Role of budding yeast Rad18 in repair of HO-induced double-strand breaks

The Rad6-Rad18 complex mono-ubiquitinates proliferating cell nuclear antigen (PCNA) at the lysine 164 residue after DNA damage and promotes DNA polymerase eta (Poleta)- and Polzeta/Rev1-dependent DNA synthesis. Double-strand breaks (DSBs) of DNA can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ), both of which require new DNA synthesis. HO endonuclease introduces DSBs into specific DNA sequences. We have shown that Polzeta and Rev1 localize to HO-induced DSBs in a Mec1-dependent manner and promote Ku-dependent DSB repair. However, Polzeta and Rev1 localize to DSBs independently of PCNA ubiquitination. Here we provide evidence indicating that Rad18-mediated PCNA ubiquitination stimulates DNA synthesis by Polzeta and Rev1 in repair of HO-induced DSBs. Ubiquitination defective PCNA mutation or rad18Delta mutation confers the same DSB repair defect as rev1Delta mutation. Consistent with a role in DSB repair, Rad18 localizes to HO-induced DSBs in a Rad6-dependent manner. Unlike Polzeta or Rev1, Poleta is dispensable for repair of HO-induced DSBs. Ku and DNA ligase IV constitute a central NHEJ pathway. We also show that Polzeta and Rev1 act in the same pathway as DNA ligase IV, suggesting that Polzeta and Rev1 are involved in DNA synthesis during NHEJ. Our results suggest that Polzeta-Rev1 accumulates at regions near DSBs independently of PCNA ubiquitination and then interacts with ubiquitinated PCNA to facilitate DNA synthesis.     

Double-strand breaks repair by gene conversion in silkworm holocentric chromosomes

Maintenance of genome stability relies on the accurate repair of DNA double-strand breaks (DSBs) that arise during DNA replication or introduced by DNA-damaging agents. Failure to repair such breaks can lead to the introduction of mutations and chromosomal translocations. Several pathways, homologous recombination, single-strand annealing and nonhomologous end-joining, are known to repair DSBs. So far in the silkworm Bombyx mori, these repair pathways have been analyzed using extrachromosomal plasmids in vitro or in cultured cells. To elucidate the precise nature of the chromosomal DSB repair pathways in cultured silkworm cells, we developed a luciferase-based assay system for measuring the frequency of chromosomal homologous recombination and SSA. An I-SceI-induced DSB, within a nonfunctional luciferase gene, could be efficiently repaired by HR. Additionally, the continuous expression of the I-SceI endonuclease in the HR reporter cell allowed us to investigate the interrelationship between HR, SSA and NHEJ. In this study, we demonstrated that chromosome DSBs were mainly repaired by NHEJ and HR, whereas SSA was unlikely to be a dominant repair pathway in cultured silkworm cell. These results indicate that the assay system presented here will be useful to analyze the mechanisms of DSB repair in insect cells.     

Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae

The ends of chromosomal DNA double-strand breaks (DSBs) can be accurately rejoined by at least two discrete pathways, homologous recombination and nonhomologous end-joining (NHEJ). The NHEJ pathway is essential for repair of specific classes of DSB termini in cells of the budding yeast Saccharomyces cerevisiae. Endonuclease-induced DSBs retaining complementary single-stranded DNA overhangs are repaired efficiently by end-joining. In contrast, damaged DSB ends (e.g., termini produced by ionizing radiation) are poor substrates for this pathway. NHEJ repair involves the functions of at least 10 genes, including YKU70, YKU80, DNL4, LIF1, SIR2, SIR3, SIR4, RAD50, MRE11, and XRS2. Most or all of these genes are required for efficient recombination-independent recircularization of linearized plasmids and for rejoining of EcoRI endonuclease-induced chromosomal DSBs in vivo. Several NHEJ mutants also display aberrant processing and rejoining of DSBs that are generated by HO endonuclease or formed spontaneously in dicentric plasmids. In addition, all NHEJ genes except DNL4 and LIF1 are required for stabilization of telomeric repeat sequences. Each of the proteins involved in NHEJ appears to bind, directly or through protein associations, with the ends of linear DNA. Enzymatic and/or structural roles in the rejoining of DSB termini have been postulated for several proteins within the group. Most yeast NHEJ genes have homologues in human cells and many biochemical activities and protein:protein interactions have been conserved in higher eucaryotes. Similarities and differences between NHEJ repair in yeast and mammalian cells are discussed.     

