Poly(ADP-ribose)polymerases are involved in microhomology mediated back-up non-homologous end joining in Arabidopsis thaliana

Besides the KU-dependent classical non-homologous end-joining (C-NHEJ) pathway, an alternative NHEJ pathway first identified in mammalian systems, which is often called the back-up NHEJ (B-NHEJ) pathway, was also found in plants. In mammalian systems PARP was found to be one of the essential components in B-NHEJ. Here we investigated whether PARP1 and PARP2 were also involved in B-NHEJ in Arabidopsis. To this end Arabidopsis parp1, parp2 and parp1parp2 (p1p2) mutants were isolated and functionally characterized. The p1p2 double mutant was crossed with the C-NHEJ ku80 mutant resulting in the parp1parp2ku80 (p1p2k80) triple mutant. As expected, because of their role in single strand break repair (SSBR) and base excision repair (BER), the p1p2 and p1p2k80 mutants were shown to be sensitive to treatment with the DNA damaging agent MMS. End-joining assays in cell-free leaf protein extracts of the different mutants using linear DNA substrates with different ends reflecting a variety of double strand breaks were performed. The results showed that compatible 5′-overhangs were accurately joined in all mutants, that KU80 protected the ends preventing the formation of large deletions and that PARP proteins were involved in microhomology mediated end joining (MMEJ), one of the characteristics of B-NHEJ.     

The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining

Classical-non-homologous end-joining (C-NHEJ) is considered the main pathway for repairing DNA double strand breaks (DSB) in mammalian cells. When C-NHEJ is defective, cells may switch DSB repair to an alternative-end-joining, which depends on PARP1 and is more erroneous. This PARP1-EJ is suggested to be active especially in tumor cells contributing to their genomic instability. Here, we define conditions under which cells would switch the repair to PARP1-EJ. Using the end jining repair substrate pEJ, we revealed that PARP1-EJ is solely used when Ku is deficient but not when either DNA-PKcs or Xrcc4 is lacking. In the latter case, DSB repair, however, could be shuttled to PARP1-EJ after additional Ku80 down-regulation, which partly rescued the DSB repair in these mutants. We demonstrate here that PARP-EJ may work on DSB ends at high fidelity manner, as evident from the unchanged efficiency upon blocking end resection by either roscovitin or mirin. Furthermore, we demonstrate for that PARP-EJ is likewise involved in the repair of multiple DSBs (I-PpoI- and IR-induced). Importantly, we identified a chromatin signature associated with the switch to PARP1-EJ which is characterized by a strong enrichment of both PARP1 and LigIII at damaged chromatin. Together, these data indicate that Ku is the main regulator for the hierarchal organization between C-NHEJ and PARP1-EJ.     

Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis^−/−, DNA ligase 4^−/− (LIG4^−/−), and LIG4^−/−/Artemis^−/− double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.     

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.     

Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis

Double-strand breaks in genomic DNA (DSB) are potentially lethal lesions which separate parts of chromosome arms from their centromeres. Repair of DSB by recombination can generate mutations and further chromosomal rearrangements, making the regulation of recombination and the choice of recombination pathways of the highest importance. Although knowledge of recombination mechanisms has considerably advanced, the complex interrelationships and regulation of pathways are far from being fully understood. We analyse the different pathways of DSB repair acting in G2/M phase nuclei of irradiated plants, through quantitation of the kinetics of appearance and loss of γ-H2AX foci in Arabidopsis mutants. These analyses show the roles for the four major recombination pathways in post-S-phase DSB repair and that non-homologous recombination pathways constitute the major response. The data suggest a hierarchical organisation of DSB repair in these cells: C-NHEJ acts prior to B-NHEJ which can also inhibit MMEJ. Surprisingly the quadruple ku80 xrcc1 xrcc2 xpf mutant can repair DSB, although with severely altered kinetics. This repair leads to massive genetic instability with more than 50% of mitoses showing anaphase bridges following irradiation. This study thus clarifies the relationships between the different pathways of DSB repair in the living plant and points to the existence of novel DSB repair processes.     

Reprint of “Functional overlaps between XLF and the ATM-dependent DNA double strand break response”

Developing B and T lymphocytes generate programmed DNA double strand breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the immunoglobulin heavy chain (IgH) class switch recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via classical non-homologous end-joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors.     

