We Current Opinion in Cell Biology 2009, 21:778–784
imagine the following sequence of events may lead to genome rearrangements: when cells replicate their DNA, replication forks stall. Stalling may be stochastic, or may be induced by unusual DNA sequences (secondary structures; [5]), by sites occupied by protein–DNA complexes [6,7], or by DNA damage [8,9]. Replication fork stalling is probably unavoidable because of the existence of protein–DNA complexes involved in gene expression (e.g., RNA polymerases; [6,7] or in chromosome duplication (e.g., centromeres, origins of replication; [7]). When forks stall, regulatory and repair mechanisms rescue the stalled fork by error-free mechanisms outlined below. When error-free fork recovery fails, the strands in the stalled fork may anneal to other sequences in the genome, thereby leading to genome rearrangements. How this ‘ectopic’ annealing of strands occurs is a matter of speculation; annealing may occur either directly from a stalled fork (something we call faulty template switching) or following a double-strand break (DSB). We discuss the forks five error-free mechanisms of recovery, and speculate on how genome rearrangements arise when these fail.
The five degrees of freedom in replication fork recovery Stalled replication forks recover by mechanisms that remain largely speculative to date, largely because of the lack of defined fork-stalling in vivo or in vitro systems. In vitro systems in bacteria hold much promise (for example, see [11,12]), though it is unclear to what extent the generality of bacterial fork behavior will apply to eukaryotic fork behavior. In eukaryotes, even such simple-sounding terms as ‘stalled fork’ and ‘lesion that stalls a fork’ do not yet have concrete molecular descriptions. Nonetheless, molecular studies of fork behavior provide early and important clues on links between replication forks and genome stability. The stalled fork’s five degrees of freedom are shown in the models in Figure 1 that derives from studies in S. cerevisae, leave out many interesting possibilities, and suggest conclusions that may extend to all eukaryotic cells. (Excellent reviews of replication more detailed than this one abound; [10,11,12].) First, stalled forks may recover without fork remodeling; the offending damage is repaired or blocking protein removed (#1). Many regulators (e.g., Mec1 and Rad53) may enforce this first option, though how they may do so is unclear [10]). The next four options have been termed ‘fork collapse’, generally referring to events that ensue after DNA www.sciencedirect.com
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Figure 1
A replication fork encounters a lesion (in this case DNA damage, triangle), and uses one of 5 options to overcome it. The fork may simple resume replication (#1); may undergo sister-strand annealing and replication to form a hemicatenane that is resolved by a host of proteins (#2); may undergo sister-strand annealing to form a regressed fork (‘chicken foot’) that allows lesion repair (#3); may undergo sister-strand annealing to form a regressed fork that allows replication of the annealed strands, followed by regression or re-invasion, forming a Holliday structure that needs to be resolved (#4); for undergoes strand breakage to form a DSB. DSB may also form from the regressed fork.
polymerases dissociate from the fork [13]). The second, third, and fourth options posit that a stalled fork undergoes a sister-strand annealing reaction, called a template switch or copy choice switch. The idea is that template switching allows either efficient repair/removal, or replication past a lesion. One form of template switching (#2) involves a ‘behind the fork’ event in which sister-strand anneal, forming a hemicatenane (a shorter one of which may form at the initiation of replication; [14,15]). A hemicatenane is a topological intertwining of one of two strands of two DNA duplexes (see blue and green strands of option #2). The hemicatenane is then resolved, most probably by the action of the helicase Sgs1 and topoisomerase Top3. Template switching might also occur at the front of the fork (#3 and #4). In these events, forks first regress to pair newly replicated sister strands as shown, forming a so-called ‘chicken foot’ structure. This regressed structure is then resolved either by fork reversal without replication (option #3), or by more complicated mechanisms (option #4) that involve replication and/or www.sciencedirect.com
strand invasion, and possibly resolution of a Holliday structure (see [12,16]). Finally, a fifth fork option involves a double-strand break, formed by cleavage of the exposed single-stranded DNA at the stalled fork or by cleavage of the regressed fork, followed by strand invasion (not shown in Figure 1). Both fork-mediated and DSB-mediated mechanisms may require homologous recombination proteins to facilitate correct pairing [14,16,51]. Genome rearrangements may arise from a failure of correct annealing during the template switch or DSB options.