NHEJ-deficient DT40 cells have increased levels of immunoglobulin gene conversion: evidence for a double strand break intermediate

Activation-induced cytidine deaminase (AID) likely initiates immunoglobulin gene-conversion (GC) by deaminating cytidines within the V-region of chicken B-cells. However, the intervening DNA lesion required to initiate GC remains elusive. GC could be initiated by a single strand break or a double strand break (DSB). To distinguish between these possibilities, we examined GC in the chicken DT40 B cell line deficient in non-homologous end joining (NHEJ). It is known that the NHEJ and homologous recombination DNA repair pathways compete for DSBs. In light of this, if a DSB is the major intermediate, deficiency in NHEJ should result in increased levels of GC. Here we show that DNA–PKcs^−/−/− and Ku70^−/− DT40 cells had 5- to 10-fold higher levels of GC relative to wildtype DT40 as measured by surface IgM reversion and sequencing of the V-region. These data suggest that a DSB is the major DNA lesion that initiates GC.     

Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance

DNA double strand breaks (DSBs) are one of the most cytotoxic forms of DNA damage and must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of plant NHEJ mutants atlig4-4 and atku80 with the neutral comet assay shows that alternative DSB repair pathways are active. Surprisingly, these kinetic measurements show that DSB repair was faster in the NHEJ mutant lines than in wild-type Arabidopsis.Here we provide the first characterization of this KU-independent, rapid DSB repair pathway operating in Arabidopsis. The alternate pathway that rapidly removes the majority of DSBs present in nuclear DNA depends upon structural maintenance of chromosomes (SMC) complex proteins, namely MIM/AtRAD18 and AtRAD21.1. An absolute requirement for SMC proteins and kleisin for rapid repair of DSBs in Arabidopsis opens new insight into the mechanism of DSB removal in plants.     

Changes in DNA double-strand break repair during aging correlate with an increase in genomic mutations

A double -strand break (DSB) is one of the most deleterious forms of DNA damage. In eukaryotic cells, two main repair pathways have evolved to repair DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is the predominant pathway of repair in the unicellular eukaryotic organism, S. cerevisiae. However, during replicative aging the relative use of HR and NHEJ shifts in favor of end-joining repair. By monitoring repair events in the HO-DSB system, we find that early in replicative aging there is a decrease in the association of long-range resection factors, Dna2-Sgs1 and Exo1 at the break site and a decrease in DNA resection. Subsequently, as aging progressed, the recovery of Ku70 at DSBs decreased and the break site associated with the nuclear pore complex at the nuclear periphery, which is the location where DSB repair occurs through alternative pathways that are more mutagenic. End-bridging remained intact as HR and NHEJ declined, but eventually it too became disrupted in cells at advanced replicative age. In all, our work provides insight into the molecular changes in DSB repair pathway during replicative aging. HR first declined, resulting in a transient increase in the NHEJ. However, with increased cellular divisions, Ku70 recovery at DSBs and NHEJ subsequently declined. In wild type cells of advanced replicative age, there was a high frequency of repair products with genomic deletions and microhomologies at the break junction, events not observed in young cells which repaired primarily by HR.     

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.     

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku^−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.     

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 °C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH).It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.     

The anaphase promoting complex promotes NHEJ repair through stabilizing Ku80 at DNA damage sites

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). The choice between these two pathways is largely influenced by cell cycle phases. HDR can occur only in S/G2 when sister chromatid can provide homologous templates, whereas NHEJ can take place in all phases of the cell cycle except mitosis. Central to NHEJ repair is the Ku70/80 heterodimer which forms a ring structure that binds DSB ends and serves as a platform to recruit factors involved in NHEJ. Upon completion of NHEJ repair, DNA double strand-encircling Ku dimers have to be removed. The removal depends on ubiquitylation and proteasomal degradation of Ku80 by the ubiquitin E3 ligases RNF8. Here we report that RNF8 is a substrate of APC^Cdh1 and the latter keeps RNF8 level in check at DSBs to prevent premature turnover of Ku80.     

CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

Topoisomerases class II (topoII) cleave and re-ligate the DNA double helix to allow the passage of an intact DNA strand through it. Chemotherapeutic drugs such as etoposide target topoII, interfere with the normal enzymatic cleavage/re-ligation reaction and create a DNA double-strand break (DSB) with the enzyme covalently bound to the 5'-end of the DNA. Such DSBs are repaired by one of the two major DSB repair pathways, non-homologous end-joining (NHEJ) or homologous recombination. However, prior to repair, the covalently bound topoII needs to be removed from the DNA end, a process requiring the MRX complex and ctp1 in fission yeast. CtIP, the mammalian ortholog of ctp1, is known to promote homologous recombination by resecting DSB ends. Here, we show that human cells arrested in G0/G1 repair etoposide-induced DSBs by NHEJ and, surprisingly, require the MRN complex (the ortholog of MRX) and CtIP. CtIP's function for repairing etoposide-induced DSBs by NHEJ in G0/G1 requires the Thr-847 but not the Ser-327 phosphorylation site, both of which are needed for resection during HR. This finding establishes that CtIP promotes NHEJ of etoposide-induced DSBs during G0/G1 phase with an end-processing function that is distinct to its resection function.     

Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.     

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.     

Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast

Mitotic cells experience double-strand breaks (DSBs) from both exogenous and endogenous sources. Since unrepaired DSBs can result in genome rearrangements or cell death, cells mobilize multiple pathways to repair the DNA damage. In the yeast Saccharomyces cerevisiae, mitotic cells preferentially use a homologous recombination repair pathway. However, when no significant homology to the DSB ends is available, cells utilize a repair process called non-homologous end joining (NHEJ), which can join ends with no homology through resection to uncover microhomologies of a few nucleotides. Although components of the homologous recombination repair system are also involved in NHEJ, the rejoining does not involve all of the homologous recombination repair genes. The SRS2 DNA helicase has been shown to be required for DSB repair when the homologous single-stranded regions are short. Here it is shown that SRS2 is also required for NHEJ, regardless of the cell mating type. Efficient NHEJ of sticky ends requires the Ku70 and Ku80 proteins and the silencing genes SIR2, SIR3 and SIR4. However, NHEJ of blunt ends, while very inefficient, is not further reduced by mutations in YKU70, SIR2, SIR3, SIR4 or SRS2, suggesting that this rejoining process occurs by a different mechanism.     

Phosphorylation of Ku dictates DNA double-strand break (DSB) repair pathway choice in S phase

Multiple DNA double-strand break (DSB) repair pathways are active in S phase of the cell cycle; however, DSBs are primarily repaired by homologous recombination (HR) in this cell cycle phase. As the non-homologous end-joining (NHEJ) factor, Ku70/80 (Ku), is quickly recruited to DSBs in S phase, we hypothesized that an orchestrated mechanism modulates pathway choice between HR and NHEJ via displacement of the Ku heterodimer from DSBs to allow HR. Here, we provide evidence that phosphorylation at a cluster of sites in the junction of the pillar and bridge regions of Ku70 mediates the dissociation of Ku from DSBs. Mimicking phosphorylation at these sites reduces Ku''s affinity for DSB ends, suggesting that phosphorylation of Ku70 induces a conformational change responsible for the dissociation of the Ku heterodimer from DNA ends. Ablating phosphorylation of Ku70 leads to the sustained retention of Ku at DSBs, resulting in a significant decrease in DNA end resection and HR, specifically in S phase. This decrease in HR is specific as these phosphorylation sites are not required for NHEJ. Our results demonstrate that the phosphorylation-mediated dissociation of Ku70/80 from DSBs frees DNA ends, allowing the initiation of HR in S phase and providing a mechanism of DSB repair pathway choice in mammalian cells.     

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.     

Interacting proteins Rtt109 and Vps75 affect the efficiency of non-homologous end-joining in Saccharomyces cerevisiae

One of the key pathways for DNA double-stranded break (DSB) repair is the non-homologous end-joining (NHEJ) pathway, which directly re-ligates two broken ends of DNA. Using a plasmid repair assay screen, we identified that the deletion strain for RTT109 had a reduced efficiency for NHEJ in yeast. This deletion strain also had a reduced efficiency to repair induced chromosomal DSBs in vivo. Tandem-affinity purification of Rtt109 recovered Vps75 as a physical interacting protein. Deletion of VPS75 was also shown to have an effect on the efficiency of NHEJ in both the plasmid repair and the chromosomal repair assays. In addition, deletion mutants for both RTT109 and VPS75 showed hypersensitivity to different DNA damaging agents. Our genetic interaction analysis supports a role for RTT109 in DNA damage repair. We propose that one function of the Rtt109-Vps75 interacting protein pair is to affect the efficiency of NHEJ in yeast. Vps75 but not Rtt109 also seem to have an effect on the efficiency of DSB repair using homologous recombination.     

The Ku-dependent non-homologous end-joining but not other repair pathway is inhibited by high linear energy transfer ionizing radiation

Ionizing radiation (IR) induced DNA double strand breaks (DSBs) are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) in mammalian cells. The NHEJ repair includes a Ku-dependent main pathway and a PARP-1-dependent complementary pathway. Compared with low linear energy transfer (LET) IR (X or gamma ray) at the same doses, high LET IR (high-charge particles) induces more cell death because of ineffective DNA repair. However, it remains unclear whether high LET IR inhibits all repair or specifically one repair pathway. By combining the assays of clonogenic survival, G2M checkpoint and gammaH2AX in the cell lines with deficiencies in different repair genes, we show here that high LET IR inhibits only the Ku-dependent main NHEJ pathway and does not inhibit either the HRR pathway or the PARP-1-dependent complementary NHEJ pathway. In addition, by developing an assay to detect small fragments of DSB (<400 bp) and by detecting the binding abilities of purified Ku and PARP to different sized dsDNA, we present a possible link for explaining the phenotypes. When compared with low LET IR at the same dose, high LET IR might induce similar yields of DNA DSBs in total but it might induce more small fragments of DNA DSBs (<40 base pairs) that prevent Ku binding efficiently to two ends of one DSB fragment at the same time, thus delaying Ku-dependent repair.