Rad51 Mutants Research Articles

Abstract A27: Assessing somatic tumor-associated RAD51 mutations and screening for novel dominant-interfering RAD51 proteins

Abstract The responsiveness of cancer cells to chemotherapy and targeted treatment, as well as the development of resistance, is determined by the state of DNA damage response pathways. Homology-directed repair (HDR) is crucial for error-free repair of DNA double-strand breaks that arise during replication stress or are induced by exogenous genotoxins. Therefore, HDR efficacy status in cancer cells could potentially be utilized as an indicator to predict tumor responsiveness to standard chemotherapies and to poly(ADP-ribose) polymerases (PARP) inhibitors. We are interested in understanding the influence of different levels of functional activity of the HDR pathway on treatment responses. To this end, we have investigated the functional consequence of RAD51 point mutations identified in tumors. RAD51 is an effector of the key tumor suppressor BRCA2 and contributes to genome integrity in actively dividing cells. RAD51 mutations are identified from Memorial Sloan Kettering clinical sequencing cohort (MSK-IMPACT), the Catalogue of Somatic Mutations in Cancer (COSMIC) and the International Fanconi Anemia Registry (IFAR). To understand the implications of these altered residues on RAD51 function in HDR, we tested repair efficiency using a previously established DR-GFP reporter assay in mammalian cells. Of eighteen novel RAD51 mutants examined, ten displayed decreased HDR efficiency, three had wild-type levels of HDR, and five had increased HDR levels. To test whether the HDR-deficient RAD51 mutants lead to sensitivity to genotoxins, we assayed the survival of corn smut U. maydis that expressed these RAD51 mutants after treatment with DNA damaging agents ultraviolet light, diepoxybutane, or methylmethane sulfonate. All ten HDR-defective mutants were found to be sensitive to one or more genotoxins. In addition, six were found to promote sensitivity to DNA damaging agents when they were expressed in wild-type cells—i.e., a dominant negative phenotype. Interestingly, these dominant negative mutants confer selective sensitivity to specific DNA damaging agent(s). The underlying mechanism is still under investigation; nevertheless, these mutations can be used as biomarkers for HDR efficiency and to identify novel interactions of RAD51. The identification of these somatic tumor-associated RAD51 mutants warrants further testing for sensitivity towards chemotherapy drugs such as PARP inhibitors and gemcitabine. Furthermore, a random mutagenesis screening is in progress to identify more novel dominant negative RAD51 mutations. The functional analysis of RAD51 is critical as readouts for HDR efficiency, which impacts personalized chemotherapeutic choices. The result of this study will hopefully expand the benefits of using PARP inhibitors not just on BRCA1/2- and RAD51 paralog-deficient tumors but also on tumors with RAD51 mutations. Citation Format: Pei Xin Lim, Jeanette Sutherland, Raymond Noonan, Alexandra Dananberg, William Holloman, Agata Smogorzewska, Maria Jasin. Assessing somatic tumor-associated RAD51 mutations and screening for novel dominant-interfering RAD51 proteins [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A27.

Yap1 and Skn7 genetically interact with Rad51 in response to oxidative stress and DNA double-strand break in Saccharomyces cerevisiae

Reactive oxygen species (ROS)-mediated DNA adducts as well as DNA strand breaks are highly mutagenic leading to genomic instability and tumorigenesis. DNA damage repair pathways and oxidative stress response signaling have been proposed to be highly associated, but the underlying interaction remains unknown. In this study, we employed mutant strains lacking Rad51, the homolog of E. coli RecA recombinase, and Yap1 or Skn7, two major transcription factors responsive to ROS, to examine genetic interactions between double-strand break (DSB) repair proteins and cellular redox regulators in budding yeast Saccharomyces cerevisiae. Abnormal expression of YAP1 or SKN7 aggravated the mutation rate of rad51 mutants and their sensitivity to DSB- or ROS-generating reagents. Rad51 deficiency exacerbated genome instability in the presence of increased levels of ROS, and the accumulation of DSB lesions resulted in elevated intracellular ROS levels. Our findings suggest that evident crosstalk between DSB repair pathways and ROS signaling proteins contributes to cell survival and maintenance of genome integrity in response to genotoxic stress.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork.

Requirement of POL3 and POL4 on non-homologous and microhomology-mediated end joining in rad50/xrs2 mutants of Saccharomyces cerevisiae.

