The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution

The efficient repair of double-strand breaks (DSBs) in genomic DNA is important for the survival of all organisms. In recent years, basic mechanisms of DSB repair in somatic plant cells have been elucidated. DSBs are mainly repaired by non-homologous end-joining (NHEJ). The repair can be associated with deletions, but also insertions due to copying genomic sequences from elsewhere into the break. Species-specific differences of NHEJ have been reported and an inverse correlation of deletion size to genome size has been postulated, indicating that NHEJ might contribute significantly to evolution of genome size. DSB repair by homologous recombination (HR) might also influence genome organization. Whereas homology present in an allelic or an ectopic position is hardly used for repair, the use of homologous sequences in close proximity to the break is frequent. A 'single-strand annealing' mechanism that leads to sequence deletions between direct repeats is particularly efficient. This might explain the accumulation of single long terminal repeats of retroelements in cereal genomes. The conservative 'synthesis-dependent strand annealing' mechanism, resulting in conversions without crossovers is also prominent and seems to be significant for the evolution of tandemly arranged gene families such as resistance genes. Induction of DSBs could be used as a means for the controlled manipulation of plant genomes in an analogous way for the use of marker gene excision and site-specific integration.     

Microhomology-dependent end joining and repair of transposon-induced DNA hairpins by host factors in Saccharomyces cerevisiae

The maize, cut-and-paste transposon Ac/Ds is mobile in Saccharomyces cerevisiae, and DNA sequences of repair products provide strong genetic evidence that hairpin intermediates form in host DNA during this transposition, similar to those formed for V(D)J coding joints in vertebrates. Both DNA strands must be broken for Ac/Ds to excise, suggesting that double-strand break (DSB) repair pathways should be involved in repair of excision sites. In the absence of homologous template, as expected, Ac excisions are repaired by nonhomologous end joining (NHEJ) that can involve microhomologies close to the broken ends. However, unlike repair of endonuclease-induced DSBs, repair of Ac excisions in the presence of homologous template occurs by gene conversion only about half the time, the remainder being NHEJ events. Analysis of transposition in mutant yeast suggests roles for the Mre11/Rad50 complex, SAE2, NEJ1, and the Ku complex in repair of excision sites. Separation-of-function alleles of MRE11 suggest that its endonuclease function is more important in this repair than either its exonuclease or Rad50-binding properties. In addition, the interstrand cross-link repair gene PSO2 plays a role in end joining hairpin ends that is not seen in repair of linearized plasmids and may be involved in positioning transposase cleavage at the transposon ends.     

Effect of double-strand break DNA sequence on the PARP-1 NHEJ pathway

Efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by non-homologous end-joining (NHEJ). We have previously described a new DSBs microhomology end-joining pathway depending on PARP-1 and the XRCC1/DNA ligase III complex. In this study we analysed, with recombinant proteins and protein extracts, the effect of DSB end sequences: (i) on the DSB synapsis activity; (ii) on the end-joining activity. We report that PARP-1 DSB synapsis activity is independent of the DSB sequence and could be detected with non-complementary DSBs. We demonstrate also that the efficiency of DSBs repair by PARP-1 NHEJ is strongly dependent on the presence of G:C base pairs at microhomology termini. These results highlight a new role of the PARP-1 protein on the synapsis of DSBs and could explain why the PARP-1 NHEJ pathway is strongly dependent on the DSBs microhomology sequence.     

Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis

DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.     

RNA-directed repair of DNA double-strand breaks

DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity.     

Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes

To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.     

Induction of base damages representing a high risk site for double-strand DNA break formation in genomic DNA by exposure of cells to DNA damaging agents

