Homologous Recombination Mutants Research Articles

Exploring DNA repair deficient CHO cell response to low dose rate radiation

IntroductionDNA double-strand breaks (DSBs) induced by ionizing radiation pose a significant threat to genome integrity, necessitating robust repair mechanisms. This study explores the responses of repair-deficient cells to low dose rate (LDR) radiation. Non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways play pivotal roles in maintaining genomic stability. The hypothesis posits distinct cellular outcomes under LDR exposure compared to acute radiation, impacting DNA repair mechanisms and cell survival. Materials and methodsChinese hamster ovary (CHO) cells, featuring deficiencies in NHEJ, HR, Fanconi Anemia, and PARP pathways, were systematically studied. Clonogenic assays for acute and LDR gamma-ray exposures, cell growth inhibition analyses, and γ-H2AX foci assays were conducted, encompassing varied dose rates to comprehensively assess cellular responses. ResultsNHEJ mutants exhibited an unexpected inverse dose rate effect, challenging conventional expectations. HR mutants displayed unique radiosensitivity patterns, aligning with responses to major DNA-damaging agents. LDR exposure induced cell cycle alterations, growth delays, and giant cell formation, revealing context-dependent sensitivities. γ-H2AX foci assays indicated DSB accumulation during LDR exposure. DiscussionThese findings challenge established paradigms, emphasizing the intricate interplay between repair pathways and dose rates. The study offers comprehensive insights into repair-deficient cell responses, urging a reevaluation of conventional dose-response models and providing potential avenues for targeted therapeutic strategies in diverse radiation scenarios.

Phenotypic Changes Produced by Endogenous DNA Damage in Yeast Mutants Deficient in Recombination and Base Excision Repair

DNA is under constant attack by exogenous and endogenous factors. Endogenous sources of DNA lesions include free radicals such as reactive oxygen and nitrogen species (RONS), as well as many naturally occurring enzymatic and chemical processes. To prevent mutations and possibly cell death these lesions must be repaired prior to DNA replication. There are five major pathways that are utilized for repair depending on the type of DNA damage. Eukaryotes utilize two pathways, homologous recombination (HR) and nonhomologous end‐joining (NHEJ), for repair of DNA double‐strand breaks (DSBs). In addition, the cells utilize base excision repair (BER) for repair of damaged or missing bases. Previous work in the lab has shown that HR‐deficient yeast mutant cell cultures exhibit high levels of G2 phase cells during normal growth, without exposure to exogenous DNA damaging agents. Inactivation of DNA damage checkpoint genes, but not spindle checkpoint genes, abolished the high G2 cell phenotype, indicating that the damage checkpoint is persistently activated in the cells. In a screen of yeast mutant library strains deficient in each of the major repair pathways, the high G2 phenotype was observed in HR‐ and BER‐deficient cells, but not in mutants specifically defective in nucleotide excision repair (NER), mismatch repair (MMR) or NHEJ.Experiments performed for the current project have assessed the consequences of inactivation of the HR and BER pathways on yeast cells in more detail. The high G2 cell phenotype was heightened in both diploid HR (rad52) and BER (apn1, ogg1) mutants relative to their haploid counterparts. Expression of DIN1 (RNR3), a DNA damage‐inducible gene, was found to be constitutively induced in the HR mutant cells. Preliminary tests of the role of free radicals in generation of endogenous signaling were performed by overexpression of Yap1p, a transcriptional activator of antioxidant enzyme genes. The high G2 cell phenotype was abolished in ogg1 mutants and strongly reduced in rad52 cells when Yap1p was overexpressed. Current work is focused on (a) analysis of DIN1 promoter activation in BER mutants, (b) assessment of the impact of neutralization of free radicals using chemical antioxidants, and (c) measurement of phosphorylated Rad53 checkpoint protein levels using Western blots to characterize the DNA damage checkpoint signaling response.

Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks

We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G2-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G2-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G2 phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4–6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.

Molecular insights into the repair mechanism of enzymatic DNA-protein cross-link damage induced by a DNMT inhibitor.

