CELLS DEVOTE SIGNIFICANTresources to the repair of double-strand DNA breaks (DSBs). If left unrepaired, such damage re-sults in the loss of chromosomes and/or the induction of cell death. If imprecisely repaired, the damage leads to mutations and chromosomal rearrangements. Here, I focus on the repair of DNA breaks by the homologous-recombination process known as gene conversion, in which the broken chromosome is patched up by copying information from a homologous chromosome or from a sister chromatid. Gene conversion is the most conservative type of repair; it is much less likely to in-troduce a mutation at the site of damage than is a process such as non-homologous end-joining, in which various deletions or small insertions are created at the site of the DSB. However, gene conversions are sometimes accompanied by crossing over of the interacting sequences, which can lead to a loss of heterozygosity in mitotic cells. Very similar events occur in meiosis, and again are initiated by DSBs, but, in this case, crossovers are the desired outcome. In mammalian cells, because of the dif-ficulties of obtaining accurate gene target-ing and the high frequency of end-fusions of transfected DNA, it has been supposed that illegitimate recombination mecha-nisms are much more efficient than are homologous-repair pathways – the oppo-site of what one finds in yeast. However, recently, Liang and co-workers1 have shown that vertebrate cells are quite proficient at homologous recombination,
when DSBs are created within chromo-somes as opposed to when a DNA frag-ment is transfected into cells. Recom-bination is also enhanced when there are no base-pair differences between the transforming linear DNA and the target, chromosomal site: the cell’s mismatch-repair machinery does not provoke rejec-tion of the incoming DNA in the absence of such differences2. Moreover, some spe-cialized cells seem very yeast-like in their ability to carry our accurate gene target-ing. The best-studied type is chicken DT40 cells, which are derived from im-mune cells that rely on gene conversion
to generate immunoglobulin diversity. DT40 cells have become an ideal system to analyse the consequences of knock-ing out genes that encode recombination proteins – in the same way that their homologs have been studied in yeast and bacteria3,4.
The origins of double-strand breaks
In the laboratory, DSBs have classically been created by X-rays. More recently, re-pair of DSBs has been studied after their induction by endonucleases during such natural processes as meiosis or during programmed chromosome rearrange-ments such as V(D)J joining of mammalian immunoglobulin genes or switching of yeast mating-type genes. DSB repair after excision of transposable elements from a chromosome has also been analysed.
Although these repair events are impor-tant in the specialized cells that experi-ence programmed DSBs, multiple path-ways of recombinational repair almost certainly arose in response to a more prevalent source of such DNA damage: the process of DNA replication itself. Al-though DNA replication is a remarkably accurate process, human cells are esti-mated to suffer ~10 such lesions every time a cell divides; this figure is based on the incidence of sister-chromatid ex-changes. Consequently, vertebrate cells that lack the Rad51 recombination protein are inviable, presumably because they cannot repair these lesions; many broken chromosomes are present in such cells4. Homologous recombination could play a second, important role at the replication
DNA recombination:
the replication connection
James E. Haber
Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characteriz-ation of the biochemical roles of recombincharacteriz-ation proteins that are highly conserved from yeast to humans.
J. E. Haberis at Brandeis University, Waltham, MA 02454-9110, USA. (a) (b) Gene conversion Break-induced replication DSB DSB Figure 1
Repair of double-strand breaks (DSBs) that arise during DNA replication. DSBs can be produced by replication across a single-stranded nick or by rupture of a DNA strand at a stalled replication fork. Newly synthesized DNA is shown in light blue. (a) If the DSB leaves sufficient duplex DNA on either side, the ends can participate in repair by gene conversion, which patches up the broken chromosome. In this process, the ends of the broken DNA molecule invade an intact template (here, the upper sister chromatid). The process involves DNA-strand-exchange proteins such as Rad51. New DNA synthesis can be initiated from the 39 ends and fills in the missing sequences. (b) If the DSB occurs near the replication fork, exonucleases can resect the broken end so that the resulting 39 end can invade the upper, intact template to re-establish a replication fork that can proceed either to the chromosome terminus or until it meets a converging replication fork.
fork: it might repair broken replication forks in order to restart DNA replication (Fig. 1). Broken forks could arise sponta-neously or as polymerases traverse pre-existing nicks. Repair can occur through gene conversion or through a recombi-nation-dependent mechanism for DNA-replication reinitiation that is known as break-induced replication (BIR). In both processes, the ends of the DNA are re-sected to produce 39 single-stranded ends that, with the aid of many recombination proteins, can carry out DNA strand ex-change: the single strand invades an in-tact duplex with the same, or a very simi-lar, DNA sequence and forms base pairs with the donor template. Strand exchange is an essential first step in initiating DNA synthesis for repair of the DSB. Here, I fo-cus on recent progress in understanding in detail how gene conversion and BIR occur and on emerging evidence that suggests that the two mechanisms are linked more closely than was previously imagined.