Some evidence for the forks five degrees of freedom There is reasonable but far from incontrovertible evidence in support of these five degrees of freedom. The research community is presently identifying specific DNA structures and specific proteins associated with those structures. Evidence on DNA structures (formed Current Opinion in Cell Biology 2009, 21:778–784
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in vivo) comes from two-dimensional agarose gels (2-D gels, [17]) or from electron microscopy (EM; see [8,18]). Two-dimensional gels provide information about where forks stall, and in combination with biochemical techniques can identify potential abnormal structures. For example, key structures identified in 2D gels are so-called ‘X-structures’. One X-structure is probably a Holliday structure, because it is resolvable by enzymes known to resolve bonafide Holliday structures (RuvC and T4 endonuclease VII; [14,16]). A second Xstructure has been identified that is not a Holliday structure, because it cannot be resolved by RuvC, and biochemical studies are consistent with it being a hemicatenane ([14]; see figure option #2). Description of specific fork structures remains a major technical challenge. EM studies can provide less ambiguous descriptions of DNA structures than 2-D gels, though EM requires the technical challenge of purifying DNA structures from cells [8,18]. Given the ambiguities of DNA structures, it is not surprising that the association of specific proteins with specific structures also remains incomplete [10,13,19]. Our current understanding of the fate of stalled replication forks in eukaryotic and prokaryotic cells derives largely from in vivo studies (in vitro studies of prokaryotic replication forks prove informative as well; [11,12]). The in vivo studies involve blocks to DNA replication because of sequence-specific fork-stalling protein complexes or DNA damage. Specific protein-induced fork blocks are referred to as replication fork barriers, or RFBs. These studies provide limited evidence bearing on the options in this model. Two studies of RFBs provide evidence that stalled forks may remain intact and simply proceed (option #1). First, in bacteria a repressor-bound complex [20] can block fork progression, and stalled forks do not appear to break or form unusual structures. Fork recovery resumes promptly upon repressor removal (by adding inducer), and in a recombination protein (RecA) independent fashion. (Prolonged fork arrest was also accomplished in bacteria with an ectopic Ter-site placed away from its normal site of replication termination; [21]. Ter-sites contain specific proteins and DNA sequences that coordinate replication termination.) A second study that supports option 1 comes from studies in budding yeast using the rDNA RFB site system. A protein called Fob1 normally binds to specific RFB sites in the rDNA locus, and blocks replication that would oppose transcription of the rDNA genes. The Fob1-RFB system thus provides for co-directional DNA replication and transcription of the rDNA genes, minimizing head-on DNA polymerase and RNA polymerase collisions. When placed outside the rDNA locus, the RFB-Fob1 system also stalls forks and allows the monitoring of fork behavior Current Opinion in Cell Biology 2009, 21:778–784
[22]. Forks stall briefly (for 30 min) at an ectopic RFB site, the Fob1-stalled forks do not form unusual DNA structures shown in Figure 1, and do not require recombination (Rad52) or regulatory checkpoint proteins (Mec1, Rad53) to resume replication. A conceptually similar fork-stalling system with a very different outcome has been developed in fission yeast [23]. In this system, the Rtf1 protein binds to a single RTS1 DNA sequence, and that protein–DNA complex blocks DNA replication. The Rtf1-RTS1 system normally provides for unidirectional replication through the mating type locus. Lambert et al. manipulated this system to achieve more efficient fork stalling by inserting a pair of RTS1 sequences in inverted orientation such that expression of the Rtf1 protein causes stalling of two converging replication