Is Non-Homologous End-Joining Really an Inherently Error-Prone Process?

DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV–dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.DNA double-strand breaks (DSBs) are highly toxic lesions. However, in certain essential physiological processes, DSBs are used to promote genetic diversity. Programmed DSBs generated by cellular enzymes are repaired by the same mechanisms as those used for stress-induced DSBs. Thus, DSB repair stands at the crossroads between genetic variability and instability.DSB repair uses two primary strategies: non-homologous end-joining (NHEJ), which is generally considered to be error-prone, and homologous recombination (HR), which is considered to be error-free. However, this view is too simplistic. Herein, we discuss several pieces of data that challenge the fallibility of NHEJ.     

Single-stranded DNA oligomers stimulate error-prone alternative repair of DNA double-strand breaks through hijacking Ku protein

In humans, DNA double-strand breaks (DSBs) are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3′ single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. Here, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. We report that under both conditions, ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process.     

RAD51 protects against nonconservative DNA double-strand break repair through a nonenzymatic function

Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.     

MRE11 is required for homologous synapsis and DSB processing in rice meiosis

Mre11, a conserved protein found in organisms ranging from yeast to multicellular organisms, is required for normal meiotic recombination. Mre11 interacts with Rad50 and Nbs1/Xrs2 to form a complex (MRN/X) that participates in double-strand break (DSB) ends processing. In this study, we silenced the MRE11 gene in rice and detailed its function using molecular and cytological methods. The OsMRE11-deficient plants exhibited normal vegetative growth but could not set seed. Cytological analysis indicated that in the OsMRE11-deficient plants, homologous pairing was totally inhibited, and the chromosomes were completely entangled as a formation of multivalents at metaphase I, leading to the consequence of serious chromosome fragmentation during anaphase I. Immunofluorescence studies further demonstrated that OsMRE11 is required for homologous synapsis and DSB processing but is dispensable for meiotic DSB formation. We found that OsMRE11 protein was located on meiotic chromosomes from interphase to late pachytene. This protein showed normal localization in zep1, Oscom1 and Osmer3, as well as in OsSPO11-1 ^RNAi plants, but not in pair2 and pair3 mutants. Taken together, our results provide evidence that OsMRE11 performs a function essential for maintaining the normal HR process and inhibiting non-homologous recombination during meiosis.     

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.     

Different DNA-PKcs functions in the repair of radiation-induced and spontaneous DSBs within interstitial telomeric sequences

Interstitial telomeric sequences (ITSs) in hamster cells are hot spots for spontaneous and induced chromosome aberrations (CAs). Most data on ITS instability to date have been obtained in DNA repair-proficient cells. The classical non-homologous end joining repair pathway (C-NHEJ), which is the principal double strand break (DSB) repair mechanism in mammalian cells, is thought to restore the morphologically correct chromosome structure. The production of CAs thus involves DNA-PKcs-independent repair pathways. In our current study, we investigated the participation of DNA-PKcs from the C-NHEJ pathway in the repair of spontaneous or radiation-induced DSBs in ITSs using wild-type and DNA-PKcs mutant Chinese hamster ovary cells. Our data demonstrate that DNA-PKcs stabilizes spontaneous DSBs within ITSs from the chromosome 9 long arm, leading to the formation of terminal deletions. In addition, we show that DNA-PKcs-dependent C-NHEJ is employed following radiation-induced DSBs in other ITSs and restores morphologically correct chromosomes, whereas DNA-PKcs independent mechanisms co-exist in DNA-PKcs proficient cells leading to an excess of CAs within ITSs.     

