Characterisation of a new reporter system allowing high throughput in planta screening for recombination events before and after controlled DNA double strand break induction

DNA double strand breaks (DSBs) are created either by DNA damaging reagents or in a programmed manner, for example during meiosis. Homologous recombination (HR) can be used to repair DSBs, a process vital both for cell survival and for genetic rearrangement during meiosis. In order to easily quantify this mechanism, a new HR reporter gene that is suitable for the detection of rare recombination events in high-throughput screens was developed in Arabidopsis thaliana. This reporter, pPNP, is composed of two mutated Pat genes and has also one restriction site for the meganuclease I-SceI. A functional Pat gene can be reconstituted by an HR event giving plants which are resistant to the herbicide glufosinate. The basal frequency of intra-chromosomal recombination is very low (10^?5) and can be strongly increased by the expression of I-SceI which creates a DSB. Expression of I-SceI under the control of the 35S CaMV promoter dramatically increases HR frequency (10,000 fold); however the measured recombinant events are in majority somatic. In contrast only germinal recombination events were measured when the meganuclease was expressed from a floral-specific promoter. Finally, the reporter was used to test a dexamethasone inducible I-SceI which could produce up to 200× more HR events after induction. This novel inducible I-SceI should be useful in fundamental studies of the mechanism of repair of DSBs and for biotechnological applications.     

Understanding the origins of UV-induced recombination through manipulation of sister chromatid cohesion

Ultraviolet light (UV) can provoke genome instability, partly through its ability to induce homologous recombination (HR). However, the mechanism(s) of UV-induced recombination is poorly understood. Although double-strand breaks (DSBs) have been invoked, there is little evidence for their generation by UV. Alternatively, single-strand DNA lesions that stall replication forks could provoke recombination. Recent findings suggest efficient initiation of UV-induced recombination in G₁ through processing of closely spaced single-strand lesions to DSBs. However, other scenarios are possible, since the recombination initiated in G₁ can be completed in the following stages of the cell cycle. We developed a system that could address UV-induced recombination events that start and finish in G₂ by manipulating the activity of the sister chromatid cohesion complex. Here we show that sister-chromatid cohesion suppresses UV-induced recombination events that are initiated and resolved in G₂. By comparing recombination frequencies and survival between UV and ionizing radiation, we conclude that a substantial portion of UV-induced recombination occurs through DSBs. This notion is supported by a direct physical observation of UV-induced DSBs that are dependent on nucleotide excision repair. However, a significant role of nonDSB intermediates in UV-induced recombination cannot be excluded.     

Understanding the origins of UV-induced recombination through manipulation of sister chromatid cohesion

Ultraviolet light (UV) can provoke genome instability, partly through its ability to induce homologous recombination (HR). However, the mechanism(s) of UV-induced recombination is poorly understood. Although double-strand breaks (DSBs) have been invoked, there is little evidence for their generation by UV. Alternatively, single-strand DNA lesions that stall replication forks could provoke recombination. Recent findings suggest efficient initiation of UV-induced recombination in G₁ through processing of closely spaced single-strand lesions to DSBs. However, other scenarios are possible, since the recombination initiated in G₁ can be completed in the following stages of the cell cycle. We developed a system that could address UV-induced recombination events that start and finish in G₂ by manipulating the activity of the sister chromatid cohesion complex. Here we show that sister-chromatid cohesion suppresses UV-induced recombination events that are initiated and resolved in G₂. By comparing recombination frequencies and survival between UV and ionizing radiation, we conclude that a substantial portion of UV-induced recombination occurs through DSBs. This notion is supported by a direct physical observation of UV-induced DSBs that are dependent on nucleotide excision repair. However, a significant role of nonDSB intermediates in UV-induced recombination cannot be excluded.     

Genetic Evidence for Single-Strand Lesions Initiating Nbs1-Dependent Homologous Recombination in Diversification of Ig V in Chicken B Lymphocytes

Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3′ single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1^p70) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate—though not the fidelity—of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3′ to 5′ single-strand–specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate—single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases.     

