Epithelial-to-mesenchymal transition and NF-kB pathways are promoted by a mutant form of DDB2, unable to bind PCNA, in UV-damaged human cells.

BACKGROUND: DNA-Damaged Binding protein 2 (DDB2) is a protein involved in the early step of Nucleotide Excision Repair. Recently, it has been reported that DDB2 is involved in epithelial-to-mesenchymal transition (EMT), key process in tumour invasiveness and metastasis formation. However, its role is not completely known. METHODS: Boyden chamber and cell adhesion assays, and ICELLigence analysis were performed to detect HEK293 adhesion and invasion. Western blotting and gelatine zymography techniques were employed to assess the EMT protein levels and MMP enzymatic activity. Immunofluorescence analysis and pull-down assays facilitated the detection of NF-kB sub-cellular localization and interaction. RESULTS: We have previously demonstrated that the loss of DDB2-PCNA binding favours genome instability, and increases cell proliferation and motility. Here, we have investigated the phenotypic and molecular EMT-like changes after UV DNA damage, in HEK293 clones stably expressing DDB2Wt protein or a mutant form unable to interact with PCNA (DDB2PCNA-), as well as in HeLa cells transiently expressing the same DDB2 constructs. Cells expressing DDB2PCNA- showed morphological modifications along with a reduced expression of E-cadherin, an increased activity of MMP-9 and an improved ability to migrate, in concomitance with a significant upregulation of EMT-associated Transcription Factors (TFs), whose expression has been reported to favour tumour invasion. We observed a higher expression of c-Myc oncogene, NF-kB, both regulating cell proliferation and metastatic process, as well as ZEB1, a TF significantly associated with tumorigenic potential and cell migratory ability. Interestingly, a novel interaction of DDB2 with NF-kB was detected and found to be increased in cells expressing the DDB2PCNA-, suggesting a direct modulation of NF-kB by DDB2. CONCLUSION: These results highlight the role of DDB2-PCNA interaction in counteracting EMT since DDB2PCNA- protein induces in HEK293 transformed cells a gain of function contributing to the acquisition of a more aggressive phenotype.

Single Cell Determination of 7,8-dihydro-8-oxo-2'-deoxyguanosine by Fluorescence Techniques: Antibody vs. Avidin Labeling.

An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG). Although several methods are available for the biochemical analysis of this molecule, its determination at the single cell level may provide significant advantages when investigating the influence of cell heterogeneity and cell type in the DNA damage response. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detection with the glycoprotein avidin has also been proposed because of a structural similarity between its natural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliability and sensitivity is not clear. In this study, we compared the immunofluorescence determination of 8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugated with the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different cell types by treatment with potassium bromate (KBrO3), a chemical inducer of reactive oxygen species (ROS). By using increasing concentrations of KBrO3, as well as different reaction conditions, our results indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greater than that attained with avidin-AF488. These findings suggest that immunofluorescence techniques are best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage.

A functional in vitro cell-free system for studying DNA repair in isolated nuclei.

Assessment of DNA repair is an important endpoint measurement when studying the biochemical mechanisms of the DNA damage response and when investigating the efficacy of chemotherapy, which often uses DNA-damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, such methods have some limitations, which are mainly due to the lack of chromatin organization in the DNA templates used. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the corresponding damage-activated cytosolic extracts, containing biotinylated dUTP, nuclei were able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 and DDB1, stimulated UVC-induced dUTP incorporation. In contrast, a DDB2PCNA- mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different types of DNA lesions, this system offers an additional tool to study DNA repair mechanisms.This article has an associated First Person interview with the first author of the paper.

Revisiting the Function of p21CDKN1A in DNA Repair: The Influence of Protein Interactions and Stability.

The p21CDKN1A protein is an important player in the maintenance of genome stability through its function as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest after genotoxic damage. In the DNA damage response, p21 interacts with specific proteins to integrate cell-cycle arrest with processes such as transcription, apoptosis, DNA repair, and cell motility. By associating with Proliferating Cell Nuclear Antigen (PCNA), the master of DNA replication, p21 is able to inhibit DNA synthesis. However, to avoid conflicts with this process, p21 protein levels are finely regulated by pathways of proteasomal degradation during the S phase, and in all the phases of the cell cycle, after DNA damage. Several lines of evidence have indicated that p21 is required for the efficient repair of different types of genotoxic lesions and, more recently, that p21 regulates DNA replication fork speed. Therefore, whether p21 is an inhibitor, or rather a regulator, of DNA replication and repair needs to be re-evaluated in light of these findings. In this review, we will discuss the lines of evidence describing how p21 is involved in DNA repair and will focus on the influence of protein interactions and p21 stability on the efficiency of DNA repair mechanisms.

Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG.

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-ß-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.

Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells.

Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here, we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase ß and Proliferating Cell Nuclear Antigen (PCNA), together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells.

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage.

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2Wt and DDB2PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity.

Exploring new potential role of DDB2 by host cell reactivation assay in human tumorigenic cells.