Break-induced replication
The first intimations of an origin-independent, recombination-dependent replication mechanism came from Mosig’s study of late DNA replication in phage T4 (Ref. 5), and our present understanding owes much to recent studies by many labs6–8– especially those by the late Tokio Kogoma, who analysed recombination-dependent replication in Escherichia coli9. In this mechanism, one end of the DSB invades the template and establishes a replication fork (Fig. 1b) that could pro-gress all the way to the end of the chromo-some or until a converging replication fork is encountered. Extensive chromo-some replication can account for DSB repair as well as for the restoration of replication7.
Voekel-Meiman and Roeder invoked the idea of BIR in Saccharomyces cerevisiae to account for what appeared to be gene conversion of most of an entire chromo-some arm in mitotic recombination10: the
centromere-proximal end of a broken chromosome could in-vade a homologous chromo-some and replication could pro-gress to the chromosome end. Malkova et al.11 showed that such events are rare when in competition with gene conver-sion but that, when gene con-version was abolished in a rad51 mutant diploid, BIR remained a viable option. In wild-type hap-loids, homologous sequences are present only at ectopic lo-cations. If a DSB is created such that sequences near the centro-mere-proximal end share hom-ology with sequences located at a different chromosome end, a nonreciprocal translocation can arise by BIR (Ref. 12). Murrow et al.13provided further strong evidence for BIR by showing that a transformed linearized DNA fragment can frequently ‘pick up’ an entire chromosome arm (several hundred kilobases) through a nonreciprocal, pre-sumably replicational, process. BIR might account for the way mammalian tumor cells that lack telomerase create nonrecipro-cal translocations and are able to maintain telomeres14. Indeed, in S. cerevisiae, the genetic re-quirements for maintaining telo-meres in the absence of telom-erase are the same as those defined for BIR (Ref. 15). Gene conversion
The DSB-repair model proposed by Szostak et al.16comprises the following: invasion of a template by two 39 single-stranded ends of the DSB; priming of new DNA synthesis from these 39 ends; and formation of Holliday junctions, the four-stranded, branched structures whose resolution can yield both cross-overs and noncrosscross-overs (Fig. 2a). In this model, the newly synthesized DNA is semiconservative – that is, one new strand is present in the donor, and one is present in the recipient.
Such a mechanism, however, does not account for several observations in organ-isms ranging from bacteria to mice. First, only a small proportion of mitotic gene conversions are associated with crossing over; this suggests either that Holliday junctions are preferentially resolved with-out exchange or that such structures do not form or are dismantled17,18. Sec-ond, heteroduplex DNA derived from 59–39 Resection
Strand invasion
New DNA synthesis
Holliday-junction resolution DSB DSB 59–39 Resection Strand invasion
New DNA synthesis
No crossing over Crossing over of flanking markers No crossing over Crossing over of
flanking markers
Strand annealing or
(a) (b)
Figure 2
Two models of gene conversion. (a) A version of the Szostak et al. model16 proposes that the two ends of
the double-strand break (DSB) are resected by 59–39 exonucleases, which allows the 39 ends to invade an intact homologous template and initiate new DNA synthesis. In the figure, the two DNA molecules are oriented such that strands of the same polarity are close together. Resolution of the pair of symmetrical Holliday junctions produces gene conversions without or with an accompanying crossover. Note that, in this model, newly synthesized DNA is found both in the donor molecule and in the recipient molecule and therefore mutations or rearrangements arising during DNA synthesis should occur in the donor and in the recipient with equal frequency. (b) A version of the synthesis-dependent strand-annealing model proposed by Nassif et al.18and modified by Ferguson and Holloman22and Pâques et al.20Strand invasion and new
DNA synthesis are initiated from either end of the DSB. Replication occurs in a D loop, and the newly synthesized strand is displaced from its template. The 39 single-stranded end at the other end of the DSB can anneal with the newly synthesized strand or it could anneal with the D loop to form a pair of Holliday junctions. The latter event allows synthesis-dependent strand annealing (SDSA) to be accom-panied by crossing over. Here, all, or nearly all, of the newly synthesized DNA is found in the recipient.
mismatches between the DSB-containing recipient locus and its donor template is rarely found in the donor, but frequently is found in the recipient. This second re-sult would not be anticipated if a ssDNA molecule produced after 59–39 resection of the ends of the DSB invaded the double-stranded donor sequence and remained uncorrected17. Third, when the donor template contains a set of repeated se-quences, as many as 50% of gene conver-sions exhibit expansion or contraction of the number of copies, and all of these rearrangements are found in the recipi-ent19,20. Consequently, several models for so-called synthesis-dependent strand annealing (SDSA) have emerged21.
