Reactive oxygen species stimulate homologous recombination in plants

Coping with the continuous production of free radicals is a daily routine of the cell. Despite their toxicity, the reactive oxygen species (ROS) are involved in dual physiological action – signal transduction and immune response. We analysed the influence of oxidative stress‐generating compounds, rose Bengal (RB), paraquat (PQ) and amino‐triazole (ATZ) on the genome stability of Arabidopsis using transgenic recombination‐monitoring plants. Homologous recombination frequencies in plants were increased upon the treatment with RB and PQ but not ATZ. Application of the N‐acetyl‐L ‐cysteine (NAC), radicals scavenging compound, decreased the DNA damage caused by RB. Interestingly, the incubation of plants with very low concentration of RB (less than 0.2 µM ) led to the subsequent increase in plant tolerance to methyl methane sulfonate (MMS): stronger plants with a lower increase of homologous recombination frequency. In contrast, the incubation of plants with 0.5 µM of RB resulted in the potentiation of the MMS effect: the weaker plants with higher frequency of recombination. The data of the present study suggest the existence of a dual concentration‐dependent role of ROS in plants.     

AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis

DNA damage is a significant problem in living organisms and DNA repair pathways have been evolved in different species to maintain genomic stability. Here we demonstrated the molecular function of AtMMS21, a component of SMC5/6 complex, in plant DNA damage response. Compared with wild type, the AtMMS21 mutant plants show hypersensitivity in the DNA damaging treatments by MMS, cisplatin and gamma radiation. However, mms21-1 is not sensitive to replication blocking agents hydroxyurea and aphidicolin. The expression of a DNA damage response gene PARP2 is upregulated in mms21-1 under normal condition, suggesting that this signaling pathway is constitutively activated in the mutant. Depletion of ATAXIA-TELANGIECTASIA MUTATED (ATM) in mms21-1 enhances its root growth defect phenotype, indicating that ATM and AtMMS21 may play additive roles in DNA damage pathway. The analysis of homologous recombination frequency showed that the number of recombination events is reduced in mms21-1 mutant. Conclusively, we provided evidence that AtMMS21 plays an important role in homologous recombination for DNA damage repair.     

Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity

The nucleoside analog ganciclovir (GCV) elicits cytotoxicity in tumor cells via a novel mechanism in which drug incorporation into DNA produces minimal disruption of replication, but numerous DNA double strand breaks occur during the second S-phase after drug exposure. We propose that homologous recombination (HR), a major repair pathway for DNA double strand breaks, can prevent GCV-induced DNA damage, and that inhibition of HR will enhance cytotoxicity with GCV. Survival after GCV treatment in cells expressing a herpes simplex virus thymidine kinase was strongly dependent on HR (>14-fold decrease in IC₅₀ in HR-deficient vs. HR-proficient CHO cells). In a homologous recombination reporter assay, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), decreased HR repair events up to 85%. SAHA plus GCV produced synergistic cytotoxicity in U251tk human glioblastoma cells. Elucidation of the synergistic mechanism demonstrated that SAHA produced a concentration-dependent decrease in the HR proteins Rad51 and CtIP. GCV alone produced numerous Rad51 foci, demonstrating activation of HR. However, the addition of SAHA blocked GCV-induced Rad51 foci formation completely and increased γH2AX, a marker of DNA double strand breaks. SAHA plus GCV also produced synergistic cytotoxicity in HR-proficient CHO cells, but the combination was antagonistic or additive in HR-deficient CHO cells. Collectively, these data demonstrate that HR promotes survival with GCV and compromise of HR by SAHA results in synergistic cytotoxicity, revealing a new mechanism for enhancing anticancer activity with GCV.     

Interplay between Fanconi anemia and homologous recombination pathways in genome integrity

The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error‐free pathway for double‐strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication‐associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability.     

The role and mechanism of long non-coding RNAs in homologous recombination repair of radiation-induced DNA damage

DNA double-strand breaks can seriously damage the genetic information that organisms depend on for survival and reproduction. Therefore, cells require a robust DNA damage response mechanism to repair the damaged DNA. Homologous recombination (HR) allows error-free repair, which is key to maintaining genomic integrity. Long non-coding RNAs (lncRNAs) are RNA molecules that are longer than 200 nucleotides. In recent years, a number of studies have found that lncRNAs can act as regulators of gene expression and DNA damage response mechanisms, including HR repair. Moreover, they have significant effects on the occurrence, development, invasion and metastasis of tumor cells, as well as the sensitivity of tumors to radiotherapy and chemotherapy. These studies have therefore begun to expose the great potential of lncRNAs for clinical applications. In this review, we focus on the regulatory roles of lncRNAs in HR repair.     