BACKGROUND: The Host Cell Reactivation assay (HCR) allows studying the DNA repair capability in different types of human cells. This assay was carried out to assess the ability in removing UV-lesions from DNA, thus verifying NER efficiency. Previously we have shown that DDB2, a protein involved in the Global Genome Repair, interacts directly with PCNA and, in human cells, the loss of this interaction affects DNA repair machinery. In addition, a mutant form unable to interact with PCNA (DDB2PCNA-), has shown a reduced ability to interact with a UV-damaged DNA plasmid in vitro. METHODS: In this work, we have investigated whether DDB2 protein may influence the repair of a UV-damaged DNA plasmid into the cellular environment by applying the HCR method. To this end, human kidney 293 stable clones, expressing DDB2Wt or DDB2PCNA-, were co-transfected with pmRFP-N2 and UV-irradiated pEGFP-reported plasmids. Moreover, the co-localization between DDB2 proteins and different NER factors recruited at DNA damaged sites was analysed by immunofluorescence and confocal microscopy. RESULTS: The results have shown that DDB2Wt recognize and repair the UV-induced lesions in plasmidic DNA transfected in the cells, whereas a delay in these processes were observed in the presence of DDB2PCNA-, as also confirmed by the different extent of co-localization of DDB2Wt and some NER proteins (such as XPG), vs the DDB2 mutant form. CONCLUSION: The HCR confirms itself as a very helpful approach to assess in the cellular context the effect of expressing mutant vs Wt NER proteins on the DNA damage response. Loss of interaction of DDB2 and PCNA affects negatively DNA repair efficiency.

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.

Biology of the cell cycle inhibitor p21(CDKN1A): molecular mechanisms and relevance in chemical toxicology.

The cell cycle inhibitor p21(CDKN1A) is a protein playing multiple roles not only in the DNA damage response, but also in many cellular processes during unperturbed cell growth. The main, well-known function of p21 is to arrest cell cycle progression by inhibiting the activity of cyclin-dependent kinases. In addition, p21 is involved in the regulation of transcription, apoptosis, DNA repair, as well as cell motility. However, p21 appears to a have a dual-face behavior because, in addition to its tumor suppressor functions, it may act as an oncogene, depending on the cell type and on the cellular localization. As a biomarker of the cell response to different toxic stimuli, p21 expression and functions have been analyzed in an impressive number of studies investigating the activity of several types of chemicals, in order to determine their possible harmful effects on human cells. Here, we review these studies in order to highlight the different roles p21 may play in the cell response to chemical exposure and to better evaluate the information provided by this biomarker.

Degradation of p21CDKN1A after DNA damage is independent of type of lesion, and is not required for DNA repair.

The inhibitor of cyclin-dependent kinases p21CDKN1A plays a fundamental role in several pathways involved in the DNA damage response, like checkpoint-mediated cell cycle arrest, transcription, apoptosis, and DNA repair. Although p21 protein level is regulated by proteasomal degradation, the relationship of this process with DNA repair pathways is not yet clear. In addition, the role of protein/protein interaction in regulating turnover of p21 protein, is controversial. Here, we show that in human fibroblasts treated with agents inducing lesions repaired through nucleotide excision repair (NER), or base excision repair (BER), p21 degradation was triggered more by the extent, than by the type of DNA damage, or consequent DNA repair pathway. In fact, lowering the amount of DNA damage resulted in an increased stability of p21 protein. Overexpression of p21 was found to obscure degradation, both for p21wt and a p21 mutant unable to bind PCNA (p21PCNA-). However, when expressed to lower levels, turnover of p21 protein after DNA damage was greatly influenced by interaction with PCNA, since p21PCNA- was more efficiently degraded than wild-type protein. Interestingly, a p21 mutant protein unable to localize in the nucleus because of mutations in the NLS region, was not degraded after DNA damage, thus indicating that nuclear localization is necessary for p21 turnover. Removal of p21 was not required for NER activity, since inhibition of p21 degradation by caffeine did not affect the UV-induced recruitment of repair proteins, such as PCNA and DNA polymerase delta, nor significantly influence DNA repair synthesis, as determined by autoradiography. These results indicate that degradation of p21 is not dependent on a particular DNA repair pathway, and is not required for efficient DNA repair.

Multiple roles of the cell cycle inhibitor p21(CDKN1A) in the DNA damage response.

Among cell cycle regulatory proteins that are activated following DNA damage, the cyclin-dependent kinase inhibitor p21(CDKN1A) plays essential roles in the DNA damage response, by inducing cell cycle arrest, direct inhibition of DNA replication, as well as by regulating fundamental processes, like apoptosis and transcription. These functions are performed through the ability of p21 to interact with a number of proteins involved in these processes. Despite an initial controversy, during the last years several lines of evidence have also indicated that p21 may be directly involved in DNA repair. In particular, the participation of p21 in nucleotide excision repair (NER), base excision repair (BER), and DNA translesion synthesis (TLS), has been suggested to occur thanks to its interaction with proliferating cell nuclear antigen (PCNA), a crucial protein involved in several aspects of DNA metabolism, and cell-cycle regulation. In this review, the multiple roles of p21 in the DNA damage response, including regulation of cell cycle, apoptosis and gene transcription, are discussed together with the most recent findings supporting the direct participation of p21 protein in DNA repair processes. In particular, spatio-temporal dynamics of p21 recruitment to sites of DNA damage will be considered together with several lines of evidence indicating a regulatory role for p21. In addition, the relevance of post-translational regulation in the fate (e.g. degradation) of p21 protein after cell exposure to DNA damaging agents will be analyzed. Both sets of evidence will be discussed in terms of the overall DNA damage response.