forks. In this setting, forks stall very efficiently (much more so that the Fob1-RFB system in budding yeast). Viability is dependent on homologous recombination, and X-structures are generated suggested that a pairing reaction is involved. This implies that fork recovery may occur via either hemicatenanes or regressed forks or a recombination intermediate. Genome rearrangements occur when forks stall, and it is unclear if failure of a specific option in Figure 1 is involved. One additional site-specific fork-stalling system deserves special mention as it provides evidence for structures that are necessary for options 3 and 4. In bacteriophage T4, Long and Kreuzer describe an elegant system that involves what they call the ‘origin fork’; after T4 infection, bidirection DNA replication occurs from a specific origin from which the two forks emerge at different times; one of the two forks replicates immediately while the other ‘origin fork’ delays and replicates later (an apparent design feature of T4 replication in place for reasons unknown to us; [26]). Long and Kreuzer study the delayed origin fork, can follow its behavior by 2-D gels, and found that certain phage proteins are required for its normal firing. They identified by 2-D gels a specific structure they propose is a regressed fork that accumulates in mutants in specific phage replication proteins. These studies as well as in vitro studies of bacteria DNA replication hold promise for increasing our understanding of eukaryotic stalled forks [11]. Quite a number of studies evaluate how DNA damage induces both fork stalling and genome instability, and those studies suggest options involving template switching (options 3 and 4). Formulation of models is very much a work in progress [18,26,27,28,29,30]. To site but a few observations, Rad5 and Srs2 may be needed to form regressed forks [31,32], Rad53 may both prevent regression and regulate Exo1’s degradation of regressed forks [29,30], and a number of proteins involved in postreplication repair (e.g., Rad18) may be involved in formation and resolution of the hemicatenanes [14,27]. www.sciencedirect.com
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Finally, the extent to which stalled replication forks break (option #5) is unknown. Stalled forks in yeast and bacteria do appear to break (for example, [7,38,39]), though to what extent a DSB pathway is used compared to the other no-DSB options is unclear. In sum, the evidence addressing the forks five degrees of freedom is clearly incomplete, and the model seems based largely on inference. A major technical problem is that neither RFB yeast systems that achieve stalled forms require checkpoint proteins (Mec1 and Rad53 in S. cerevisiae) for their stability. Development of an RFB system that requires Mec1 and Rad53 for stability, as do DNA damage-induced forks [24,25], would clearly be of value. Other features implicit in this model, DNA polymerase coupling and template switching, are discussed next, after which we conclude with discussion of genome instability.
DNA polymerase uncoupling The coupling between leading and lagging strand DNA polymerases is implicit in some aspects of the models shown; for example, damage that stalls the DNA polymerase on one strand only forms a stalled fork if the other DNA polymerase remains coupled to the stalled one. It seems reasonable to speculate that replication polymerases retain some coupling to minimize the amount of single-stranded DNA were leading or lagging DNA polymerases to stall. There is some evidence, for example, that some replication proteins do couple some proteins to a stalled fork (Mrc1/Tof1 in yeast, DnaB helicase in bacteria; [19,33]). This coupling seems to odds, however, with elegant experiments in bacteria and SV40 that demonstrate that leading and lagging strand synthesis, which is normally coupled, can uncouple when the leading strand or lagging strand is blocked by damage [8,9]. We do not know the answer to this conondrum; it may be as simple as suggesting that experiments that use DNA damage generate lesions in close proximity on both strands.