DNA-protein cross-links (DPCs) are a special type of DNA damage that is formed when a protein that participates in DNA transactions is irreversibly and covalently linked to DNA bases. DPCs are formed after exposure to chemicals, anticancer drugs, and ionizing radiation resulting in enzymatic and non-enzymatic DPCs. Up to now, the exact repair mechanism of DPCs has not been fully identified. For this reason, the outputs of the current study provide molecular insights about the repair mechanism of a specific type of enzymatic DPCs formed by DNA-trapped DNMT. In the current study, cells treated with 5-azadC show a clear cytotoxic effect with LD20 ranging from 0.4 to 5 µM. The analysis of DPCs by fluorescence labeling reveals that 5-azadC induces DPCs in a dose-dependent manner. Moreover, cells that are deficient in homologous recombination (HR) pathway (RAD51D and XRCC3) were 2-4 folds sensitive to 5-azadC compared to wild type. In contrast, cells deficient in nucleotide excision repair (NER) pathway (XPD and XPF) and FANC pathway (Fanc A, B, and C) didn’t show any sensitivity to 5-azadC doses. Unexpectedly, mutation in nonhomologous end-joining (NHEJ) gene (DNA-PKcs) gives cells a great survival. Also, double-strand breaks (DSBs) were significantly detected in HR mutant (RAD51D) compared with a wild type indicating that the trapped DNMT by 5-azadC hamper the progression of stalled replication fork generating DSBs. Further investigations are required to understand the accurate mechanism by which cells can repair DPCs that will provide good knowledge regarding the targeting of DPCs in cancer treatment.

Role of homologous recombination genesRAD51,RAD52, andRAD59in the repair of lesions caused by γ-radiation to cycling and G2/M-arrested cells ofCandida albicans

We have analysed the role of homologous recombination (HR) genes on the repair of double-strand breaks induced by γ-ionising radiation in Candida albicans. Depletion of either CaRad51 or CaRad52 caused a dramatic drop in the number of survivors compared with wild type, whereas depletion of CaRad59 caused a moderate decrease. Besides, compared with Saccharomyces cerevisiae, C.albicans relies more on HR proteins for repair of ionising radiation lesions. Pulse-field electrokaryotypes of survivors identified genetic alterations mainly in the form of aneuploidy in HR mutants and chromosome length polymorphism and ectopic translocation in wild type. Increasing irradiation (4 to 80krad) of both cycling and nocodazole-treated (G2/M-arrested) cells revealed a gradual loss of chromosomes, larger chromosomes being more affected than smaller ones. For cycling wild-type cells, shattered chromosomes were progressively restored following incubation in yeast extract, peptone, dextrose medium, but not in phosphate-buffered saline, and this accompanied by a moderate increase in colony-forming units, suggesting that repair was followed by replication of survivors. Irradiated G2/M arrested cells from wild type but not from HR mutants partially restored the chromosome ladder following incubation (4-8hr) in yeast peptone dextrose-nocodazole. However, HR mutants showed a chromosome shattering pattern similar to wild type, an indication that lesions other than double-strand breaks, likely single-strand break, are responsible for their drastically reduced survivability.

Role of Homologous Recombination Genes in Repair of Alkylation Base Damage by Candida albicans.

Candida albicans mutants deficient in homologous recombination (HR) are extremely sensitive to the alkylating agent methyl-methane-sulfonate (MMS). Here, we have investigated the role of HR genes in the protection and repair of C. albicans chromosomes by taking advantage of the heat-labile property (55 °C) of MMS-induced base damage. Acute MMS treatments of cycling cells caused chromosome fragmentation in vitro (55 °C) due to the generation of heat-dependent breaks (HDBs), but not in vivo (30 °C). Following removal of MMS wild type, cells regained the chromosome ladder regardless of whether they were transferred to yeast extract/peptone/dextrose (YPD) or to phosphate buffer saline (PBS); however, repair of HDB/chromosome restitution was faster in YPD, suggesting that it was accelerated by metabolic energy and further fueled by the subsequent overgrowth of survivors. Compared to wild type CAI4, chromosome restitution in YPD was not altered in a Carad59 isogenic derivative, whereas it was significantly delayed in Carad51 and Carad52 counterparts. However, when post-MMS incubation took place in PBS, chromosome restitution in wild type and HR mutants occurred with similar kinetics, suggesting that the exquisite sensitivity of Carad51 and Carad52 mutants to MMS is due to defective fork restart. Overall, our results demonstrate that repair of HDBs by resting cells of C. albicans is rather independent of CaRad51, CaRad52, and CaRad59, suggesting that it occurs mainly by base excision repair (BER).