Roles of reactive oxygen species in UVA‐induced oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid‐melanin as studied by differential spectrophotometric method

Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA‐induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA‐induced degradation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA)‐melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA‐induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole‐5,6‐quinone‐2‐carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole‐2,3,5‐tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA‐melanin was confirmed by direct measurements of singlet oxygen phosphorescence.     

Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination

Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It’s well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR.     

幽门螺杆菌对胃癌前期阶段胃上皮DNA同源重组修复关键蛋白的影响

目的 通过检测胃癌前期阶段幽门螺杆菌（Helicobacter pylori,H. pylori）阳性和阴性患者胃黏膜组织中DNA损伤标志物H2AX及同源重组（homologous recombination,HR）修复关键蛋白MRE11、Rad51、CtIP表达水平,评价H. pylori感染在胃癌前期阶段对HR精确修复的影响。方法 选择2017年3月至9月行胃镜及病理检测的165例H. pylori阳性和阴性患者,取胃黏膜上皮组织,石蜡包埋切片,行HE染色,根据世界卫生组织标准和更新的悉尼标准,划分病理类型。然后应用免疫组织化学染色方法检测H. pylori和DNA损伤标记蛋白及HR修复关键蛋白表达水平,并行统计学分析。结果 胃黏膜上皮细胞中H2AX的表达,在CSG、CAG和IM阶段,H. pylori阳性组表达高于阴性组（Mann-Whitney U=1116.5,P=0.001;Mann-Whitney U=185.0,P=0.018;Mann-Whitney U=214.5,P=0.041）,在Dys阶段,H. pylori阳性组和阴性组差异无统计学意义（Mann-Whitney U=35.5,P=0.964）;MRE11的表达,在CSG、CAG阶段,H. pylori阳性组表达高于阴性组（Mann-Whitney U=1117.0,P=0.001;Mann-Whitney U=201.0,P=0.002）,在IM、Dys阶段,H. pylori阳性组和阴性组差异无统计学意义（Mann-Whitney U=171.0,P=0.568;Mann-Whitney U=41.5,P=0.616）;Rad51的表达,在CSG、IM阶段,H. pylori阳性组表达低于阴性组（Mann-Whitney U=490.0,P=0.002;Mann-Whitney U=73.0,P=0.007）,在CAG、Dys阶段,H. pylori阳性组和阴性组差异无统计学意义（Mann-Whitney U=101.0,P=0.404;Mann-Whitney U=24.0,P=0.291）;CtIP的表达,在CSG、IM阶段,H. pylori阳性组表达低于阴性组（Mann-Whitney U=593.0,P=0.044;Mann-Whitney U=58.5,P=0.001）,在CAG、Dys阶段,H. pylori阳性组和阴性组差异无统计学意义（Mann-Whitney U=84.0,P=0.136;Mann-Whitney U=18.5,P=0.102）。结论 在胃癌前期发展阶段,H. pylori感染导致人体胃上皮细胞DNA损伤,却抑制部分HR修复通道关键蛋白表达,从而可能抑制精确的HR修复,增加细胞恶变几率。     

ATM-dependent chromatin remodeler Rsf-1 facilitates DNA damage checkpoints and homologous recombination repair

As a member of imitation switch (ISWI) family in ATP-dependent chromatin remodeling factors, RSF complex consists of SNF2h ATPase and Rsf-1. Although it has been reported that SNF2h ATPase is recruited to DNA damage sites (DSBs) in a poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent manner in DNA damage response (DDR), the function of Rsf-1 is still elusive. Here we show that Rsf-1 is recruited to DSBs confirmed by various cellular analyses. Moreover, the initial recruitment of Rsf-1 and SNF2h to DSBs shows faster kinetics than that of γH2AX after micro-irradiation. Signals of Rsf-1 and SNF2h are retained over 30 min after micro-irradiation, whereas γH2AX signals are gradually reduced at 10 min. In addition, Rsf-1 is accumulated at DSBs in ATM-dependent manner, and the putative pSQ motifs of Rsf-1 by ATM are required for its accumulation at DSBs. Furtheremore, depletion of Rsf-1 attenuates the activation of DNA damage checkpoint signals and cell survival upon DNA damage. Finally, we demonstrate that Rsf-1 promotes homologous recombination repair (HRR) by recruiting resection factors RPA32 and Rad51. Thus, these findings reveal a new function of chromatin remodeler Rsf-1 as a guard in DNA damage checkpoints and homologous recombination repair.     

