Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.     

DNA polymerase β-dependent cell survival independent of XRCC1 expression

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1^+/+ and Xrcc1^−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.     

Different methylation and MicroRNA expression pattern in male and female familial breast cancer

Epigenetic regulation, has been very scarcely explored in familial breast cancer (BC). In the present study RASSF1A and RAR beta promoter methylation and miR17, miR21, miR 124, and let‐7a expression were investigated to highlight possible differences of epigenetic regulation between male and female familial BC, also in comparison with sporadic BC. These epigenetic alterations were studied in 56 familial BC patients (27 males and 29 females) and in 16 female sporadic cases. RASSF1A resulted more frequently methylated in men than women (76% vs. 28%, respectively, P = 0.0001), while miR17 and let‐7a expression frequency was higher in women than in men (miR17: 66% in women vs. 41% in men, P < 0.05; let‐7a: 45% in women vs. 15% in men, P = 0.015). RASSF1A methylation affected 27.6% of familial BC while 83% of familial cases showed high expression of the gene (P = 0.025); on the contrary, only 17% of familial BC presented RAR beta methylation and 55% of familial cases overexpressed this gene (P = 0.005). Moreover, miR17, miR21, and let‐7a resulted significantly overexpressed in familial compared to sporadic BC. RASSF1A overexpression (86% vs. 65%, P = 0.13) and RAR beta overexpression (57% vs. 32%, P = 0.11) were higher in BRCA1/2 carriers even if not statistical significance was reached. BRCA mutation carriers also demonstrated significant overexpression of: miR17 (93% vs. 35%, P = 0.0001), let‐7a (64% vs. 16%, P = 0.002), and of miR21 (100% vs. 65%, P = 0.008). In conclusion, the present data suggest the involvement of RASSF1A in familial male BC, while miR17 and let‐7a seem to be implied in familial female BC. J. Cell. Physiol. 228: 1264–1269, 2013. © 2012 Wiley Periodicals, Inc.     

Impact of Single Nucleotide Polymorphisms of Base Excision Repair Genes on DNA Damage and Efficiency of DNA Repair in Recurrent Depression Disorder

Elevated level of DNA damage was observed in patients with depression. Furthermore, single nucleotide polymorphisms (SNPs) of base excision repair (BER) genes may modulate the risk of this disease. Therefore, the aim of this study was to delineate the association between DNA damage, DNA repair, the presence of polymorphic variants of BER genes, and occurrence of depression. The study was conducted on peripheral blood mononuclear cells of 43 patients diagnosed with depression and 59 controls without mental disorders. Comet assay was used to assess endogenous (oxidative) DNA damage and efficiency of DNA damage repair (DRE). TaqMan probes were employed to genotype 12 SNPs of BER genes. Endogenous DNA damage was higher in the patients than in the controls, but none of the SNPs affected its levels. DRE was significantly higher in the controls and was modulated by BER SNPs, particularly by c.977C>G–hOGG1, c.972G>C–MUTYH, c.2285T>C–PARP1, c.580C>T–XRCC1, c.1196A>G–XRCC1, c.444T>G–APEX1, c.-468T>G–APEX1, or c.*50C>T–LIG3. Our study suggests that both oxidative stress and disorders in DNA damage repair mechanisms contribute to elevated levels of DNA lesions observed in depression. Lower DRE can be partly attributed to the presence of specific SNP variants.     

Disruption of PARP1 function inhibits base excision repair of a sub-set of DNA lesions

The repair of endogenously induced DNA damage is essential to maintain genomic integrity. It has been shown that XRCC1 and PARP1 are involved in the repair of base lesions and SSBs, although the exact mode of action has yet to be determined. Here we show that XRCC1 is involved in the repair of base lesions and SSBs independent of the cell cycle. However, the rate of repair of damage requiring XRCC1 does reflect the damage complexity. The repair of induced DNA damage occurs by PARP1-dependent and PARP1-independent sub-pathways of BER. It is suggested that the repair of SSBs and purine base damage is by a sub-pathway of BER that requires both XRCC1 and PARP1. Repair of pyrimidine base damage may require XRCC1 but does not require PARP1 activity. Therefore, although BER of simple lesions occurs rapidly, pathway choice and the involvement of PARP1 are highly dependent on the types of lesion induced.     

XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein-protein interactions.

The major human AP endonuclease APE1 (HAP1, APEX, Ref1) initiates the repair of abasic sites generated either spontaneously, from attack of bases by free radicals, or during the course of the repair of damaged bases. APE1 therefore plays a central role in the base excision repair (BER) pathway. We report here that XRCC1, another essential protein involved in the maintenance of genome stability, physically interacts with APE1 and stimulates its enzymatic activities. A truncated form of APE1, lacking the first 35 amino acids, although catalytically proficient, loses the affinity for XRCC1 and is not stimulated by XRCC1. Chinese ovary cell lines mutated in XRCC1 have a diminished capacity to initiate the repair of AP sites. This defect is compensated by the expression of XRCC1. XRCC1, acting as both a scaffold and a modulator of the different activities involved in BER, would provide a physical link between the incision and sealing steps of the AP site repair process. The interaction described extends the coordinating role of XRCC1 to the initial step of the repair of DNA abasic sites.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

BRCA1 Pathway Function in Basal-Like Breast Cancer Cells

Sporadic basal-like cancers (BLCs) are a common subtype of breast cancer that share multiple biological properties with BRCA1-mutated breast tumors. Despite being BRCA1^+/+, sporadic BLCs are widely viewed as phenocopies of BRCA1-mutated breast cancers, because they are hypothesized to manifest a BRCA1 functional defect or breakdown of a pathway(s) in which BRCA1 plays a major role. The role of BRCA1 in the repair of double-strand DNA breaks by homologous recombination (HR) is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. Therefore, it is suspected that sporadic BLCs exhibit a defect in HR. To test this hypothesis, multiple DNA damage repair assays focused on several types of repair were performed on a group of cell lines classified as sporadic BLCs and on controls. The sporadic BLC cell lines failed to exhibit an overt HR defect. Rather, they exhibited defects in the repair of stalled replication forks, another BRCA1 function. These results provide insight into why clinical trials of poly(ADP-ribose) polymerase (PARP) inhibitors, which require an HR defect for efficacy, have been unsuccessful in sporadic BLCs, unlike cisplatin, which elicits DNA damage that requires stalled fork repair and has shown efficacy in sporadic BLCs.     

Full-exon pyrosequencing screening of BRCA germline mutations in Mexican women with inherited breast and ovarian cancer

Hereditary breast cancer comprises 10% of all breast cancers. The most prevalent genes causing this pathology are BRCA1 and BRCA2 (breast cancer early onset 1 and 2), which also predispose to other cancers. Despite the outstanding relevance of genetic screening of BRCA deleterious variants in patients with a history of familial cancer, this practice is not common in Latin American public institutions. In this work we assessed mutations in the entire exonic and splice-site regions of BRCA in 39 patients with breast and ovarian cancer and with familial history of breast cancer or with clinical features suggestive for BRCA mutations by massive parallel pyrosequencing. First we evaluated the method with controls and found 41-485 reads per sequence in BRCA pathogenic mutations. Negative controls did not show deleterious variants, confirming the suitability of the approach. In patients diagnosed with cancer we found 4 novel deleterious mutations (c.2805_2808delAGAT and c.3124_3133delAGCAATATTA in BRCA1; c.2639_2640delTG and c.5114_5117delTAAA in BRCA2). The prevalence of BRCA mutations in these patients was 10.2%. Moreover, we discovered 16 variants with unknown clinical significance (11 in exons and 5 in introns); 4 were predicted as possibly pathogenic by in silico analyses, and 3 have not been described previously. This study illustrates how massive pyrosequencing technology can be applied to screen for BRCA mutations in the whole exonic and splice regions in patients with suspected BRCA-related cancers. This is the first effort to analyse the mutational status of BRCA genes on a Mexican-mestizo population by means of pyrosequencing.     

