Accumulation Of Single-strand Breaks Research Articles

Acetaminophen‐induced Liver Injury Induces DNA Damage and Activation of Ataxia Telangiectasia Mutated in Mice

Acetaminophen (APAP) is a common analgesic/antipyretic that is widely used throughout the world. At therapeutic doses it is safe but at higher dosages APAP causes acute liver failure (ALF). Drug‐induced liver toxicity is the most common cause of liver damage, accounting for 50% of the cases of liver failure. With the only efficacious form of treatment being liver transplant, it is imperative to find alternative forms of treatment. Gaining a greater understanding of the molecular pathology contributing to the progression of liver failure, we allow insights on methods to enhance patient care and quality of life. Acute liver injury from APAP is associated with increased levels of inflammation, oxidative stress, and hepatic cell death. High levels of oxidative stress damages multiple systems within the cell, most notability the DNA. In response to DNA damage, DNA damage response (DDR) markers are activated leading to cell cycle arrest, DNA repair and the eventual death of the cell if damage is unable to be repaired. Our hypothesis is that the accumulation of DNA damage results in the activation of the DDR, which then aids in driving further progression of APAP‐induced liver injury.MethodsC57Bl/6 mice were injected with APAP (300mg/kg) do induce liver injury. Liver tissue was collected after 1, 2, 4, 6, 12, and 24 hours. Histology was performed on paraffin‐embedded sections using antibodies specific for phospho‐ATM (ser 1981) and γH2AX (ser 139). Replication protein A (RPA), Ataxia Telangiectasia mutated (ATM) and 53BP were assessed by qPCR. Phospho‐ATM and RPA were assessed by immunoblotting.ResultsAPAP injected mice showed a marked increase in the accumulation of DNA damage when compared to saline injected mice in the liver. Markers for DNA double strand breaks (DSB), γH2AX and 53BP, were shown to be elevated 2 hours after APAP injection followed by a gradual decrease. RPA, a single stranded DNA‐binding protein, was also shown to be elevated 4 hours after APAP injection indicating an accumulation of single strand breaks. ATM, a DDR maker, and the phosphorylated form of ATM were shown to also be elevated after APAP injection after onset of DSB, with peak expression at 4 hours.ConclusionDuring APAP‐induced liver injury, there is an elevated level of DNA damage found within the liver. This elevated amount of DNA damage results in the activation of ATM and the DDR. ATM‐mediated signaling may be a potential driver for hepatocellular death and ALF. Agents designed to inhibit ATM‐mediated signaling or the DDR response need to be assessed as potential therapeutic targets for the treatment of APAP‐induced liver damage.Support or Funding InformationThis study was funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This work was completed with support from the Veterans Health Administration and with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

Noncanonical Functions of the Human Ribosomal Repeat

Ribosomal genes encode ribosomal RNA (rRNA), which is an integral part of ribosomes. The main function of ribosomal genes in the cell is the synthesis of rRNA. However, ribosomal genes can also perform other functions in the body. It was found that DNA of ribosomal genes (rDNA) is an active biomolecule, which can be attributed to the family of DAMPs (danger-associated molecular patterns). Three unusual characteristics of rDNA confer to it the properties of a DAMP molecule: (1) high content of unmethylated CpG motifs—ligands of DNA sensing TLR9; (2) low oxidation potential; and (3) resistance to fragmentation under the accumulation of single-strand breaks in rDNA chains. Owing to these properties, rDNA fragments are accumulated as a part of circulating extracellular DNA and stimulate the TLR9–MyD88–NF-kB signaling pathway in various cells of the body. Oxidized rDNA permeates into the cells, where it can stimulate other DNA sensors (AIM2, RIG1, STING). Extracellular oxidized rDNA reaches the structures of the nucleolus and affects the level of rRNA in the cell. The body defends itself against the excess of extracellular rDNA by producing antibodies to rDNA, which form much stronger complexes with rDNA than common antibodies to double-stranded DNA. It is reasonable to further study extracellular rDNA as a potential target in the treatment of autoimmune, oncological, and cardiovascular diseases.