DNA Repair Deficient Chinese Hamster Ovary Cells Exhibiting Differential Sensitivity to Charged Particle Radiation under Aerobic and Hypoxic Conditions

It has been well established that hypoxia significantly increases both cellular and tumor resistance to ionizing radiation. Hypoxia associated radiation resistance has been known for some time but there has been limited success in sensitizing cells to radiation under hypoxic conditions. These studies show that, when irradiated with low linear energy transfer (LET) gamma-rays, poly (ADP-ribose), polymerase (PARP), Fanconi Anemia (FANC), and mutant Chinese Hamster Ovary (CHO) cells respond similarly to the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair mutant CHO cells. Comparable results were observed in cells exposed to 13 keV/μm carbon ions. However, when irradiated with higher LET spread out Bragg peak (SOBP) carbon ions, we observed a decrease in the oxygen enhancement ratio (OER) in all the DNA of repair mutant cell lines. Interestingly, PARP mutant cells were observed as having the largest decrease in OER. Finally, these studies show a significant increase in the relative biological effectiveness (RBE) of high LET SOBP carbon and iron ions in HR and PARP mutants. There was also an increase in the RBE of NHEJ mutants when irradiated to SOBP carbon and iron ions. However, this increase was lower than in other mutant cell lines. These findings indicate that high LET radiation produces unique types of DNA damage under hypoxic conditions and PARP and HR repair pathways play a role in repairing this damage.

THE ROLE OF PROTEIN CHAPERONES IN THE SURVIVAL FROM ANTHRACYCLINE-INDUCED OXIDATIVE STRESS IN SACCHAROMYCES CEREVISIAE.

Several S. cerevisiae deletion strains involving heat-shock response factors were among the most sensitive mutants identified in a previous genetic screen for doxorubicin hypersensitivity. These strains included ydj1Δ, ssz1Δ and zuo1Δ mutants. In addition, new1Δ, whose function was unknown, also displayed significant sensitivity to anthracyclines. We further investigated the basis for the sensitivity of these mutants. We determined that heat-shock could partially rescue the sensitivity of the strains to doxorubicin, including the homologous recombination mutant rad52Δ, which is sensitive to doxorubicin-mediated DNA double strand breaks (DSBs). However, none of the heat-shock response mutants were sensitive to DSBs, but were highly sensitive to reactive oxygen species (ROS) generated by quinone-ring-containing agents, such as anthracyclines and menadione. A fluorescent-based assay indicates that doxorubicin causes protein aggregation. Interestingly, the disaggregase mutant hsp104Δ is not sensitive to anthracyclines or menadione suggesting that Hsp104p does not play a role in disaggregating doxorubicin-induced protein aggregates. However New1p, which has been recently shown to be a novel disaggregase, is essential for cell viability after exposure to anthracyclines and menadione and it is not involved in thermotolerance. Our data suggest that in S. cerevisiae, doxorubicin produces protein aggregation through ROS and requires Ydj1p and New1p for resolution.

Distinct cellular phenotype linked to defective DNA interstrand crosslink repair and homologous recombination.

Repair of DNA interstrand crosslinks (ICLs) predominantly involves the Fanconi anemia (FA) pathway and homologous recombination (HR). The HR repair system eliminates DNA double strand breaks (DSBs) that emerge during ICLs removal. The current study presents a novel cell line, CL-V8B, representing a new complementation group of Chinese hamster cell mutants hypersensitive to DNA crosslinking factors. CL-V8B exhibits increased sensitivity to various DNA-damaging agents, including compounds leading to DSBs formation (bleomycin and 6-thioguanine), and is extremely sensitive to poly (ADP-ribose) polymerase inhibitor (>400-fold), which is typical for HR-defective cells. In addition, this cell line exhibits a reduced number of spontaneous and induced sister chromatid exchanges, which suggests likely impairment of HR in CL-V8B cells. However, in contrast to other known HR mutants, CL-V8B cells do not show defects in Rad51 foci induction, but only slight alterations in the focus formation kinetics. CL-V8B is additionally characterized by a considerable chromosomal instability, as indicated by a high number of spontaneous and MMC-induced chromosomal aberrations, and a twice as large proportion of cells with abnormal centrosomes than that in the wild type cell line. The molecular defect present in CL-V8B does not affect the efficiency and stabilization of replication forks. However, stalling of the forks in response to replication stress is observed relatively rarely, which suggests an impairment of a signaling mechanism. Exposure of CL-V8B to crosslinking agents results in S-phase arrest (as in the wild type cells), but also in larger proportion of G2/M-phase cells and apoptotic cells. CL-V8B exhibits similarities to HR- and/or FA-defective Chinese hamster mutants sensitive to DNA crosslinking agents. However, the unique phenotype of this new mutant implies that it carries a defect of a yet unidentified gene involved in the repair of ICLs.

Homologous Recombination Defective Arabidopsis Mutants Exhibit Enhanced Sensitivity to Abscisic Acid.