Non-homologous end joining (NHEJ) directly joins two broken DNA ends without sequence homology. A distinct pathway called microhomology-mediated end joining (MMEJ) relies on a few base pairs of homology between the recombined DNA. The majority of DNA double-strand breaks caused by endogenous oxygen species or ionizing radiation contain damaged bases that hinder direct religation. End processing is required to remove mismatched nucleotides and fill in gaps during end joining of incompatible ends. POL3 in Saccharomyces cerevisiae encodes polymerase δ that is required for DNA replication and other DNA repair processes. Our previous results have shown that POL3 is involved in gap filling at 3' overhangs in POL4-independent NHEJ. Here, we studied the epistatic interaction between POL3, RAD50, XRS2 and POL4 in NHEJ using a plasmid-based endjoining assay in yeast. We demonstrated that either rad50 or xrs2 mutation is epistatic for end joining of compatible ends in the rad50 pol3-t or xrs2 pol3-t double mutants. However, the pol3-t and rad50 or pol3-t and xrs2 mutants caused an additive decrease in the end-joining efficiency of incompatible ends, suggesting that POL3 and RAD50 or POL3 and XRS2 exhibit independent functions in NHEJ. In the rad50 pol4 mutant, end joining of incompatible ends was not detected. In the rad50 or xrs2 mutants, NHEJ events did not contain any microhomology at the rejoined junctions. The pol3-t mutation restored MMEJ in the rad50 or xrs2 mutant backgrounds. Moreover, we demonstrated that NHEJ of incompatible ends required RAD50 and POL4 more than POL3. In conclusion, POL3 and POL4 have differential functions in NHEJ, independent of the RAD50-mediated repair pathway.

Telomere dynamics in the lower plant Physcomitrella patens.

A comparative approach in biology is needed to assess the universality of rules governing this discipline. In plant telomere research, most of the key principles were established based on studies in only single model plant, Arabidopsis thaliana. These principles include the absence of telomere shortening during plant development and the corresponding activity of telomerase in dividing (meristem) plant cells. Here we examine these principles in Physcomitrella patens as a representative of lower plants. To follow telomerase expression, we first characterize the gene coding for the telomerase reverse transcriptase subunit PpTERT in P. patens, for which only incomplete prediction has been available so far. In protonema cultures of P. patens, growing by filament apical cell division, the proportion of apical (dividing) cells was quantified and telomere length, telomerase expression and activity were determined. Our results show telomere stability and demonstrate proportionality of telomerase activity and expression with the number of apical cells. In addition, we analyze telomere maintenance in mre11, rad50, nbs1, ku70 and lig4 mutants of P. patens and compare the impact of these mutations in double-strand-break (DSB) repair pathways with earlier observations in corresponding A. thaliana mutants. Telomere phenotypes are absent and DSB repair kinetics is not affected in P. patens mutants for DSB factors involved in non-homologous end joining (NHEJ). This is compliant with the overall dominance of homologous recombination over NHEJ pathways in the moss, contrary to the inverse situation in flowering plants.

Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae

Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death.

Rad51-Rad52 mediated maintenance of centromeric chromatin in Candida albicans.

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-ACaCse4 levels at the programmed stall sites of early replicating centromeres.