The R280H X-ray cross-complementing 1 germline variant induces genomic instability and cellular transformation

X-ray repair cross complementing protein 1 (XRCC1) plays an important role in base excision DNA repair (BER) as a scaffolding protein for BER enzymes. BER is one of the basic DNA repair pathways repairing greater than 20,000 endogenous lesions per cell per day. Proper functioning of XRCC1, one of the most important players in BER, was suggested to be indispensable for effective DNA repair. Despite accumulating evidence of an important role that XRCC1 plays in maintaining genomic stability, the relationship between one of its most predominant variants, R280H (rs25489), and cancer prevalence remains ambiguous. In the current study we functionally characterized the effect of the R280H variant expression on immortal non-transformed mouse mammary epithelial C127 and human breast epithelial MCF10A cells. We found that expression of R280H results in increased focus formation in mouse C127 cells and induces cellular transformation in human MCF10A cells. Cells expressing R280H showed significantly increased levels of chromosomal aberrations and accumulate double strand breaks in the G1 cell cycle phase. Our results confirm a possible link between R280H and genomic instability and suggest that individuals carrying this mutation may be at increased risk of cancer development.     

The PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2′-deoxycytidine lesions

Decitabine (5-aza-2′-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1–DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML.     

Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair

Single-strand break repair (SSBR) and base excision repair (BER) of modified bases and abasic sites share several players. Among them is XRCC1, an essential scaffold protein with no enzymatic activity, required for the coordination of both pathways. XRCC1 is recruited to SSBR by PARP-1, responsible for the initial recognition of the break. The recruitment of XRCC1 to BER is still poorly understood. Here we show by using both local and global induction of oxidative DNA base damage that XRCC1 participation in BER complexes can be distinguished from that in SSBR by several criteria. We show first that XRCC1 recruitment to BER is independent of PARP. Second, unlike SSBR complexes that are assembled within minutes after global damage induction, XRCC1 is detected later in BER patches, with kinetics consistent with the repair of oxidized bases. Third, while XRCC1-containing foci associated with SSBR are formed both in eu- and heterochromatin domains, BER complexes are assembled in patches that are essentially excluded from heterochromatin and where the oxidized bases are detected.     

The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF^FBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF^FBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF^FBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF^FBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development.     

Impact of genetic polymorphisms in base excision repair genes on the risk of breast cancer in a Korean population

The contribution of single nucleotide polymorphisms (SNPs) in base excision repair (BER) genes to the risk of breast cancer (BC) was evaluated by focusing on two key genes: apurinic/apyrimidinic endonuclease 1 (APEX1) and 8-oxoguanine DNA glycosylase (OGG1). Genetic variations in the genes encoding these DNA repair enzymes may alter their functions and increase susceptibility to carcinogenesis. The aim of this study was to analyze polymorphisms in two BER genes, exploring their associations and particularly the combined effects of these variants on BC risk in a Korean population. Three SNPs of two BER genes were genotyped using the Illumina GoldenGate™ method. In total, 346 BC patients and 361 cancer-free controls were genotyped for these BER gene polymorphisms and analyzed for their correlation with BC risk in multiple logistic regression models. Multiple logistic regression models adjusted for age, family history of BC, and body mass index were used. The APEX1 Asp148Glu polymorphism was weakly associated with BC risk. The combined analysis among the BER genes, however, showed significant effects on BC risk. The APEX1 Asp148Glu carrier, in combination with OGG1 rs2072668 and OGG1 Ser326Cys, was strongly associated with an increased risk of BC. Moreover, the combination of the C–C haplotype of OGG1 with the APEX1 Asp148Glu genotype was also associated with an additive risk effect of BC [ORs = 2.44, 2.87, and 3.50, respectively]. The combined effect of APEX1 Asp148Glu was found to be associated with an increased risk of BC. These results suggest that the combined effect of different SNPs within BER genes may be useful in predicting BC risk.     

Dynamic light scattering study of base excision DNA repair proteins and their complexes

Base excision repair (BER) involves many enzymes acting in a coordinated fashion at the most common types of DNA damage. The coordination is facilitated by interactions between the enzymes and accessory proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1). Here we use dynamic light scattering (DLS) technique to determine the hydrodynamic sizes of several BER enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), XRCC1 and PARP1, present alone or in the equimolar mixtures with each other. From the DLS data combined with glutaraldehyde cross-linking experiments and previous quantitative binding data the oligomeric states of BER proteins and their complexes are estimated. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner. The DLS technique is applied for the first time to measure the hydrodynamic sizes of PARP1 molecules covalently bound with poly(ADP-ribose) (PAR) synthesized upon the automodification reaction. PARP1 has been detected to form huge conglomerates stabilized by Mg²⁺ coordinated bonds with PAR polymers.