Abscisic acid (ABA) acts as an important plant hormone in regulating various aspects of plant growth and developmental processes particularly under abiotic stress conditions. An increased ABA level in plant cells inhibits DNA replication and cell division, causing plant growth retardation. In this study, we have investigated the effects of ABA on the growth responses of some major loss-of-function mutants of DNA double-stand break (DSB) repair genes in Arabidopsis during seed germination and early stages of seedling growth for understanding the role of ABA in the induction of genome instability in plants. A comparative analysis of ABA sensitivity of wild-type Arabidopsis and the knockout mutant lines related to DSB sensors, including atatm, atatr, the non-homologous end joining (NHEJ) pathway genes, and mutants related to homologous recombination (HR) pathway genes showed relatively enhanced sensitivity of atatr and HR-related mutants to ABA treatment. The expression levels of HR-related genes were increased in wild-type Arabidopsis (Col-0) during seed germination and early stages of seedling growth. Immunoblotting experiments detected phosphorylation of histone H2AX in wild-type (Col-0) and DSB repair gene mutants after ABA treatment, indicating the activation of DNA damage response due to ABA treatment. Analyses of DSB repair kinetics using comet assay under neutral condition have revealed comparatively slower DSB repair activity in HR mutants. Overall, our results have provided comprehensive information on the possible effect of ABA on DNA repair machinery in plants and also indicated potential functional involvement of HR pathway in repairing ABA induced DNA damage in Arabidopsis.

Sequential and counter-selectable cassettes for fission yeast.

BackgroundFission yeast is one of the most commonly used model organisms for studying genetics. For selection of desirable genotypes, antibiotic resistance cassettes are widely integrated into the genome near genes of interest. In yeasts, this is achieved by PCR amplification of the cassette flanked by short homology sequences, which can be incorporated by homology directed repair. However, the currently available cassettes all share the same tef promoter and terminator sequences. It can therefore be challenging to perform multiple genetic modifications by PCR-based targeting, as existing resistance cassettes in strains can be favored for recombination due to shared homology between the cassettes.ResultsHere we have generated new selection cassettes that do not recombine with those traditionally used. We achieved this by swapping the tef promoter and terminator sequences in the established antibiotic resistance MX6 cassette series for alternative promoters and/or terminators. The newly created selection cassettes did not recombine with the tef-containing MX6 cassettes already present in the genome, allowing for sequential gene targeting using the PCR-based method. In addition, we have generated a series of plasmids to facilitate the C-terminal tagging of genes with desired epitopes. We also utilized the anti-selection gene HSV-TK, which results in cell death in strains grown on the drug 5-Fluoro-2’-deoxyuridine (FdU, Floxuridin or FUDR). By fusing an antibiotic resistance gene to HSV-TK, we were able to select on the relevant antibiotic as well as counter-select on FdU media to confirm the desired genomic modification had been made. We noted that the efficiency of the counter selection by FdU was enhanced by treatment with hydroxyurea. However, a number of DNA replication checkpoint and homologous recombination mutants, including rad3∆, cds1∆, rad54∆ and rad55∆, exhibited sensitivity to FdU even though those strains did not carry the HSV-TK gene. To remove counter-selectable markers, we introduced the Cre-loxP irreversible recombination method. Finally, utilizing the negative selectable markers, we showed efficient induction of point mutations in an endogenous gene by a two-step transformation method.ConclusionsThe plasmid constructs and techniques described here are invaluable tools for sequential gene targeting and will simplify construction of fission yeast strains required for study.

Role of various DNA repair pathways in chromosomal inversion formation in CHO mutants

Purpose: In an effort to better understand the formation of chromosomal inversions, we investigated the role of various DNA repair pathways, including the non-homologous end joining (NHEJ), homologous recombination (HR), and Fanconi Anemia (FA) repair pathways for the formation of radiation induced chromosomal inversions.Materials and methods: CHO10B2 wild type, CHO DNA repair-deficient, and CHO DNA repair-deficient corrected mutant cells were synchronized into G1 phase and exposed to gamma-rays. First post-irradiation metaphase cells were analyzed for chromosomal inversions by a differential chromatid staining technique involving a single cycle pre-irradiation ethynyl-uridine treatment and statistic calculations.Results: It was observed that inhibition of the NHEJ pathway resulted in an overall decrease in the number of radiation-induced inversions, roughly a 50% decrease when compared to the CHO wild type. Interestingly, inhibition of the FA pathway resulted in an increase in both the number of spontaneous inversions and the number of radiation-induced inversions observed after exposure to 2 Gy of ionizing radiation. It was observed that FA-deficient cells contained roughly 330% (1.24 inversions per cell) more spontaneous inversions and 20% (0.4 inversions per cell) more radiation-induced inversions than the wild-type CHO cell lines. The HR mutants, defective in Rad51 foci, showed similar number of spontaneous and radiation-induced inversion as the wild-type cells. Gene complementation resulted in both spontaneous and radiation-induced inversions resembling the CHO wild-type cells.Conclusions: We have concluded that the NHEJ repair pathway contributes to the formation of radiation-induced inversions. Additionally, through an unknown molecular mechanism it appears that the FA signal pathway prevents the formation of both spontaneous and radiation induced inversions.