RAD52-Dependent Synthetic Lethality Eradicates Leukemia Stem Cells

Abstract 3▪▪This icon denotes a clinically relevant abstractWe showed that imatinib-naive and imatinib-treated BCR-ABL1 kinase-positive chronic myeloid leukemia in chronic phase (CML-CP) Lin−CD34+CD38− stem cells (LSCs) and Lin−CD34+CD38+ leukemia progenitor cells (LPCs) accumulate high numbers of the reactive oxygen species (ROS)-induced DNA double-strand breaks (DSBs) which, if not repaired, are lethal (Nieborowska-Skorska et al., Blood, 2012). Genome-wide microarray screen indicated that LSCs overexpress numerous genes responsible for homologous recombination repair (HRR) of DSBs. Direct targeting of RAD51, a key element in HRR, by small molecule inhibitor exerted anti-CML effect, but normal cells were also affected, indicating that RAD51 is not a preferable target. In normal cells HRR depends primarily on BRCA1/BRCA2-RAD51 pathway, while RAD52-RAD51 pathway may serve as a back-up. However BRCA1 protein is downregulated in CML, and RAD51(F259V) mutant (RAD52 binding-deficient) induced apoptosis and reduced cell growth in BCR-ABL1 –dependent manner, thus highlighting the potential role of alternative RAD52-driven HRR in CML. The absence of RAD52 (Rad52−/−) inhibited BCR-ABL1 –mediated cell cycle progression and clonogenic activity, induced apoptosis, reduced Lin−Kit+Sca1+CD34−Flt3− long-term LSCs and Lin−Kit+Sca1+CD34+Flt3− short-term LSCs, and abrogated leukemogenesis in murine model of CML. At the same time RAD52 was expendable in normal cells. In addition, RAD52 was essential to prevent accumulation of ROS-induced lethal DSBs in BCR-ABL1 –positive murine LSCs. Thus it appears that RAD52 is necessary to repair numerous ROS-induced DSBs in LSCs to promote leukemogenesis. BCR-ABL1 kinase interacts with and phosphorylates RAD52 on Y104, but phosphorylation-deficient RAD52(Y104F) mutant did not exert any negative effect on BCR-ABL1 kinase-driven leukemogenesis. This observation suggests that RAD52 activity is preserved in imatinib-treated cells. On the other hand, DNA binding-deficient RAD52(F79A) and RAD52(K102A) mutants (disrupt DNA binding domain I and II, respectively) inhibited clonogenic potential of Lin−CD34+ CML-CP cells and BCR-ABL1 –positive Lin−Kit+Sca1+ murine LSCs, but not normal counterparts. Therefore, RAD52-mediated DNA binding activity appears to be essential for BCR-ABL1 kinase-driven leukemogenesis, but not normal hematopoiesis. RAD52 DNA I binding groove containing F79 forms a pocket/niche. Peptide aptamer containing amino acid residues surrounding RAD52 F79, but not that with F79A substitution, inhibited RAD52 foci, RAD52-dependent RAD51 foci and HRR activity, resulting in elevation of the number of lethal DSBs and abrogation of clonogenic activity of BCR-ABL1-positive murine leukemia cells. Aptamer F79 reduced the growth of LSCs and LPCs from CML-CP and more aggressive CML-accelerated phase (CML-AP), depleted quiescent LSCs by attrition and eradicated BCR-ABL1 leukemia from SCID mice. Moreover, aptamer F79 enhanced the anti-CML effects of imatinib. The effect of aptamer F79 in BCR-ABL1 leukemia cells depended on downregulation of BRCA1 implicating “synthetic lethality”. At the same time the aptamer did not exert any measurable negative effects on normal cells and tissues. In conclusion, we postulate that CML-CP/AP cells are “addicted” to RAD52 and that targeting of DNA binding domain of RAD52 may induce “synthetic lethality” to eliminate proliferating LSCs/LPCs and to deplete quiescent LSCs. Moreover, anti-RAD52 F79 aptamer exerted anti-tumor activity in acute myeloid leukemia primary cells, and in cell lines derived from carcinomas of breast, ovary and pancreas displaying BRCA1 and/or BRCA2 deficiency. Thus, BRCA1/BRCA2ness-driven “addiction” to RAD52 may be selectively targeted to achieve “synthetic lethality” in wide-range of tumors while being harmless to normal cells. Disclosures:Copland:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

RAD51 Mutants Cause Replication Defects and Chromosomal Instability

RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements.

Differing Requirements for RAD51 and DMC1 in Meiotic Pairing of Centromeres and Chromosome Arms in Arabidopsis thaliana

During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains.

RecQ5 Interacts with Rad51 and Is Involved in Resistance of <i>Drosophila</i> to Cisplatin Treatment

RecQ5 is a member of the RecQ family of DNA helicases. There are 5 RecQ members in humans. Defects in 3 of them, i.e., BLM, WRN, and RTS, cause Bloom, Werner, and Rothmund-Thomson syndromes, respectively. RECQL1 and RECQL5 have not been associated with any human disease, and their precise roles are unknown. Our previous study suggests that the lack of RecQ5, which is the Drosophila homolog of RECQL5, leads to the accumulation of DNA double-stranded breaks (DSBs). It is possible that RecQ5 is involved in DSB repair. However, little is known about this possible function of RecQ5 in DSB repair. Here, we report that Rad51 protein, which plays a critical role in DSB repair, interacted with RecQ5 in vitro and in vivo in Drosophila. The Rad51 protein interacted with the C-terminal region of RecQ5, as shown by the yeast two-hybrid method. Moreover, the C-terminal region of the RecQ5 protein and the central region of Rad51 interacted directly and specifically when examined by the glutathione-S-transferase pull-down method. Consistent with these results, when RecQ5 and Rad51 were co-expressed in Drosophila cells in culture, they became co-localized in nuclei and could be co-immunoprecipitated. Furthermore, RecQ5-deficient flies (recq5) were more sensitive to the chemotherapeutic agent cisplatin compared with wild-type ones. Also, Rad51 mutants (rad51) were more sensitive to cisplatin, with sensitivity similar to that of recq5 rad51 double mutants. These data suggest that RecQ5 and Rad51 in Drosophila functioned for survival after the flies had been treated with cisplatin.