DNA repair is known as a defense mechanism against genotoxic insults. However, the most lethal type of DNA damages, double-strand DNA breaks (DSBs), can be produced by DNA repair. We have previously demonstrated that when long patch base excision repair attempts to repair a synthetic substrate containing two uracils, the repair produces DSBs (Vispe, S. and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392 and Vispe, S., Ho, E. L., Yung, T. M., and Satoh, M. S. (2003) J. Biol. Chem. 278, 35279-35285). In this synthetic substrate, the two uracils are located on the opposite DNA strands (separated by an intervening sequence stable at 37 degrees C) and represent a high risk site for DSB formation. It is not clear, however, whether similar high risk sites are also induced in genomic DNA by exposure to DNA damaging agents. Thus, to investigate the mechanisms of DSB formation, we have modified the DSB formation assay developed previously and demonstrated that high risk sites for DSB formation are indeed generated in genomic DNA by exposure of cells to alkylating agents. In fact, genomic DNA containing alkylated base damages, which could represent high risk sites, are converted into DSBs by enzymes present in extracts prepared from cells derived from clinically normal individuals. Furthermore, DSBs are also produced by extracts from cells derived from ataxia-telangiectasia patients who show cancer proneness due to an impaired response to DSBs. These results suggest the presence of a novel link between base damage formation and DSBs and between long patch base excision repair and human diseases that occur due to an impaired response to DSB.     

The influence of heterochromatin on DNA double strand break repair: Getting the strong, silent type to relax

DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the major DNA double strand break (DSB) pathways in mammalian cells, whilst ataxia telangiectasia mutated (ATM) lies at the core of the DSB signalling response. ATM signalling plays a major role in modifying chromatin structure in the vicinity of the DSB and increasing evidence suggests that this function influences the DSB rejoining process. DSBs have long been known to be repaired with two (or more) component kinetics. The majority (～85%) of DSBs are repaired with fast kinetics in a predominantly ATM-independent manner. In contrast, ～15% of radiation-induced DSBs are repaired with markedly slower kinetics via a process that requires ATM and those mediator proteins, such as MDC1 or 53BP1, that accumulate at ionising radiation induced foci (IRIF). DSBs repaired with slow kinetics predominantly localise to the periphery of genomic heterochromatin (HC). Indeed, there is mounting evidence that chromatin complexity and not damage complexity confers slow DSB repair kinetics. ATM's role in HC-DSB repair involves the direct phosphorylation of KAP-1, a key HC formation factor. KAP-1 phosphorylation (pKAP-1) arises in both a pan-nuclear and a focal manner after radiation and ATM-dependent pKAP-1 is essential for DSB repair within HC regions. Mediator proteins such as 53BP1, which are also essential for HC-DSB repair, are expendable for pan-nuclear pKAP-1 whilst being essential for pKAP-1 formation at IRIF. Data suggests that the essential function of the mediator proteins is to promote the retention of activated ATM at DSBs, concentrating the phosphorylation of KAP-1 at HC DSBs. DSBs arising in G2 phase are also repaired with fast and slow kinetics but, in contrast to G0/G1 where they all DSBs are repaired by NHEJ, the slow component of DSB repair in G2 phase represents an HR process involving the Artemis endonuclease. Results suggest that whilst NHEJ repairs the majority of DSBs in G2 phase, Artemis-dependent HR uniquely repairs HC DSBs. Collectively, these recent studies highlight not only how chromatin complexity influences the factors required for DSB repair but also the pathway choice.     

Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae

Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.     

Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks

The Drosophila element Mos1 is a class II transposon, which moves by a 'cut-and-paste' mechanism and can be experimentally mobilized in the Caenorhabditis elegans germ line. Here, we triggered the excision of identified Mos1 insertions to create chromosomal breaks at given sites and further manipulate the broken loci. Double-strand break (DSB) repair could be achieved by gene conversion using a transgene containing sequences homologous to the broken chromosomal region as a repair template. Consequently, mutations engineered in the transgene could be copied to a specific locus at high frequency. This pathway was further characterized to develop an efficient tool--called MosTIC--to manipulate the C. elegans genome. Analysis of DSB repair during MosTIC experiments demonstrated that DSBs could also be sealed by end-joining in the germ line, independently from the evolutionarily conserved Ku80 and ligase IV factors. In conjunction with a publicly available Mos1 insertion library currently being generated, MosTIC will provide a general tool to customize the C. elegans genome.     

Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.     