Interaction of p21(CDKN1A) with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair.

The cell-cycle inhibitor p21(CDKN1A) has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA.

An improved method for the detection of nucleotide excision repair factors at local UV DNA damage sites.

Among different DNA repair processes that cells use to face with DNA damage, nucleotide excision repair (NER) is particularly important for the removal of a high variety of lesions, including those generated by some antitumor drugs. A number of factors participating in NER, such as the TFIIH complex and the endonuclease XPG are also involved in basal processes, e.g. transcription. For this reason, localization of these factors at DNA damage sites may be difficult. Here we have applied a mild digestion of chromatin with DNase I to improve the in situ extraction necessary to detect chromatin-bound proteins by immunofluorescence. We have compared this method with different extraction protocols and investigated its application on different cell types, and with different antibodies. Our results show that a short DNase I treatment before the immunoreaction, enhances the fluorescence signal of NER proteins, such as XPG, DDB2 and XPC. In addition, our findings indicate that the antibody choice is a critical factor for accurate localization of DNA repair proteins at DNA damage sites. In conclusion, a mild DNA digestion with DNase I improves the immunofluorescence detection of the recruitment of NER factors at local DNA damage sites by enhancing accessibility to the antibodies, independently of the cell type.

Anthocyanidins decrease endothelin-1 production and increase endothelial nitric oxide synthase in human endothelial cells.

Epidemiological and intervention studies correlate anthocyanin-rich beverages and a low incidence of coronary heart diseases. Since endothelin-1 (ET-1) and nitric oxide (NO) produced by endothelial NO synthase (eNOS) are vascular tension regulators secreted by endothelial cells, we studied the influence of two anthocyanidins, namely cyanidin (CY) and delphinidin (DP), on the regulation of ET-1 and eNOS in cultured human umbilical vein endothelial cells (HUVECs). Aglycon anthocyanidin forms, such as CY and DP, may be present in vivo after the first deglycosylation step occurring in the jejunum and in the liver. DP showed a major action compared to CY inducing a significant dose-dependent inhibitory effect on both protein and mRNA levels of ET-1. CY and DP both increased the protein level of eNOS, but DP showed the major effect raising eNOS protein in a dose-dependent manner. To correlate the vasoprotective effect of CY and DP with their antioxidant activity, we analysed also the antioxidant effect of anthocyanidins both in vitro and in HUVECs. In particular, we examined the effect of anthocyanidins on endothelial heme oxygenase-1 (HO-1), an inducible stress protein. In all tests, DP showed a higher antioxidant activity than CY. Finally, the antiproliferative effect induced by DP was detected in HUVECs. DP and CY differ in the number and position of hydroxyl groups in their structure; therefore, the greater biological activity by DP, compared with CY, seems to be due to the presence of the three hydroxyl groups on the B ring in the molecular structure of DP.

p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates.

The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.

p21CDKN1A does not interfere with loading of PCNA at DNA replication sites, but inhibits subsequent binding of DNA polymerase delta at the G1/S phase transition.

The ability of the cyclin-dependent kinase (CDK) inhibitor p21CDKN1A to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G1 phase cell cycle arrest induced by p21GFP occurred also at the G1/S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21PCNA-) or CDK (p21CDK-) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21(wt)HA and p21CDK-HA, but not p21PCNA-, co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase delta (pol delta). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol delta to PCNA at the G1/S phase transition.

A DDB2 mutant protein unable to interact with PCNA promotes cell cycle progression of human transformed embryonic kidney cells.

DNA damage binding protein 2 (DDB2) is a protein involved in the early step of DNA damage recognition of the nucleotide excision repair (NER) process. Recently, it has been suggested that DDB2 may play a role in DNA replication, based on its ability to promote cell proliferation. We have previously shown that DDB2 binds PCNA during NER, but also in the absence of DNA damage; however, whether and how this interaction influences cell proliferation is not known. In this study, we have addressed this question by using HEK293 cell clones stably expressing DDB2(Wt) protein, or a mutant form (DDB2(Mut)) unable to interact with PCNA. We report that overexpression of the DDB2(Mut) protein provides a proliferative advantage over the wild type form, by influencing cell cycle progression. In particular, an increase in the number of S-phase cells, together with a reduction in p21(CDKN1A) protein level, and a shorter cell cycle length, has been observed in the DDB2(Mut) cells. These results suggest that DDB2 influences cell cycle progression thanks to its interaction with PCNA.

Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria.

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.