pairing and replication, the replication complex appears to dissociate from the first chromosome and resume replication after it pairs with allelic sequences on the second homologous chromosome. In a separate study, Schmidt et al. provided evidence for what we believe may be a similar template-switch reaction, this time involving non-allelic sequences and forming genome rearrangements [36]. The simplest model of such template-switch events envisions the 30 end of the polymer that DNA polymerase is extending dissociating from one sequence and reannealing to another, presumably in a single cell cycle. Molecular details that underlie such a template switch are unknown, and are difficult to study given the rarity of events. One feature that can be analyzed is the role of sequence homology in template switching. Several studies suggest to us that extensive sequence homology may not be required. For example, a human genome disorder that arises from multiple complex rearrangements was proposed to involve switching between short sequence homologies (<10 bp; [3]). In yeast Rattray et al. provide evidence that single-strand annealing and polymerase extension in vivo may also involve only short sequence homologies [37]. Finally, we find intrachromosomal rearrangements between repeat sequences that may involve a template-switch reaction, and appear to involve only limited sequence homology (Admire et al., 2006; unpublished results).
The role of DSBs versus template switch in fork recovery Most models of genome instability do favor a DSB intermediate, as opposed to a template-switch mechanism. What difference would it make whether a DSB or a template-switch-based mechanism were involved in fork recovery and in genome rearrangements? One idea is that a template-switch mechanism, without a DSB intermediate, might favor the correct pairing to nearby sister strands simply by greater geometrical constraint than that afforded were a DSB intermediate involved.
Template switching One major feature of replication fork recovery proposes template switches (Figure 1), and some genome rearrangements come about by a faulty template switch. There is now substantial evidence for template-switchlike events in bacteria [34] and in budding yeast [35,36], though molecular details remain obscure. In yeast, Smith et al. [35] showed that template switching occurs during repair of a DSB. They introduced a chromosome fragment with a DSB into a cell that contains two chromosomes with homology to the introduced chromosome fragment. The DSB of the chromosome fragment pairs with one of the homologous chromosomes to begin ‘repair’ of the DSB. Repair involves formation of a replication fork in a process referred to as break-induced replication (BIR). Smith et al. found that after the initial www.sciencedirect.com
The role of sequence homology and location in genome rearrangements Whatever the mechanism, rearrangements in budding yeast occur frequently between two sequences sharing sequence homology, ranging from several bases to several hundred bases [40,41,53]. A recent study in yeast suggests quite reasonably that the genetic requirements for rearrangements involving extensive sequence homology differ from those involved in short or no sequence homology [53]. Rearrangements in mammalian cells may also involve two sequences having only short homologies [42], though rearrangements arising from fusions with longer stretches occur experimentally [43] and probably in genomic disorders (e.g., involving copy number variability; [4]). Current Opinion in Cell Biology 2009, 21:778–784
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Co-localization of two sequences that fuse is also probably a relevant variable. One study reported a higher frequency of co-localization of RET and ELE1 genes in thyroid cell as opposed to epithelial cells, and fusion of these two sequences is commonly found in thyroid cancer [44]. Specific sequences in yeast also co-localize and preferentially recombine [45,46], and in bacteria sequences that are closer also recombine more preferentially [47]. How might two sequences co-localize? Replication factories [48] may bring together two sequences that are not directly adjacent.