Expression and regulation of RAD51 mediate cellular responses to chemotherapeutics

There is evidence that RAD51 expression associates with resistance to commonly used chemotherapeutics. Our previous work demonstrated that inhibitors of thymidylate synthase (TS) induced RAD51-dependent homologous recombination (HR), and depleting the RAD51 recombinase sensitized cells to TS inhibitors. In this study, the consequences of RAD51 over-expression were studied. Over-expression of wild-type RAD51 (∼6-fold above endogenous RAD51) conferred resistance to TS inhibitors. In contrast, over-expression of a mutant RAD51 (T309A) that is incapable of being phosphorylated rendered cells more chemosensitive. Moreover, over-expression of the T309A mutant acted in a dominant negative manner over endogenous RAD51 by causing the reduced localization of RAD51 foci following treatment with TS inhibitors. To measure the effect of mutant RAD51 on the cellular response to other DNA damaging chemotherapeutics, the topoisomerase poison etoposide was utilized. Cells over-expressing wild-type RAD51 showed reduced DNA strand breaks, while cells over-expressing the mutant RAD51 showed more than twice as many strand breaks, suggesting that the mutant RAD51 was actively inhibiting strand break resolution. To directly demonstrate an effect on HR, wild-type RAD51 and T309A mutant RAD51 were transiently expressed in HeLa cells that contained an HR reporter construct. HR events provoked by DNA breaks induced by the I-SceI endonuclease increased in cells expressing wild-type RAD51 and decreased in cells expressing the T309A mutant. Collectively, the data suggest that interference with the activation of RAD51-mediated HR represents a potentially useful anticancer target for combination therapies.

Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination

The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site.

Effect of rad50 mutation on illegitimate recombination in Saccharomyces cerevisiae

Genes in the RAD52 epistasis group are involved in repairing DNA double-stranded breaks via homologous recombination. We have previously shown that RAD50 is involved in mitotic nonhomologous integration but not in homologous integration. However, the role of Rad50 in nonhomologous integration has not previously been examined. In the current work, we report that the rad50∆ mutation caused a tenfold decrease in the frequency of nonhomologous integration with the majority of nonhomologous integrants showing an unstable Ura(+) phenotype. Sequencing analysis of the integration target sites showed that integration events of both ends of the integrating vector in the rad50∆ mutant occurred at different chromosomal locations, resulting in large deletions or translocations on the genomic insertion sites. Interestingly, 47% of events in the rad50∆ mutant were integrated into repetitive sequences including rDNA locus, telomeres and Ty elements and 27% of events were integrated into non-repetitive sequences as compared to 11% of events integrated into rDNA and 70% into non-repetitive sequences in the wild-type cells. These results showed that deletion of RAD50 significantly changes the distribution of different classes of integration events, suggesting that Rad50 is required for nonhomologous integration at non-repetitive sequences more so than at repetitive ones. Furthermore, Southern analysis indicated that half of the events contained deletions at one or at both ends of the integrating DNA fragment, suggesting that Rad50 might have a role in protecting free ends of double-strand breaks. In contrast to the rad50∆ mutant, the rad50S mutant (separation of function allele) slightly increases the frequency of nonhomologous integration but the distribution of integration events is similar to that of wild-type cells with the majority of events integrated into a chromosomal locus. Our results suggest that deletion of RAD50 may block the major pathway of nonhomologous integration into a non-repetitive chromosomal locus and Rad50 may be involved in tethering two ends of the integrating DNA into close proximity that facilitates nonhomologous integration of both ends into a single chromosomal locus.

Targeting Phosphotyrosine-315 In RAD51 Recombinase to Prevent BCR-ABL1 - Mediated Unfaithful Homeologous Recombination Repair.