Sp1-independent downregulation of NHEJ in response to BER deficiency

Base excision repair (BER) is the major repair pathway that removes DNA single strand breaks (SSBs) arising spontaneously due to the inherent instability of DNA. Unrepaired SSBs promote cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, in response to persistent DNA strand breaks, ATM-dependent degradation of transcription factor Sp1 leads to downregulation of BER genes expression, further accumulation of SSBs and renders cells susceptible to elimination via apoptosis. In contrast, many cancer cells are not able to block replication and to downregulate the expression of Sp1 in response to DNA damage. However, knockdown of BER in cancer cells leads to the accumulation of DNA double strand breaks (DSBs), suggesting deficiency in non-homologous end joining (NHEJ) repair of DSBs. Here we investigated whether DNA repair deficiency caused by knockdown of the XRCC1 gene expression in proliferating cells results in downregulation of NHEJ genes expression. We find that knockdown of the XRCC1 gene expression does not cause degradation of Sp1, but leads to downregulation of Lig4/XRCC4 and Ku70/80 at the transcription and protein levels. We thus propose the existence of Sp1-independent backup mechanism that in response to BER deficiency downregulates NHEJ in proliferating cells.

Abnormal leaf development of rpt5a mutant under zinc deficiency reveals important role of DNA damage alleviation for normal leaf development

Leaf development in plants, including dorsoventral (adaxial–abaxial) patterning, is tightly regulated. The involvement of several subunits of the 26S proteasome in adaxial–abaxial polarity establishment has been reported. In the present study, we revealed that in Arabidopsis thaliana, a mutation in RPT5A, a subunit of 26S proteasome, causes abnormally narrow true leaves under zinc deficiency. mRNA accumulations of DNA damage marker genes in leaves were elevated by zinc deficiency. PARP2, a single-strand break (SSB) inducible gene, was more strongly induced by zinc deficiency in rpt5a mutants compared with the wild type. A comet assay indicated that SSB is enhanced in mutants grown under the zinc deficiency condition. These results suggest that SSB accumulation is accompanied by abnormal leaf development. To test if DNA damage is a sole cause of abnormal leaf development, we treated the wild type grown under normal zinc conditions with zeocin, a DNA damage-inducing reagent, and found that narrow leaves developed, suggesting that DNA damage is sufficient to induce the development of abnormally narrow leaves. Taken together with the observation of the abnormal leaf morphology of our mutant plant under zinc deficiency, we demonstrated that the alleviation of DNA damage is important for normal leaf development.

DNA single-strand break-induced DNA damage response causes heart failure

The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure.

Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells.

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity.

Abstract 15981: DNA Single-strand Break-induced DNA Damage Response Causes Heart Failure

Introduction: The DNA damage response (DDR) pathway is activated upon DNA damage. In mitotic cells, the DDR plays essential role in maintaining genomic stability and preventing cancer formation. DNA damage and activation of the DDR are also observed in the post-mitotic cardiomyocytes of patients with end-stage heart failure, however, their roles in the pathogenesis of heart failure remains elusive. Methods and Results: We performed transverse aortic constriction (TAC) operation to produce mice model of pressure-overload induced heart failure. Alkaline- and neutral- comet assay revealed that unrepaired DNA single-strand break (SSB), not double-strand break, is accumulated in cardiomyocytes of the failing heart. Mice with cardiomyocyte-specific deletion of XRCC1, a scaffold protein essential for SSB repair, exhibited more severe heart failure and higher mortality after TAC operation. Knockdown of Xrcc1 using siRNA produced SSB accumulation in cardiomyocytes and SSB accumulation induced persistent DDR through activation of ataxia telangiectasia mutated (ATM) kinase. Activated ATM also induced nuclear translocation of NF-κB and increased the expression of inflammatory cytokines. Activation of DDR, nuclear translocation of NF-κB, and increased expression of inflammatory cytokines were also observed in the failing heart and were enhanced in the heart of cardiomyocyte-specific XRCC1 knockout mice. Conclusions: Unrepaired DNA SSB accumulates in post-mitotic cardiomyocytes and plays a pathogenic role in pressure overload-induced heart failure. Approaches that promote efficient SSB repair or suppress aberrant activation of DDR pathway may become a novel therapeutic strategy against heart failure.

Abstract 53: Pathogenic role of DNA Single Strand Break Accumulation in Heart Failure

Various environmental stress including reactive oxygen species (ROS) causes nuclear DNA damage. Increased production of ROS is observed in the failing heart and is considered as one of the causes of heart failure. Accumulating evidences suggest the presence of DNA damage in the failing heart, however, mechanistic link that connects DNA damage and heart failure remains elusive. Here, we show that DNA single strand break (SSB) accumulates in the failing heart and that SSB accumulation induces cell-autonomous inflammation through activation of DNA damage response (DDR) signaling pathway. Using alkaline- and neutral comet assay, we found that SSB is increased in the failing heart of pressure overload. Using in vitro model, we found that SSB accumulation activates ataxia talengiectasia mutated (ATM) kinase, which in turn induces nuclear translocation of NF-kB and increases the expression of inflammatory cytokines. Our findings suggest that SSB accumulation in cardiomyocytes plays an important role in the pathogenesis of heart failure by activating DDR pathway and subsequent cell-autonomous inflammation. SSB accumulation is supposed to be characteristic to post-mitotic cells like cardiomyocytes because unrepaired SSB usually develops into DNA double strand break and lead to catastrophic cellular death in mitotic cells. Approaches targeting efficient SSB repair or DDR pathway may become a novel therapeutic strategy against heart failure.