Mammalian meiosis involves DNA double-strand breaks with 3′ overhangs

Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3′ single-strand overhangs that are active in homology search, repair and formation of recombinant molecules. Are 3′ overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme. We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules. Signals showing 3′ overhangs were observed during wild-type mouse spermatogenesis, but not in Spo11 ^?/? males, which lack meiotic DSBs. In Atm ^?/? mice, abundant positively stained spermatocytes were present, indicating an accumulation of non-repaired DSBs, suggesting the involvement of ATM in repair of meiotic DSBs. Thus the processing of DSBs into 3′ overhangs is common to meiotic cells in mammals and yeast, and probably in all eukaryotes.     

Dominant negative mutant of Plasmodium Rad51 causes reduced parasite burden in host by abrogating DNA double‐strand break repair

Malaria parasites survive through repairing a plethora of DNA double‐stranded breaks (DSBs) experienced during their asexual growth. In Plasmodium Rad51 mediated homologous recombination (HR) mechanism and homology‐independent alternative end‐joining mechanism have been identified. Here we address whether loss of HR activity can be compensated by other DSB repair mechanisms. Creating a transgenic Plasmodium line defective in HR function, we demonstrate that HR is the most important DSB repair pathway in malarial parasite. Using mouse malaria model we have characterized the dominant negative effect of PfRad51^K143R mutant on Plasmodium DSB repair and host–parasite interaction. Our work illustrates that Plasmodium berghei harbouring the mutant protein (PfRad51^K143R) failed to repair DSBs as evidenced by hypersensitivity to DNA‐damaging agent. Mice infected with mutant parasites lived significantly longer with markedly reduced parasite burden. To better understand the effect of mutant PfRad51^K143R on HR, we used yeast as a surrogate model and established that the presence of PfRad51^K143R completely inhibited DNA repair, gene conversion and gene targeting. Biochemical experiment confirmed that very low level of mutant protein was sufficient for complete disruption of wild‐type PfRad51 activity. Hence our work provides evidence that HR pathway of Plasmodium could be efficiently targeted to curb malaria.     

MRE11 and COM1/SAE2 are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena

Programmed DNA double-strand breaks (DSBs) are generated during meiosis to initiate homologous recombination. Various aspects of DSB formation, signaling, and repair are accomplished or governed by Mre11, a component of the MRN/MRX complex, partially in cooperation with Com1/Sae2/CtIP. We used Tetrahymena to study evolutionarily conserved and changed functions of Mre11 and Com1. There is a difference between organisms with respect to the dependency of meiotic DSB formation on Mre11. By cytology and an electrophoresis-based assay for DSBs, we found that in Tetrahymena Mre11p is not required for the formation and ATR-dependent signaling of DSBs. Its dispensability is also reflected by wild-type-like DSB-dependent reorganization of the meiotic nucleus and by the phosphorylation of H2A.X in mre11∆ mutant. However, mre11∆ and com1∆ mutants are unable to repair DSBs, and chromosome pairing is reduced. It is concluded that, while MRE11 has no universal role in DNA damage signaling, its requirement for DSB repair is conserved between evolutionarily distant organisms. Moreover, reduced chromosome pairing in repair-deficient mutants reveals the existence of two complementing pairing processes, one by the rough parallel arrangement of chromosomes imposed by the tubular shape of the meiotic nucleus and the other by repair-dependent precise sequence matching.     

Bacterial NHEJ: a never ending story

Double‐strand breaks (DSBs) are the most detrimental DNA damage encountered by bacterial cells. DBSs can be repaired by homologous recombination thanks to the availability of an intact DNA template or by Non‐Homologous End Joining (NHEJ) when no intact template is available. Bacterial NHEJ is performed by sets of proteins of growing complexity from Bacillus subtilis and Mycobacterium tuberculosis to Streptomyces and Sinorhizobium meliloti. Here, we discuss the contribution of these models to the understanding of the bacterial NHEJ repair mechanism as well as the involvement of NHEJ partners in other DNA repair pathways. The importance of NHEJ and of its complexity is discussed in the perspective of regulation through the biological cycle of the bacteria and in response to environmental stimuli. Finally, we consider the role of NHEJ in genome evolution, notably in horizontal gene transfer.     