AbstractAbstract 1190 Background:CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast phase (CML-BP) and cell lines transformed by non-mutated BCR-ABL1 kinase or tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) (Nowicki et al., Blood, 2006; Cramer et al., Cancer Res., 2008). DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS and radiation induced DSBs generated chromosomal aberrations in BCR-ABL1-positive leukemia cells (Koptyra et al., Leukemia, 2008), which may contribute to the malignant progression to CML-BP. We also showed that homologous recombination repair (HomoRR) driven by RAD51 recombinase is one of the major DSB repair mechanisms, which is stimulated by BCR-ABL1 (Slupianek et al., Molecular Cell, 2001). Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. Here we investigated if BCR-ABL1 can modulate RAD51 recombinase to corrupt the fidelity of recombination and if this process can be targeted to prevent genomic instability in leukemia cells. Result:To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease–inducible DSB site and a repair template displaying 1% sequence divergence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells, BCR-ABL1-32Dcl3 cells and BCR-ABL1(T315I)-32Dcl3 cells. BCR-ABL1 and BCR-ABL1(T315I) kinase caused about a 3-fold increase in HomeoRR activity implicating its role in the accumulation of chromosomal aberrations in CML cells. The magnitude of HomeoRR stimulation depended on BCR-ABL1 expression levels. RAD51 recombinase, a key regulator of recombination repair, forms a complex with BCR-ABL1 which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain of BCR-ABL1, but does not depend on its kinase activity. In fact, BCR-ABL1(K1172R) kinase-dead mutant formed complexes more abundantly with RAD51 than the kinase-active BCR-ABL1. BCR-ABL1-RAD51 complex formation resulted in direct phosphorylation of RAD51 on Y315 [RAD51(phosphoY315)], which is located in the vicinity of PP motifs in the C-terminal portion of RAD51. Phosphorylation-less C-terminal RAD51(Y315F) mutant formed a stronger complex with BCR-ABL1 than the wild-type form. Altogether, it appears that RAD51 PP bind to the SH3 domain of BCR-ABL1 kinase followed by quick phosphorylation of RAD51 on Y315 and disassembly of RAD51(phosphoY315) from the complex. Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. In fact RAD51(phosphoY315) was found in the nuclei of BCR-ABL1 leukemia cells, but not of parental cells and formed numerous foci on DSBs. Phosphorylation-less RAD51(Y315F) mutant abrogated foci formation and inhibited HomeoRR. Thus, BCR-ABL1 – RAD51(phosphoY315) pathway appears to promote unfaithful HomeoRR and chromosomal instability. To test if RAD51(phosphoY315) can be targeted to prevent unfaithful HomeoRR in leukemia cells, a peptide aptamer strategy was applied. Aptamer mimicking the RAD51(phosphoY315) fragment, but not that with the Y315F phosphorylation-less substitution inhibited RAD51 foci formation and HomeoRR activity in BCR-ABL1 leukemia cells. Conclusion:In summary, the BCR-ABL1-RAD51 axis may promote accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR-ABL1 kinase. We hypothesize that targeting BCR-ABL1-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BP progression. Furthermore, RAD51 and recombination is also affected by other oncogenic tyrosine kinases (OTKs) such as TEL-ABL1, TEL-PDGFR, ZNF198-FGFR1, TEL-JAK2, JAK2[V617F], NPM-ALK, IGF-1R, EGFR and FLT3-ITD suggesting that abrogation of OTK-mediated aberrant modulation of RAD51 may inhibit genetic instability and tumor progression. Disclosures:No relevant conflicts of interest to declare.

Distinct Roles of the ATR Kinase and the Mre11-Rad50-Nbs1 Complex in the Maintenance of Chromosomal Stability in Arabidopsis

Signaling of chromosomal DNA breaks is of primary importance for initiation of repair and, thus, for global genomic stability. Although the Mre11-Rad50-Nbs1 (MRN) complex is the first sensor of double-strand breaks, its role in double-strand break (DSB) signaling is not fully understood. We report the absence of γ-ray-induced, ATM/ATR-dependent histone H2AX phosphorylation in Arabidopsis thaliana rad50 and mre11 mutants, confirming that the MRN complex is required for H2AX phosphorylation by the ATM and ATR kinases in response to irradiation-induced DSB in Arabidopsis. rad50 and mre11 mutants spontaneously activate a DNA damage response, as shown by the presence of γ-H2AX foci and activation of cell cycle arrest in nonirradiated plants. This response is ATR dependent as shown both by the absence of these spontaneous foci and by the wild-type mitotic indices of double rad50 atr and mre11 atr plants. EdU S-phase labeling and fluorescence in situ hybridization analysis using specific subtelomeric probes point to a replicative S-phase origin of this chromosome damage in the double mutants and not to telomere destabilization. Thus, the data presented here show the exclusive involvement of ATR in DNA damage signaling in MRN mutants and provide evidence for a role for ATR in the avoidance of S-phase DNA damage.