Clinical significance of PARP-1 inhibitors in cancer chemotherapy

Poly (ADPribose) polymerase 1 (PARP-1) protein plays an important role in the repair of single strand breaks (SSBs) in the DNA. This is mediated by base excision repair (BER) of SSBs. PARP-1 inhibition allows accumulation of SSBs and consequently double strand breaks (DSBs). PARP inhibitors disrupt the BER and causes cell damage and death, especially in breast cancer susceptibility proteins 1 (BRCA 1) or BRCA2mutated cell in which homologous recombination (HR) pathway is defective. PARP-1 is effective as a single agent in BRCA-1 and BRCA-2 mutated ovarian and breast cancer. In addition, a number of studies have shown beneficial effects with PARP-1 inhibitors in combination with cytotoxic agents that target SSBs, especially in case of triple negative breast cancers (TNBC). Here we have reviewed potential clinical applications of PARP-1 inhibitors and issues associated with it for successful cancer chemotherapy.

PARP Inhibitors in Cancer Therapy: Magic Bullets but Moving Targets

The pharmacological inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) have reached the first milestone toward their inclusion in the arsenal of anti-cancer drugs by showing consistent benefits in clinical trials against BRCA-mutant cancers that are deficient in the homologous recombination repair (HRR) of DNA double strand breaks (DSB) (1, 2). PARP inhibitors (PARPi) also potentiate therapeutic efficacy of ionizing radiation and some chemotherapeutic agents (1). These effects of PARPi were initially linked to inhibition of the role of PARP-1 in base excision repair (BER) of DNA damaged by endogenous or exogenous agents, resulting in accumulation of single strand breaks (SSB), which upon conversion to toxic DSB lesions would kill cancer cells deficient in DSB repair (1, 3, 4). However, PARPi lethality in HRR-deficient cancers can also be explained by other mechanisms not involving a direct effect of PARPi on BER [reviewed in Ref. (5, 6)]. In addition, therapeutic benefits of PARPi with agents such as carboplatin in HRR-proficient and -deficient tumors [reviewed in Ref. (1, 7)], simply cannot be explained by BER inhibitory effect of PARPi. Therefore, PARPi are like magic bullets that can kill cancer cells under different circumstances, but to comprehend their global scope and limitations, here we discuss the full range of their targets and the possible impact of broad specificity of current PARPi during prolonged therapy of cancer patients.

8-Oxoguanine causes neurodegeneration during MUTYH-mediated DNA base excision repair

8-Oxoguanine (8-oxoG), a common DNA lesion caused by reactive oxygen species, is associated with carcinogenesis and neurodegeneration. Although the mechanism by which 8-oxoG causes carcinogenesis is well understood, the mechanism by which it causes neurodegeneration is unknown. Here, we report that neurodegeneration is triggered by MUTYH-mediated excision repair of 8-oxoG-paired adenine. Mutant mice lacking 8-oxo-2'-deoxyguanosine triphosphate-depleting (8-oxo-dGTP-depleting) MTH1 and/or 8-oxoG-excising OGG1 exhibited severe striatal neurodegeneration, whereas mutant mice lacking MUTYH or OGG1/MUTYH were resistant to neurodegeneration under conditions of oxidative stress. These results indicate that OGG1 and MTH1 are protective, while MUTYH promotes neurodegeneration. We observed that 8-oxoG accumulated in the mitochondrial DNA of neurons and caused calpain-dependent neuronal loss, while delayed nuclear accumulation of 8-oxoG in microglia resulted in PARP-dependent activation of apoptosis-inducing factor and exacerbated microgliosis. These results revealed that neurodegeneration is a complex process caused by 8-oxoG accumulation in the genomes of neurons and microglia. Different signaling pathways were triggered by the accumulation of single-strand breaks in each type of DNA generated during base excision repair initiated by MUTYH, suggesting that suppression of MUTYH may protect the brain under conditions of oxidative stress.