SDSA can arise through a variation of the Szostak et al.16model: the two semi-conservatively replicated strands are un-wound from their donor templates and annealed. This places all the newly syn-thesized DNA in the repaired recipient locus, eliminates the Holliday junctions and, thus, precludes crossing over17. The annealing of newly synthesized strands could readily account for changes in the number of repeated sequences that are found only in the recipient locus. Alter-natively, Nassif et al.18suggest that the newly synthesized strands could be con-tinuously unwound from a migrating small replication bubble similar to that pro-posed after in vitro studies of DNA repli-cation (Fig. 2b). Usually, the newly synthe-sized strand anneals with the second end of the DSB, but Ferguson and Holloman22 recognized that the second end can also engage the replication bubble itself and, thereby, cause formation of a Holliday junction that can be resolved by cross-ing over. The proportion of crossover-associated events could be a function of the frequency with which this alternative structure forms (indeed in yeast meiosis, where crossing over is more frequent, mu-tations in five genes reduce the frequency of exchange to the level at which cross-ing over occurs in growcross-ing cells)23. Repair-replication-fork capture
Increasingly, BIR and gene conversion seem to be closely related processes. Gene conversion, as in the case of BIR, can begin with the invasion of a donor template by one end of a DSB. Invasion establishes a replication fork, and both leading- and lagging-strand DNA synthe-sis occur. Gene conversion can result if the fork engages the second end of the DSB, which could anneal either to the dis-placed template strand in the migrating replication fork (Fig. 3a, Part 3) or to the newly synthesized strand as it is unwound
from the replication fork (Fig. 3a, Part 4). In the latter case, no crossing over is associ-ated with gene conversion; in the former case, resolution of the intermediate struc-ture produces gene conversions with or without associated crossing over. If the second end is lost during mitosis or is degraded – for example, at chromosome ends in cells that lack telomerase – BIR occurs. In this case, replication proceeds to the end of the chromosome (Fig. 3b). Note that, if mitosis occurs prior to the repair of a DSB, the centromereless termi-nal fragment is likely to be lost, and the re-maining centromere-containing broken chromosome can be repaired only by BIR.
Recently, my group found that the re-pair replication fork involves leading- and lagging-strand DNA polymerases and sev-eral associated factors24. However, repli-cation during recombination might lack some components of the replication fork, such as an efficient helicase that could load only at origins of DNA replication. This might explain why repair replication is more likely to result in displacement of newly synthesized strands. It might also explain why gene conversion in yeast be-comes less efficient as the length of DNA to be copied increases from a few base pairs to 10 kb – a length that would not be a problem for the normal replication
(b) (a) 1 2 3 or or 4 5 1 2 3 4 Figure 3
Break-induced replication (BIR) and gene conversion can emerge from the same process. As in the model shown in Fig. 2b, repair of the double-strand break (DSB) begins with strand invasion from one end. A modified replication fork is established, involving both leading-and lagging-strleading-and synthesis. (a) In gene conversion, the second end of the DSB engages the replication structure and anneals, which terminates DNA synthesis and produces a patch of newly synthesized DNA. This can happen by annealing of the second end of the DSB with either the displaced template strand in the migrating replication bubble (Part 3) or the newly synthesized DNA as it unwinds (Part 4). In the former case, gene conversion can be accompanied by crossing over. (b) In BIR, the migrating replication structure progresses all the way to the end of the chromosome or until it meets a converging replication fork.
fork20. The model that we envisage also takes into account other genetic results by proposing that the two newly synthesized DNA strands are unwound and annealed at the recipient locus (Fig. 3b). This step might require a helicase for branch migr-ation; such a helicase would be similar to the RuvAB proteins of E. coli, which bind to and cause the migration of Holliday junctions. The RuvAB proteins play an im-portant role in origin-independent, recom-bination-dependent DNA synthesis9. Of course, the replication-fork-capture DSB-repair mechanism might compete with one or more of the mechanisms described above. The choice of mechanism could be influenced by whether both ends of the DSB have perfect homology to the donor. Genetic characterization of proteins involved in homologous recombination
Our understanding of the proteins in-volved in homologous recombinational re-pair of DSBs comes mostly from work in bacteria and bacteriophage, and work in yeast. In bacteria, the key recombination protein is RecA, which catalyses DNA strand exchange with a homologous du-plex molecule25. In bacteria that lack RecA, nearly all recombination is eliminated.