A role for XLF in DNA repair and recombination in human somatic cells

Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. A core C-NHEJ component is the DNA ligase IV co-factor, Cernunnos/XLF (hereafter XLF). In patients, mutations in XLF cause predicted increases in radiosensitivity and deficits in immune function, but also cause other less well-understood pathologies including neural disorders. To characterize XLF function(s) in a defined genetic system, we used a recombinant adeno-associated virus-mediated gene targeting strategy to inactivate both copies of the XLF locus in the human HCT116 cell line. Analyses of XLF-null cells (which were viable) showed that they were highly sensitive to ionizing radiation and a radiomimetic DNA damaging agent, etoposide. XLF-null cells had profound DNA DSB repair defects as measured by in vivo plasmid end-joining assays and were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus, our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs demonstrated that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly, mutant forms of XLF bearing point mutations at amino acid positions L115 and L179, also completely complemented the null phenotype suggesting, in contrast to predictions to the contrary, that these mutations do not abrogate XLF function. Finally, we demonstrate that the absence of XLF causes a small, but significant, increase in homologous recombination, implicating XLF in DSB pathway choice regulation. We conclude that human XLF is a non-essential, but critical, C-NHEJ-repair factor.     

Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks

In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs.     

The Exonuclease Homolog OsRAD1 Promotes Accurate Meiotic Double-Strand Break Repair by Suppressing Nonhomologous End Joining

During meiosis, programmed double-strand breaks (DSBs) are generated to initiate homologous recombination, which is crucial for faithful chromosome segregation. In yeast, Radiation sensitive1 (RAD1) acts together with Radiation sensitive9 (RAD9) and Hydroxyurea sensitive1 (HUS1) to facilitate meiotic recombination via cell-cycle checkpoint control. However, little is known about the meiotic functions of these proteins in higher eukaryotes. Here, we characterized a RAD1 homolog in rice (Oryza sativa) and obtained evidence that O. sativa RAD1 (OsRAD1) is important for meiotic DSB repair. Loss of OsRAD1 led to abnormal chromosome association and fragmentation upon completion of homologous pairing and synapsis. These aberrant chromosome associations were independent of OsDMC1. We found that classical nonhomologous end-joining mediated by Ku70 accounted for most of the ectopic associations in Osrad1. In addition, OsRAD1 interacts directly with OsHUS1 and OsRAD9, suggesting that these proteins act as a complex to promote DSB repair during rice meiosis. Together, these findings suggest that the 9-1-1 complex facilitates accurate meiotic recombination by suppressing nonhomologous end-joining during meiosis in rice.Meiosis comprises two successive cell divisions after a single S phase, generating four haploid products. To ensure proper chromosome segregation at the first meiotic division, crossovers (COs) are formed between homologous chromosomes (Kleckner, 2006). CO formation requires faithful repair of programmed DNA double-strand breaks (DSBs) introduced by the protein SPO11 (Keeney et al., 1997; Shinohara et al., 1997).Mitotic cells employ two basic strategies for DSB repair: homologous recombination (HR) and classical nonhomologous end-joining (C-NHEJ; Deriano and Roth, 2013). HR requires an undamaged template sequence for repair, while the C-NHEJ pathway involves direct ligation of the broken ends in a Ku-dependent manner. Both HR and C-NHEJ safeguard genome integrity during mitosis (Ceccaldi et al., 2016; Symington and Gautier, 2011). However, DSBs are preferentially repaired by HR during meiosis, because only this pathway generates COs. C-NHEJ competes with HR and creates de novo mutations in the gametes, indicating that this activity should be restricted during meiotic DSB repair. Previous studies have identified several factors essential for preventing C-NHEJ in meiosis (Goedecke et al., 1999; Martin et al., 2005; Adamo et al., 2010; Lemmens et al., 2013).Although the mechanism inhibiting C-NHEJ during meiosis is still elusive, regulators guaranteeing the success of the HR pathway have been extensively studied. Radiation sensitive1 (RAD1) is an evolutionarily conserved protein whose best-known function is checkpoint signaling. RAD1, a member of the ring-shaped RAD9-RAD1-HUS1 (9-1-1) complex, plays a crucial role in activating the pachytene checkpoint, a surveillance mechanism for monitoring the progression of meiotic HR in many organisms (Lydall et al., 1996; Hong and Roeder, 2002; Eichinger and Jentsch, 2010).In addition to their well-known roles in checkpoint signaling, members of 9-1-1 complex may also play a direct role in facilitating DSB repair and HR during meiosis. RAD1 is associated with both synapsed and unsynapsed chromosomes during prophase I in mouse (Freire et al., 1998). The homolog of RAD1 in Saccharomyces cerevisiae is Rad17, and rad17 mutant exhibits persistent Rad51 foci (Shinohara et al., 2003). Moreover, mutations in Rad17 lead to a reduced frequency of interhomolog recombination, aberrant synapsis, increased rates of ectopic recombination events, and illegitimate repair from the sister chromatids during meiosis (Grushcow et al., 1999). Recently, Rad17 was shown to be necessary for the efficient assembly of ZMM proteins (Shinohara et al., 2015). Apart from RAD1, the other partners of 9-1-1 were also shown to be involved in DSB repair. HUS1 is proved essential for meiotic DSB repair in Drosophila (Peretz et al., 2009). Moreover, Hus1 inactivation in mouse testicular germ cells results in persistent meiotic DNA damage, chromosomal defects, and germ cell depletion (Lyndaker et al., 2013). Nevertheless, little is known about the role of 9-1-1 proteins in higher plants. In Arabidopsis (Arabidopsis thaliana), mutants of RAD9 show increased sensitivity to genotoxic agents and delayed general repair of mitotic DSBs (Heitzeberg et al., 2004). A recent study indicates that HUS1 is involved in DSB repair of both mitotic and meiotic cells in rice (Che et al., 2014).In this study, we showed that OsRAD1 was required for the accurate repair of DSBs in rice during meiosis. Importantly, we demonstrated that the defective meiotic DSB repair in the Osrad1 mutants could be partially suppressed by blocking the C-NHEJ pathway. We also investigated the relationship between OsRAD1 and other key recombination proteins. Together, our findings indicated that the 9-1-1 complex plays a crucial role in the meiotic DSB repair mechanism.     