Studies of genome instability in budding yeast Genome instability systems have been described in budding yeast [1,5,23,36,37,41,49,50,53]. These systems usually possess specific DNA sequence features causing instability (e.g., instability of palindromes; [5], inverted repeats; [49,50]). How instability occurs relevant to the models shown in the figure is often not obvious from these studies. We are left wondering exactly how defects in replication fork recovery are converted into rearranged genomes. We conclude with discussion of two studies that may address key issues relevant to the model in the figure. One study by Pannunzio, Bailis and colleagues provides, in our view, a suggestion that DSBs may not be intermediates in some genome rearrangements [52]. The authors divided the HIS3 gene into two DNA fragments that were inserted at two sites on different chromosomes. The HIS3 gene fragments share 300 bases of sequence overlap, allowing their recombination to form an intact HIS3 gene. Engineered adjacent to each sequence was a DNA sequence that can be cleaved by the HO endonuclease (a site-specific endonuclease that is normally involved in a gene conversion involved in mating type switching). They then isolated His+ cells, either formed spontaneously or after formation of a DSB at the HO site. The His+ cells contained translocations that fused the HIS3 fragments, as expected. Induction of the DSB increased the frequency of His+ translocations dramatically, as expected. What was striking to us is that the genetics of the spontaneous His+ and DSB-mediated His+ events were completely different (the spontaneous events had no requirement for Mre11, Rad1, Rad59 nor Ku70, while the DSB-mediated events required these proteins. Both events did require RAD52, a key strand annealing protein). That the genetics of spontaneous and DSB-induced events differed so dramatically suggests to us that spontaneous events are not proceeding by a DSBmediated mechanism; perhaps they proceed by a forkmediated template-switch process instead. We finish by citing another study by Payen, Fischer and colleagues that is of interest for a several reasons. They developed a genome instability assay that detects segmental duplications [41]. Their system uses the fact that Current Opinion in Cell Biology 2009, 21:778–784
two genes encode identically acting ribosomal proteins, deletion of one results in very poor growth, and subsequent duplication of the existing gene restores growth. They selected for growth of a strain deleted for one of the two genes, and found that many survivors contained segmental duplications containing the remaining gene. The segmental duplications involve either intra or intermolecular events, and duplications of different sizes. (The authors favor the view that segmental duplications occur via DSBs, though fork-mediated mechanisms seem possible as well.) Interestingly, the Rad52 strand annealing protein is required when duplications involve two DNA sequences that lie far apart (on the same chromosome or interchromosomally), but Rad52 was not required for the fusion of sequences that were closer together on the same chromosome. (Rad52-dependent fusions involved longer sequence homologies while Rad52-independent fusions involved shorter sequence homologies.) This suggests to us that, in this system, rearrangements involving nearby sequences may occur by a fundamentally different mechanism (faulty template switch?) than sequences that lie further apart.
Conclusion Studies of replication fork biology and genome instability are progressing at a rapid pace. Understanding how normal replication fork recovery mechanisms rescue a stalled fork is key to understanding how genome rearrangements arise. Either DSB or fork-mediated (template switch) events may drive genome rearrangements. Finally, the mechanisms underlying replication fork biology and genome instability may be crucial to the oncogenic process [54–56].
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Kolodner RD, Putnam CD, Myung K: Maintenance of genome stability in Saccharomyces cerevisiae. Science 2002, 297(5581):552-557.
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Sharp AJ, Hansen S, Selzer RR, Cheng Z, Regan R, Hurst JA, Stewart H, Price SM, Blair E, Hennekam RC et al.: Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nat Genet 2006, 38(9):1038-1042.
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Lee JA, Carvalho CM, Lupski JR: A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell 2007, 131(7):1235-1247. An oft-cited study of the structure of genome rearrangements in a human disorder. The authors invoke a template-switch model, in which DNA polymerase must ‘hop’ 3 or 4 times between sequences up to 0.5 MB apart to sequences of little homology, to make sense of the rearrangement structures. 4.
Mefford HC, Eichler EE: Duplication hotspots, rare genomic disorders, and common disease. Curr Opin Genet Dev 2009, 19(3):196-204.
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Narayanan V, Mieczkowski PA, Kim H, Petes TD, Lobachev KS: The pattern of gene amplification is determined by the chromosomal location of hairpin-capped breaks. Cell 2006, 125(7):1283-1296. www.sciencedirect.com
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Ivessa AS, Lenzmeier BA, Bessler JB, Goudsouzian LK, Schnakenberg SL, Zakian VA: The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein–DNA complexes. Mol Cell 2003, 12(6):1525-1536. A classic study showing that in budding yeast, the replication apparatus may stall about once every 10 kb (if the Rrm3 DNA helicase is defective).
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Cordeiro-Stone M, Makhov AM, Zaritskaya LS, Griffith JD: Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand. J Mol Biol 1999, 289(5):1207-1218. A key study, using SV40 replication in vitro, defined plasmid templates with damage, and EM to study structures, that DNA replication can be uncoupled.