Eukaryotes have one or more evolution-arily conserved homologs of RecA. One homolog, Rad51, is expressed in all cells; another homolog, Dmc1, is expressed spe-cifically in meiotic cells. Rad51p and prob-ably Dmc1p carry out strand exchange in vitro and have properties that are similar but not identical to those of the bacterial protein21,26. Eukaryotes that lack Dmc1 exhibit severe defects in meiotic recom-bination27,28. In somatic vertebrate cells, Rad51 is essential for cell growth, and studies in DT40 cells reveal that many un-repaired lesions are presumably a con-sequence of DSBs created during DNA replication4.
Despite the universality of Rad51 pro-teins, Rad51p is not indispensable for all forms of DSB-induced recombination in S. cerevisiae. Rad51p is as essential as Rad52p for some processes, such as DSB-induced gene conversion involving two homologous chromosomes. It is, however, much less important, or even dispensable, for other types of events (e.g. spontaneous recombination and some types of known DSB-induced event)21,29; most notably, BIR can take place in the absence of Rad51p but not in the absence of Rad52p (Ref. 11). How strand invasion at the start of DNA replication can occur in the absence of a Rad51 homolog remains a mystery. The phenotype of a rad51 deletion in S. cere-visiae is very similar to those of deletions of rad54, rad55 and rad57 (Ref. 15 and L. Signon, A. Malkova and J. E. Haber, unpublished).
The most critical recombination protein in S. cerevisiae seems to be Rad52p, which is evolutionarily conserved in eukaryotes but not in bacteria. Nearly all types of DSB repair are eliminated in the absence of Rad52p. Curiously, the phenotype of a Rad52 knockout in vertebrate cells is not nearly as severe as that of the Rad51 knockout; but other as-yet-unidentified homologs of Rad52 might exist30. Indeed, Schizosaccharomyces pombe appears to possess two Rad52 homologs. Although deletion mutants that lack either gene are viable, the double mutant is lethal – cells appear to be blocked in late S phase (H. Okayama, pers. commun.). This ob-servation also makes more notable the reported absence of a rad52 homolog in Caenorhabditis elegans. Recently, a rad52 homolog, RAD59, was shown to affect some types of RAD51p-independent re-combination in S. cerevisiae31.
Rad55p and Rad57p share some hom-ology with Rad51p. Deletions of the yeast genes have a recombination defect
only at low temperature, and this defect can be suppressed by overexpression of rad51 (Ref. 32). These proteins might help to load Rad51p onto ssDNA, much as the RecF, RecO and RecR proteins appear to do in E. coli. Vertebrate cells also have several additional proteins that share some homology with Rad51: Rad51B, Rad51C, XRCC2 and XRCC3. These proteins are also implicated in re-pair of DNA damage and might carry out roles similar to those of yeast Rad55p and Rad57p. In DT40 cells, these homo-logs are not essential but are important for recombination and repair (E. Sonoda and S. Takeda, pers. commun.).
Rad54p is a member of the large, Swi2/Snf2 family of putative chromatin-remodeling proteins, members of which share homology with helicases, but the precise role of Rad54p in recombination is unclear. As in yeast, the absence of Rad54p in vertebrates causes increased radiation sensitivity and a marked reduc-tion in homologous gene targeting3,33. Yeast Rad54p has a homolog, Tid1p (also known as Rdh54p), and vertebrate homo-logs might also exist. Tid1p preferen-tially affects recombination between hom-ologous chromosomes (as opposed to sister chromatids); this role is reminis-cent of the interhomolog-specific role of Dmc1p in meiosis34,35. Tid1p physically interacts with Dmc1p and, apparently less strongly, with Rad51p (Ref. 36). How it par-ticipates in mitotic recombination remains obscure. In diploids, the recombination defect in the tid1 rad54 double mutant is as severe as that in the rad52 mutant. Biochemical properties of proteins that catalyse homologous recombination
The most critical recombination pro-tein, Rad52p, forms multimeric rings that can both bind to DNA ends and promote the annealing of complementary DNA RPA Rad51 Rad52 Rad55 Rad57 Rad54 Figure 4
Reconstruction of early steps of recombination in vitro. Eukaryotic proteins can stimulate strand exchange between a single-stranded circular molecule and a homologous linear dsDNA, similar to the way this process is carried out by RecA and other bacterial proteins25,26. Loading of
Rad51 protein to create a filament along ssDNA is facilitated by replication factor A (RPA) and by the Rad55–Rad57 heterodimer. Early steps in strand exchange also depend on Rad52, which interacts with both Rad51 and RPA, and on Rad54, which interacts with Rad51. Strand exchange takes place within the Rad51 filament.