Abstract 1774: Dihydroartemisinin (DHA): A potent enhancer of PARP inhibitors and DNA-damaging agents activity in human tumor models

Abstract Synthetic lethality (SL) is a cellular condition in which two or more non-allelic and non-essential mutations, which are not lethal on their own, become deadly when present within the same cell. PARP inhibitors (PARPi) represent a paradigmatic example of therapeutic application of the SL approaches. In cancer cells with BRCA1 or BRCA2 loss of function, inhibition of PARP1 activity leads to an accumulation of single strand breaks converted to double strand breaks but cannot be repaired by homologous recombination. However, preclinical and preliminary clinical evidences suggest a potentially broader scope for PARPi. Currently, several PARPi are under different phases of clinical investigation as monotherapy or in combination with DNA damaging agents. Although PARPi in monotherapy are quite safe, their combination with cytotoxic agents leads to exacerbation of the typical cytotoxic-related side effects, thus reducing the potentiality to improve the therapeutic index (T.I.) of these combinations. The study aim was to assess if the combination of PARPi and DNA-damaging agents with the widely used antimalarial agent DHA could improve both their antitumor activity and T.I. We have preliminary observed, in different cancer cells, that DHA was an inducer of endoplasmic reticulum (ER) stress (increased expression of typical ER stress-related genes), activation of checkpoint kinases and transcriptional up-regulation of different p53-target genes and of mitochondrial membrane depolarization. A consequence is PARP activation that, in absence of other factors, leads the cells to recover from damage and to their rescue. On the contrary, we observed that the simultaneous use of DHA with a PARPi or cytotoxic agents causes a massive cell death. More specifically a strong synergism was observed on tumor cells, when DHA was combined with different drugs: doxorubicin and platinum compounds on NCI-H460, temozolomide (TMZ) on MDA-MB436 and CAPAN-1. In addition, a synergistic interaction was identified between DHA and the PARPi (ABT-888 or AZD-2281) on various tumor cells (e.g., A431, HCT116, SW620, MDA-MB436, NCI-H460, A2780/DDP, CAPAN-1). In vivo, the combination of DHA (delivered at 200 mg/kg p.o. according to the schedule qdx5/w) with ABT-888 or AZD-2281, resulted in a significantly higher tumor growth inhibition than with monotherapy in NCI-H460 NSCLC, HCT116 colon ca. and MDA-MB436 triple-negative breast cancer BRCA1 negative xenografted in nude mice. DHA was also able to synergize in vivo with the alkylating agent TMZ against MDA-MB436 tumor model. No toxic effects of administrations of DHA in combination with chemotherapeutics in terms of body weight recordings were found throughout the experiments.Taken together, these results clearly pinpoint the clinical potential offered by DHA in the SL approaches in developing less toxic and more efficacious therapies to treat cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1774. doi:1538-7445.AM2012-1774

NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa− mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa− embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa− primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa− MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa− embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa− embryos. However, immortalized Itpa− MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa− MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Evidence of altered DNA integrity in the brain regions of suicidal victims of Bipolar Depression

Deoxyribonucleic acid (DNA) integrity plays a significant role in cell function. There are limited studies with regard to the role of DNA damage in bipolar affective disorder (BP). In the present study, we have assessed DNA integrity, conformation, and stability in the brain region of bipolar depression (BD) patients (n=10) compared to age-matched controls (n=8). Genomic DNA was isolated from 10 postmortem BD patients’ brain regions (frontal cortex, Pons, medulla, thalamus, cerebellum, hypothalamus, Parietal, temporal, occipital lobe, and hippocampus) and from the age-matched control subjects. DNA from the frontal cortex, pons, medulla, and thalamus showed significantly higher number of strand breaks in BD (P<0.01) compared to the age-matched controls. However, DNA from the hippocampus region was intact and did not show any strand breaks. The stability studies also indicated that the melting temperature and ethidium bromide binding pattern were altered in the DNA of BD patients’ brain regions, except in the hippocampus. The conformation studies showed B-A or secondary B-DNA conformation (instead of the normal B-DNA) in BD patients’ brain regions, with the exception of the hippocampus. The levels of redox metals such as Copper (Cu) and Iron (Fe) were significantly elevated in the brain regions of the sufferers of BD, while the Zinc (Zn) level was decreased. In the hippocampus, there was no change in the Fe or Cu levels, whereas, the Zn level was elevated. There was a clear correlation between Cu and Fe levels versus strand breaks in the brain regions of the BD. To date, as far as we are aware, this is a new comprehensive database on stability and conformations of DNA in different brain regions of patients affected with BD. The biological significance of these findings is discussed here.