Role of the AtRad1p endonuclease in homologous recombination in plants

Using a specific recombination assay, we show in the plant Arabidopsis thaliana that AtRad1 protein plays a role in the removal of non-homologous tails in homologous recombination. Recombination in the presence of non-homologous overhangs is reduced 11-fold in the atrad1 mutant compared with the wild-type plants. AtRad1p is the A. thaliana homologue of the human Xpf and Saccharomyces cerevisiae Rad1 proteins. Rad1p is a subunit of the Rad1p/Rad10p structure-specific endonuclease that acts in nucleotide excision repair and inter-strand crosslink repair. This endonuclease also plays a role in mitotic recombination to remove non-homologous, 3′-ended overhangs from recombination intermediates. The Arabidopsis atrad1 mutant (uvh1), unlike rad1 mutants known from other eukaryotes, is hypersensitive to ionizing radiation. This last observation may indicate a more important role for the Rad1/Rad10 endonuclease in recombination in plants. This is the first direct demonstration of the involvement of AtRad1p in homologous recombination in plants.     

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.     

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.     

Functional validation of miRNAs targeting genes of DNA double-strand break repair to radiosensitize non-small lung cancer cells

DNA-Double strand breaks (DSBs) generated by radiation therapy represent the most efficient lesions to kill tumor cells, however, the inherent DSB repair efficiency of tumor cells can cause cellular radioresistance and impact on therapeutic outcome. Genes of DSB repair represent a target for cancer therapy since their down-regulation can impair the repair process making the cells more sensitive to radiation. In this study, we analyzed the combination of ionizing radiation (IR) along with microRNA-mediated targeting of genes involved in DSB repair to sensitize human non-small cell lung cancer (NSCLC) cells. MicroRNAs are natural occurring modulators of gene expression and therefore represent an attractive strategy to affect the expression of DSB repair genes. As possible IR-sensitizing targets genes we selected genes of homologous recombination (HR) and non-homologous end joining (NHEJ) pathway (i.e. RAD51, BRCA2, PRKDC, XRCC5, LIG1). We examined these genes to determine whether they may be real targets of selected miRNAs by functional and biological validation. The in vivo effectiveness of miRNA treatments has been examined in cells over-expressing miRNAs and treated with IR. Taken together, our results show that hsa-miR-96-5p and hsa-miR-874-3p can directly regulate the expression of target genes. When these miRNAs are combined with IR can decrease the survival of NSCLC cells to a higher extent than that exerted by radiation alone, and similarly to radiation combined with specific chemical inhibitors of HR and NHEJ repair pathway.