Genetic interactions among homologous recombination mutants in Candida albicans

rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes.

Potentially lethal damage repair in drug arrested G2-phase cells after radiation exposure.

Potentially lethal damage (PLD) repair has been defined as that property conferring the ability of cells to recover from DNA damage depending on the postirradiation environment. Using a novel cyclin dependent kinase 1 inhibitor RO-3306 to arrest cells in the G2 phase of the cell cycle, examined PLD repair in G2 in cultured Chinese hamster ovary (CHO) cells. Several CHO-derived DNA repair mutant cell lines were used in this study to elucidate the mechanism of DNA double-strand break repair and to examine PLD repair during the G2 phase of the cell cycle. While arrested in G2 phase, wild-type CHO cells displayed significant PLD repair and improved cell survival compared with cells released immediately from G2 after irradiation. Both the radiation-induced chromosomal aberrations and the delayed entry into mitosis were also reduced by G2-holding PLD recovery. The PLD repair observed in G2 was observed in nonhomologous end-joining (NHEJ) mutant cell lines but absent in homologous recombination mutant cell lines. From the survival curves, G2-NHEJ mutant cell lines were found to be very sensitive to gamma-ray exposure when compared to G2/homologous recombination mutant cell lines. Our findings suggest that after exposure to ionizing radiation during G2, NHEJ is responsible for the majority of non-PLD repair, and conversely, that the homologous recombination is responsible for PLD repair in G2.

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.

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.

Nse1-dependent recruitment of Smc5/6 to lesion-containing loci contributes to the repair defects of mutant complexes

Of the three structural maintenance of chromosomes (SMC) complexes, Smc5/6 remains the most poorly understood. Genetic studies have shown that Smc5/6 mutants are defective in homologous recombination (HR), and consistent with this, Smc5/6 is enriched at lesions. However, Smc5/6 is essential for viability, but HR is not, and the terminal phenotype of null Smc5/6 mutants is mitotic failure. Here we analyze the function of Nse1, which contains a variant RING domain that is characteristic of ubiquitin ligases. Whereas deletion of this domain causes DNA damage sensitivity and mitotic failure, serine mutations in conserved cysteines do not. However, these mutations suppress the DNA damage sensitivity of Smc5/6 hypomorphs but not that of HR mutants and remarkably decrease the recruitment of Smc5/6 to loci containing lesions marked for HR-mediated repair. Analysis of DNA repair pathways in suppressed double mutants suggests that lesions are channeled into recombination-dependent and error-free postreplication repair. Thus the HR defect in Smc5/6 mutants appears to be due to the presence of dysfunctional complexes at lesions rather than to reflect an absolute requirement for Smc5/6 to complete HR.

Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate

DNA-methylating agents of the SN2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines. These adducts are repaired by base excision repair (BER). Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e.g. apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs). Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS). We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PKCS mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS. On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment. Since DSBs are expected to be formed specifically in the S-phase, we assessed the formation and kinetics of repair of DSBs by γH2AX quantification in a cell cycle specific manner. In the cytotoxic dose range of MMS a significant amount of γH2AX foci was induced in S, but not G1- and G2-phase cells. A major fraction of γH2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs. DSB formation following MMS treatment was also demonstrated by the neutral comet assay. Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair, while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype. Moreover, DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment. Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs. Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following SN2-alkylating agents.

Inactivation of dhaD and dhaK abolishes by-product accumulation during 1,3-propanediol production in Klebsiella pneumoniae

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P(BAD) promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.

Brca2/Xrcc2 dependent HR, but not NHEJ, is required for protection against O 6-methylguanine triggered apoptosis, DSBs and chromosomal aberrations by a process leading to SCEs

O 6-methylguanine (O 6MeG) is a highly critical DNA adduct induced by methylating carcinogens and anticancer drugs such as temozolomide, streptozotocine, procarbazine and dacarbazine. Induction of cell death by O 6MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O 6MeG. Here, we compared the response of cells mutated in HR and NHEJ proteins to temozolomide and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (DNA-PK cs mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of O 6-methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific hypersensitivity was due to O 6MeG lesions. MNNG provoked H2AX phosphorylation 24–48 h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The γH2AX foci formed in response to O 6MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O 6MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O 6MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving cancer therapy with methylating agents.