SLFN11 inhibits checkpoint maintenance and homologous recombination repair

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA‐damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1‐dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA–ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA‐damaging agents. Finally, we demonstrate that the RPA1‐binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA‐damaging therapeutic agents.     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

The effects of AtRad52 over‐expression on homologous recombination in Arabidopsis

AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene‐targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52‐1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over‐expression of this variant increased HR in the nucleus, compared with the native AtRad52‐1A: it increased intra‐chromosomal recombination and synthesis‐dependent strand‐annealing HR repair rates; but conversely, it repressed the single‐strand annealing pathway. The effect of AtRad52‐1A over‐expression on gene‐targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene‐targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52‐1A over‐expression and target/donor‐specific RNAi. This suggests that sequence‐specific small RNAs might be involved in AtRad52‐1A‐mediated HR.     

Arabidopsis RecQI4A suppresses homologous recombination and modulates DNA damage responses

The DNA damage response and DNA recombination are two interrelated mechanisms involved in maintaining the integrity of the genome, but in plants they are poorly understood. RecQ is a family of genes with conserved roles in the regulation of DNA recombination in eukaryotes; there are seven members in Arabidopsis. Here we report on the functional analysis of the Arabidopsis RecQl4A gene. Ectopic expression of Arabidopsis RecQl4A in yeast RecQ-deficient cells suppressed their hypersensitivity to the DNA-damaging drug methyl methanesulfonate (MMS) and enhanced their rate of homologous recombination (HR). Analysis of three recQl4A mutant alleles revealed no obvious developmental defects or telomere deregulation in plants grown under standard growth conditions. Compared with wild-type Arabidopsis, the recQl4A mutant seedlings were found to be hypersensitive to UV light and MMS, and more resistant to mitomycin C. The average frequency of intrachromosomal HR in recQl4A mutant plants was increased 7.5-fold over that observed in wild-type plants. The data reveal roles for Arabidopsis RecQl4A in maintenance of genome stability by modulation of the DNA damage response and suppression of HR.     

Cyclo(phenylalanine‐proline) induces DNA damage in mammalian cells via reactive oxygen species

Cyclo(phenylalanine‐proline) is produced by various organisms such as animals, plants, bacteria and fungi. It has diverse biological functions including anti‐fungal activity, anti‐bacterial activity and molecular signalling. However, a few studies have demonstrated the effect of cyclo(phenylalanine‐proline) on the mammalian cellular processes, such as cell growth and apoptosis. In this study, we investigated whether cyclo(phenylalanine‐proline) affects cellular responses associated with DNA damage in mammalian cells. We found that treatment of 1 mM cyclo(phenylalanine‐proline) induces phosphorylation of H2AX (S139) through ATM‐CHK2 activation as well as DNA double strand breaks. Gene expression analysis revealed that a subset of genes related to regulation of reactive oxygen species (ROS) scavenging and production is suppressed by the cyclo(phenylalanine‐proline) treatment. We also found that cyclo(phenylalanine‐proline) treatment induces perturbation of the mitochondrial membrane, resulting in increased ROS, especially superoxide, production. Collectively, our study suggests that cyclo(phenylalanine‐proline) treatment induces DNA damage via elevation of ROS in mammalian cells. Our findings may help explain the mechanism underlying the bacterial infection‐induced activation of DNA damage response in host mammalian cells.     

The sugar beet mitochondrial genome: A complex organisation generated by homologous recombination

Summary The complete physical map of the mitochondrial genome of the Owen cytoplasm of sugar beet has been determined from overlapping cosmid clones. The genome is 386 kb in size and has a multicircular organisation generated by homologous recombination across repeated DNA elements. The location of the rRNA genes and several polypeptide genes has been determined. In addition the mitochondrial genome was found to contain a sequence of chloroplast DNA including part of the 16 S rRNA gene.     

Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene

The Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA-damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA-damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for efficient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.