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Pages V, Fuchs RP: Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo. Science 2003, 300(5623):1300-1303. A beautiful study using multiple technical tricks and mutants to show that, in bacteria, a lesion that blocks the leading strand (and lagging strand— data not shown) causes uncoupling of DNA synthesis, with absolutely no detectable delay in replication. 10. Friedel AM, Pike BL, Gasser SM: ATR/Mec1: coordinating fork stability and repair. Curr Opin Cell Biol 2009, 21(2):237-244. 11. Heller RC, Marians KJ: Replisome assembly and the direct restart of stalled replication forks. Nat Rev Mol Cell Biol 2006, 7(12):932-943. 12. Atkinson J, McGlynn P: Replication fork reversal and the maintenance of genome stability. Nucleic Acids Res 2009, 37(11):3475-3492. An absolutely marvelous review that describes fully the issues of replication fork biology, complete with diagrams of strands going this way and that make sense and are easy to follow.
13. Cobb JA, Bjergbaek L, Shimada K, Frei C, Gasser SM: DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1. EMBO J 2003, 22(16):43254336. The study often credited, among others, with defining what a collapsed fork does; DNA polymerases dissociate (regulated by Mec1) and Mcm helicases (regulated by Rad53). They performed ChIP experiments on synchronized cells after HU-depletion, generating stalled forks in specific regions of the genome. 14. Haber JE, Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Foiani M, MaffiolettiG, Lucca C, Baryskhnikova A, Chiolo I: Rad51dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev 2005, 19(3):339350. 15. Lopes M, Cotta-Ramusino C, Liberi G, Foiani M: Branch migrating sister chromatid junctions form at replication origins through Rad51/Rad52-independent mechanisms. Mol Cell 2003, 12(6):1499-1510. 16. Zou H, Rothstein R: Holliday junctions accumulate in replication mutants via a RecA homolog-independent mechanism. Cell 1997, 90(1):87-96. 17. Friedman KL, Brewer BJ: Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol 1995, 262:613-627. 18. Sogo JM, Lopes M, Foiani M: Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 2002, 297(5581):599-602. A landmark study using EM to study the stalled DNA structures from yeast cells. 19. Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K: S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 2003, 424(6952):1078-1083. 20. Possoz C, Filipe SR, Grainge I, Sherratt DJ: Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo. EMBO J 2006, 25(11): 2596-2604. www.sciencedirect.com
21. Bidnenko V, Ehrlich SD, Michel B: Replication fork collapse at replication terminator sequences. EMBO J 2002, 21(14):38983907. 22. Calzada A, Hodgson B, Kanemaki M, Bueno A, Labib K: Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev 2005, 19(16): 1905-1919. 23. Lambert S, Watson A, Sheedy DM, Martin B, Carr AM: Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier. Cell 2005, 121(5):689-702. This study describes a RFB system in fission yeast that may yield deep insight into replication fork biology. In this study, the RTS system was maniupulated to cause profound stalls of forks, leading to genome rearrangements. 24. Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Plevani P, MuziFalconi M, Newlon CS, Foiani M: The DNA replication checkpoint response stabilizes stalled replication forks. Nature 2001, 412(6846):557-561. 25. Tercero JA, Diffley JF: Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 2001, 412(6846):553-557. 26. Long DT, Kreuzer KN: Regression supports two mechanisms of fork processing in phage T4. Proc Natl Acad Sci U S A 2008, 105(19):6852-6857. A remarkable system described in this review. The ‘origin fork’ may indeed be a very informative model for understanding fork behavior in more genetically complex eukaryotic systems. In particular, the authors find a specific 2-D gel structure that appears to be an intact regressed fork. 27. Branzei D, Vanoli F, Foiani M: SUMOylation regulates Rad18 mediated template switch. Nature 2008, 456(7224):915-920. One of what will be many studies struggling to understand post-replication repair and their links to specific DNA structures. 