Aflatoxin B1‐Associated DNA Adducts Stall S Phase and Stimulate Rad51 foci in Saccharomyces cerevisiae

AFB1 is a potent recombinagen in budding yeast. AFB1 exposure induces RAD51 expression and triggers Rad53 activation in yeast cells that express human CYP1A2. It was unknown, however, when and if Rad51 foci appear. Herein, we show that Rad53 activation correlates with cell-cycle delay in yeast and the subsequent formation of Rad51 foci. In contrast to cells exposed to X-rays, in which Rad51 foci appear exclusively in G2 cells, Rad51 foci in AFB1-exposed cells can appear as soon as cells enter S phase. Although rad51 and rad4 mutants are mildly sensitive to AFB1, chronic exposure of the NER deficient rad4 cells to AFB1 leads to increased lag times, while rad4 rad51 double mutants exhibit synergistic sensitivity and do not grow when exposed to 50 μM AFB1. We suggest RAD51 functions to facilitate DNA replication after replication fork stalling or collapse in AFB1-exposed cells.

Ctp1 and the MRN-Complex Are Required for Endonucleolytic Rec12 Removal with Release of a Single Class of Oligonucleotides in Fission Yeast

DNA double-strand breaks (DSBs) are formed during meiosis by the action of the topoisomerase-like Spo11/Rec12 protein, which remains covalently bound to the 5′ ends of the broken DNA. Spo11/Rec12 removal is required for resection and initiation of strand invasion for DSB repair. It was previously shown that budding yeast Spo11, the homolog of fission yeast Rec12, is removed from DNA by endonucleolytic cleavage. The release of two Spo11 bound oligonucleotide classes, heterogeneous in length, led to the conjecture of asymmetric cleavage. In fission yeast, we found only one class of oligonucleotides bound to Rec12 ranging in length from 17 to 27 nucleotides. Ctp1, Rad50, and the nuclease activity of Rad32, the fission yeast homolog of Mre11, are required for endonucleolytic Rec12 removal. Further, we detected no Rec12 removal in a rad50S mutant. However, strains with additional loss of components localizing to the linear elements, Hop1 or Mek1, showed some Rec12 removal, a restoration depending on Ctp1 and Rad32 nuclease activity. But, deletion of hop1 or mek1 did not suppress the phenotypes of ctp1Δ and the nuclease dead mutant (rad32-D65N). We discuss what consequences for subsequent repair a single class of Rec12-oligonucleotides may have during meiotic recombination in fission yeast in comparison to two classes of Spo11-oligonucleotides in budding yeast. Furthermore, we hypothesize on the participation of Hop1 and Mek1 in Rec12 removal.

Inactivation of the Sas2 histone acetyltransferase delays senescence driven by telomere dysfunction

Changes in telomere chromatin have been linked to cellular senescence, but the underlying mechanisms and impact on lifespan are unclear. We found that inactivation of the Sas2 histone acetyltransferase delays senescence in Saccharomyces cerevisiae telomerase (tlc1) mutants through a homologous recombination-dependent mechanism. Sas2 acetylates histone H4 lysine 16 (H4K16), and telomere shortening in tlc1 mutants was accompanied by a selective and Sas2-dependent increase in subtelomeric H4K16 acetylation. Further, mutation of H4 lysine 16 to arginine, which mimics constitutively deacetylated H4K16, delayed senescence and was epistatic to sas2 deletion, indicating that deacetylated H4K16 mediates the delay caused by sas2 deletion. Sas2 normally prevents the Sir2/3/4 heterochromatin complex from leaving the telomere and spreading to internal euchromatic loci. Senescence was delayed by sir3 deletion, but not sir2 deletion, indicating that senescence delay is mediated by release of Sir3 specifically from the telomere repeats. In contrast, sir4 deletion sped senescence and blocked the delay conferred by sas2 or sir3 deletion. We thus show that manipulation of telomere chromatin modulates senescence caused by telomere shortening.