Role of Double-Strand Break End-Tethering during Gene Conversion in Saccharomyces cerevisiae

Correct repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Whereas gene conversion (GC)-mediated repair is mostly error-free, repair by break-induced replication (BIR) is associated with non-reciprocal translocations and loss of heterozygosity. We have previously shown that a Recombination Execution Checkpoint (REC) mediates this competition by preventing the BIR pathway from acting on DSBs that can be repaired by GC. Here, we asked if the REC can also determine whether the ends that are engaged in a GC-compatible configuration belong to the same break, since repair involving ends from different breaks will produce potentially deleterious translocations. We report that the kinetics of repair are markedly delayed when the two DSB ends that participate in GC belong to different DSBs (termed Trans) compared to the case when both DSB ends come from the same break (Cis). However, repair in Trans still occurs by GC rather than BIR, and the overall efficiency of repair is comparable. Hence, the REC is not sensitive to the “origin” of the DSB ends. When the homologous ends for GC are in Trans, the delay in repair appears to reflect their tethering to sequences on the other side of the DSB that themselves recombine with other genomic locations with which they share sequence homology. These data support previous observations that the two ends of a DSB are usually tethered to each other and that this tethering facilitates both ends encountering the same donor sequence. We also found that the presence of homeologous/repetitive sequences in the vicinity of a DSB can distract the DSB end from finding its bona fide homologous donor, and that inhibition of GC by such homeologous sequences is markedly increased upon deleting Sgs1 but not Msh6.     

Effect of gravity stress on fidelity of DNA double-strand break repair.

DNA double strand break (DSB) causes many cytotoxic effects such as cellular lethality, somatic mutation, and carcinogenesis. Fidelity of DSB repair is a important factor that determines the quality of genomic stability. It is known that the most of DSBs are properly repaired on the earth, however, little is known whether those are rejoined at the same fidelity even under the space environment. One of the DSB repair pathway, homologous recombination (HR), allows the cells to repair their DSBs with error free. Therefore, the efficiency of HR is a good index to assess the fidelity of DSB repair. In order to clarify the effect of gravity stress on HR pathway, we established a cell line that can detect a site-specific DNA repair via HR. The cells carrying a reporter construct for HR were incubated under hypergravity condition after induction of site specific DSB. Our preliminary results suggest that the gravity stress may affect the HR efficiency.     

Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events.

In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the MAT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2- or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and filling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.     

Chromosomal aberrations induced by double strand DNA breaks

It has been suggested that introduction of double strand DNA breaks (DSBs) into mammalian chromosomes can lead to gross chromosomal rearrangements through improper DNA repair. To study this phenomenon, we employed a model system in which a double strand DNA break can be produced in human cells in vivo at a predetermined location. The ensuing chromosomal changes flanking the breakage site can then be cloned and characterized. In this system, the recognition site for the I-SceI endonuclease, whose 18 bp recognition sequence is not normally found in the human genome, is placed between a strong constitutive promoter and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker. We found that the most common mutation following aberrant DSB repair was an interstitial deletion; these deletions typically showed features of non-homologous end joining (NHEJ), such as microhomologies and insertions of direct or inverted repeat sequences. We also detected more complex rearrangements, including large insertions from adjacent or distant genomic regions. The insertion events that involved distant genomic regions typically represented transcribed sequences, and included both L1 LINE elements and sequences known to be involved in genomic rearrangements. This type of aberrant repair could potentially lead to gene inactivation via deletion of coding or regulatory sequences, or production of oncogenic fusion genes via insertion of coding sequences.     

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.     

Malaria parasites utilize both homologous recombination and alternative end joining pathways to maintain genome integrity

Malaria parasites replicate asexually within their mammalian hosts as haploid cells and are subject to DNA damage from the immune response and chemotherapeutic agents that can significantly disrupt genomic integrity. Examination of the annotated genome of the parasite Plasmodium falciparum identified genes encoding core proteins required for the homologous recombination (HR) pathway for repairing DNA double-strand breaks (DSBs), but surprisingly none of the components of the canonical non-homologous end joining (C-NHEJ) pathway were identified. To better understand how malaria parasites repair DSBs and maintain genome integrity, we modified the yeast I-SceI endonuclease system to generate inducible, site-specific DSBs within the parasite’s genome. Analysis of repaired genomic DNA showed that parasites possess both a typical HR pathway resulting in gene conversion events as well as an end joining (EJ) pathway for repair of DSBs when no homologous sequence is available. The products of EJ were limited in number and identical products were observed in multiple independent experiments. The repair junctions frequently contained short insertions also found in the surrounding sequences, suggesting the possibility of a templated repair process. We propose that an alternative end-joining pathway rather than C-NHEJ, serves as a primary method for repairing DSBs in malaria parasites.