The Relationship between the Aging- and Photo-Dependent T414G Mitochondrial DNA Mutation with Cellular Senescence and Reactive Oxygen Species Production in Cultured Skin Fibroblasts

Mutations in the mitochondrial genome (mtDNA) are thought to be one of the causes of age-dependent cellular decline through their detrimental effects on respiration or reactive oxygen species (ROS) production. However, for many mutations, this link has not been clearly established. This study aimed to further investigate the phenotypic importance of a T414G mutation within the control region of mtDNA, previously shown to accumulate in both chronologically and photoaged human skin. We demonstrate that during dermal skin fibroblast replication in vitro in five separate cultures obtained from elderly individuals, the T414G mutant load can either increase or decrease during progressive cell division, implying the absence of consistent selection against the mutation in this context. In support of this, by utilizing a cell-sorting approach, we demonstrate that the level of the T414G mutation does not directly correlate with increased or decreased mtDNA copy number, or markers of cellular ageing including lipofuscin accumulation or ROS production. By consequence, the mutation can be distributed with a bias towards either the proliferating or senescent cell populations depending on the cell line. In conclusion, we propose that this particular mutation may have little effect on ROS production and the onset of cellular senescence in cultured fibroblasts.

Regulation of the activity and expression of ERK8 by DNA damage

We have investigated the agonists that activate transfected extracellular signal-regulated kinase 8 (ERK8) in cells, and have found that the most potent activators are hydrogen peroxide, DNA alkylating and cross-linking agents and the poly (ADP-ribose) polymerase inhibitor KU-0058948. The feature shared by all these agents is that they lead to the accumulation of single strand breaks in DNA, suggesting a role for ERK8 in the response to, or repair of, DNA single strand breaks. The DNA alkylating agent MMS also induced the disappearance of endogenous ERK8 by a proteasome-dependent mechanism.

Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs

Oxidative base lesions, such as 8-oxoguanine (8-oxoG), accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known which form of DNA is involved, whether nuclear or mitochondrial, nor is it known how the death order is executed. We established cells which selectively accumulate 8-oxoG in either type of DNA by expression of a nuclear or mitochondrial form of human 8-oxoG DNA glycosylase in OGG1-null mouse cells. The accumulation of 8-oxoG in nuclear DNA caused poly-ADP-ribose polymerase (PARP)-dependent nuclear translocation of apoptosis-inducing factor, whereas that in mitochondrial DNA caused mitochondrial dysfunction and Ca2+ release, thereby activating calpain. Both cell deaths were triggered by single-strand breaks (SSBs) that had accumulated in the respective DNAs, and were suppressed by knockdown of adenine DNA glycosylase encoded by MutY homolog, thus indicating that excision of adenine opposite 8-oxoG lead to the accumulation of SSBs in each type of DNA. SSBs in nuclear DNA activated PARP, whereas those in mitochondrial DNA caused their depletion, thereby initiating the two distinct pathways of cell death.

Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1(-/-) DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1(-/-) cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.

APE1 overexpression in XRCC1-deficient cells complements the defective repair of oxidative single strand breaks but increases genomic instability

XRCC1 protein is essential for mammalian viability and is required for the efficient repair of single strand breaks (SSBs) and damaged bases in DNA. XRCC1-deficient cells are genetically unstable and sensitive to DNA damaging agents. XRCC1 has no known enzymatic activity and is thought to act as a scaffold protein for both SSB and base excision repair activities. To further define the defects leading to genetic instability in XRCC1-deficient cells, we overexpressed the AP endonuclease APE1, shown previously to interact with and be stimulated by XRCC1. Here, we report that the overexpression of APE1 can compensate for the impaired capability of XRCC1-deficient cells to repair SSBs induced by oxidative DNA damage, both in vivo and in whole-cell extracts. We show that, for this kind of damage, the repair of blocked DNA ends is rate limiting and can be performed by APE1. Conversely, APE1 overproduction resulted in a 3-fold increase in the sensitivity of XRCC1-deficient cells to an alkylating agent, most probably due to the accumulation of SSBs. Finally, the overproduction of APE1 results in increases of 40% in the frequency of micronuclei and 33% in sister chromatid exchanges of XRCC1− cells. These data suggest that the spontaneous generation of AP sites could be at the origin of the SSBs responsible for the spontaneous genetic instability characteristic of XRCC1-deficient cells.

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap α-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.