Early steps of double-strand break repair in Bacillus subtilis

All organisms rely on integrated networks to repair DNA double-strand breaks (DSBs) in order to preserve the integrity of the genetic information, to re-establish replication, and to ensure proper chromosomal segregation. Genetic, cytological, biochemical and structural approaches have been used to analyze how Bacillus subtilis senses DNA damage and responds to DSBs. RecN, which is among the first responders to DNA DSBs, promotes the ordered recruitment of repair proteins to the site of a lesion. Cells have evolved different mechanisms for efficient end processing to create a 3′-tailed duplex DNA, the substrate for RecA binding, in the repair of one- and two-ended DSBs. Strand continuity is re-established via homologous recombination (HR), utilizing an intact homologous DNA molecule as a template. In the absence of transient diploidy or of HR, however, two-ended DSBs can be directly re-ligated via error-prone non-homologous end-joining. Here we review recent findings that shed light on the early stages of DSB repair in Firmicutes.     

Capture of linear fragments at a double-strand break in yeast

Double-strand breaks (DSBs) are dangerous chromosomal lesions that must be efficiently repaired in order to avoid loss of genetic information or cell death. In all organisms studied to date, two different mechanisms are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Previous studies have shown that during DSB repair, non-homologous exogenous DNA (also termed ‘filler DNA’) can be incorporated at the site of a DSB. We have created a genetic system in the yeast Saccharomyces cerevisiae to study the mechanism of fragment capture. Our yeast strains carry recognition sites for the HO endonuclease at a unique chromosomal site, and plasmids in which a LEU2 gene is flanked by HO cut sites. Upon induction of the HO endonuclease, a linear extrachromosomal fragment is generated in each cell and its incorporation at the chromosomal DSB site can be genetically monitored. Our results show that linear fragments are captured at the repaired DSB site at frequencies of 10⁻⁶ to 10⁻⁴ per plated cell depending on strain background and specific end sequences. The mechanism of fragment capture depends on the NHEJ machinery, but only partially on the homologous recombination proteins. More than one fragment can be used during repair, by a mechanism that relies on the annealing of small complementary sequences. We present a model to explain the basis for fragment capture.     

Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.     

Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.     

Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.     

Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells

DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G₂ and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G₁/G₀ (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G₁/G₀ cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G₁/G₀ and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.     

Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

Background  

DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage.     

Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

Background  

Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.     

Saccharomyces cerevisiae-based system for studying clustered DNA damages

DNA-damaging agents can induce clustered lesions or multiply damaged sites (MDSs) on the same or opposing DNA strands. In the latter, attempts to repair MDS can generate closely opposed single-strand break intermediates that may convert non-lethal or mutagenic base damage into double-strand breaks (DSBs). We constructed a diploid S. cerevisiae yeast strain with a chromosomal context targeted by integrative DNA fragments carrying different damages to determine whether closely opposed base damages are converted to DSBs following the outcomes of the homologous recombination repair pathway. As a model of MDS, we studied clustered uracil DNA damages with a known location and a defined distance separating the lesions. The system we describe might well be extended to assessing the repair of MDSs with different compositions, and to most of the complex DNA lesions induced by physical and chemical agents.     

Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

V(D)J recombination and class switch recombination are the two DNA rearrangement events used to diversify the mouse and human antibody repertoires. While their double strand breaks (DSBs) are initiated by different mechanisms, both processes use non-homologous end joining (NHEJ) in the repair phase. DNA mismatch repair elements (MSH2/MSH6) have been implicated in the repair of class switch junctions as well as other DNA DSBs that proceed through NHEJ. MSH2 has also been implicated in the regulation of factors such as ATM and the MRN (Mre11, Rad50, Nbs1) complex, which are involved in V(D)J recombination. These findings led us to examine the role of MSH2 in V(D)J repair. Using MSH2-/- and MSH2+/+ mice and cell lines, we show here that all pathways involving MSH2 are dispensable for the generation of an intact pre-immune repertoire by V(D)J recombination. In contrast to switch junctions and other DSBs, the usage of terminal homology in V(D)J junctions is not influenced by MSH2. Thus, whether the repair complex for V(D)J recombination is of a canonical NHEJ type or a separate microhomology-mediated-end joining (MMEJ) type, it does not involve MSH2. This highlights a distinction between the repair of V(D)J recombination and other NHEJ reactions.