strands37; human RAD52 has similar properties38. Rad52p associates with Rad51p38–40and facilitates Rad51p-medi-ated strand exchange41,42. Rad52p also interacts with the trimeric ssDNA-binding complex replication factor A (RPA)43. RPA is essential for DNA replication, but a separation-of-function mutation in the largest subunit, rfa1-t11, nearly abolishes both gene conversion and single-strand annealing but does not impair repli-cation44. Rad51p and/or Rad52p geneti-cally and physigeneti-cally interact with several other important recombination proteins: Rad54p (Ref. 45), plus a Rad55p–Rad57p heterodimer46,47. None of these proteins appears to have a role in non-homologous end-joining.
The standard model for studies of RecA/Rad51-mediated strand exchange involves the transfer of one strand of lin-ear duplex DNA to a RecA (or Rad51)-coated single-stranded circle (Fig. 4). In various combinations, the other recombi-nation proteins improve Rad51p-mediated strand exchange in vitro, but how they all act in vivo – either in concert as part of a ‘recombinosome’46or sequentially – has not been established.
In vitro, yeast and human Rad51, as in the case of RecA, form filaments on ssDNA that then interact with homologous du-plex DNA. The filament extends the length of the dsDNA helix by 50%48, and recent NMR studies by Nishinaka et al.49, both on the bacterial protein and on the yeast protein, suggest that there are in fact two extended states, which have different heli-cal pitches that are dictated by the puck-ering of the deoxyribose sugar. Strand ex-change might therefore be driven by this rotation of the DNA inside the filament. Monitoring of recombination in vivo
Recently, cytological studies have ex-amined the requirement for the formation of subnuclear complexes (foci) by Rad51p during meiosis in budding yeast. These foci are presumably sites of DNA-strand exchange. Deletion of rad52, rad55 or rad57 prevents formation of foci by Rad51p both in normal meiosis and after exposure to ionizing radiation50. This ap-pears to be a very useful new approach to examining the early sequence of events that follows the appearance of DSBs.
Another valuable approach to study-ing the mechanisms of DSB repair and to learning about the in vivo roles of recom-bination proteins is to monitor the repair of a specific DSB induced synchronously in a large population of cells. One sys-tem that has been intensively studied is HO-endonuclease-induced switching of
mating-type genes in S. cerevisiae21. This gene-conversion process is surprisingly slow (~1 hr in duration) and permits the identification of several intermediate steps, including the 59–39 resection of DNA ends and the start of new DNA synthesis after strand invasion. Analysis of tem-perature-sensitive mutants of the genes that encode DNA polymerases and of many associated factors showed that the clamp protein PCNA and the clamp-loading replication factor C complex are required. Replication itself requires both PCNA-associated polymerases, Pold and Pole; by contrast, these two DNA polym-erases appear to play at least partially redundant roles in repair24. Most surpris-ingly, inactivation of Pola or primase, which are responsible for lagging-strand synthesis, nearly eliminated DSB repair – even in cells held in G1, in which DNA polymerases do not assemble at origins of replication and, hence, are not trapped at blocked replication forks24. The dis-covery that DSB repair requires leading-strand and lagging-leading-strand DNA polym-erases adds weight to a growing body of data that supports the new models of re-combination discussed above (Fig. 3a).
The dialog provoked by in vivo and in vitro experiments should catalyse further refinements in our understanding of these recombination processes and of their im-portance in sustaining normal replication, as well as the repair of other instances of broken chromosomes. But there is much left to do. We still do not have an in vitro system that will carry out a com-plete recombination reaction. Moreover, genes involved in such processes as the resolution of Holliday junctions remain to be found in eukaryotes, and there are probably several helicases and other important recombination components that have not been identified by genetic or biochemical approaches.
A clearer vision of the way that re-combination proceeds will also have a significant impact on several other im-portant problems in mammalian biology – for example, the problems of how to combat the immortalization of tumor cells that lack telomerase and how to improve the accuracy of gene targeting. References
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