28. Lopes M, Foiani M, Sogo JM: Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions. Mol Cell 2006, 21(1):15-27. 29. Cotta-Ramusino C, Fachinetti D, Lucca C, Doksani Y, Lopes M, Sogo J, Foiani M: Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells. Mol Cell 2005, 17(1):153-159. Together with the findings from ref [30], this EM study of DNA structures suggests how specific proteins are linked to specific DNA structures; Rad53 allows regressed fork formation, and Exo1 acts on regressed forks. 30. Segurado M, Diffley JF: Separate roles for the DNA damage checkpoint protein kinases in stabilizing DNA replication forks. Genes Dev 2008, 22(13):1816-1827. This study shows that mutations in exo1 have the remarkable ability to suppress the HU-sensitivity of rad53 mutants. This finding has at least two implications; first it suggests regulation of Exo1 by Rad53, and second it links Exo1 and Rad53 to regresssed forks (see ref [29]). 31. Blastya´k A, Pinte´r L, Unk I, Prakash L, Prakash S, Haracska L: Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression. Mol Cell 2007, 28(1):167-175. 32. Kerrest A, Anand RP, Sundararajan R, Bermejo R, Liberi G, Dujon B, Freudenreich CH, Richard GF: SRS2 and SGS1 prevent chromosomal breaks and stabilize triplet repeats by restraining recombination. Nat Struct Mol Biol 2009, 16(2):159167. 33. Indiani C, Langston LD, Yurieva O, Goodman MF, O’Donnell M: Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase. Proc Natl Acad Sci U S A 2009, 106(15):6031-6038. 34. Lovett ST: Encoded errors: mutations and rearrangements mediated by misalignment at repetitive DNA sequences. Mol Microbiol 2004, 52(5):1243-1253. 35. Smith CE, Llorente B, Symington LS: Template switching during break-induced replication. Nature 2007, 447(7140):102-105. Study shows how DNA polymerase must switch between allelic sequences on two homologs as it carries out replication, starting from a DSB (A BIR event). Current Opinion in Cell Biology 2009, 21:778–784
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48. Kitamura E, Blow JJ, Tanaka TU: Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories. Cell 2006, 125(7):1297-1308. 49. Admire A, Shanks L, Danzl N, Wang M, Weier U, Stevens W, Hunt E, Weinert T: Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast. Genes Dev 2006, 20(2):159-173. 50. Lemoine FJ, Degtyareva NP, Lobachev K, Petes TD: Chromosomal translocations in yeast induced by low levels of DNA polymerase a model for chromosome fragile sites. Cell 2005, 120(5):587-598. 51. Mimitou EP, Symington LS: Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 2008, 455(7214):770-774. A soon to be classic study of how a DSB is degraded first by Sae2directed mechanism, and then Exo1 and Sgs1. 52. Pannunzio NR, Manthey GM, Bailis AM: RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae. DNA Repair (Amst) 2008, 7(5):788-800. A study of genome instability described in this review. The genetic data suggest to us that genome rearrangements that are spontaneous occur by a different mechanism (fork-mediated?) compared to DSB-mediated events. The author focuses on the role of Rad59 in mediating fusion between short sequences of homology. 53. Putnam CD, Hayes TK, Kolodner RD: Specific pathways prevent duplication-mediated genome rearrangements. Nature 2009, 460(7258):984-989. A study of genome rearrangements using paralogous sequences, defining the events and the genetic requirements. The results from this study were compared to the genetic requirements for rearrangements involving singlecopy sequences; they are different. 54. Halazonetis TD, Gorgoulis VG, Bartek J: An oncogene-induced DNA damage model for cancer development. Science 2008, 319(5868):1352-1355. 55. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV, Kolettas E, Niforou K, Zoumpourlis VC et al.: Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006, 444(7119):633-637. 56. Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, Venere M, Ditullio RA Jr, Kastrinakis NG, Levy B: Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005, 434(7035):907-913.
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