45- https://doi.org/10.1002/cac2.12088
Correlation between the genetic variants of base excision repair (BER) pathway genes and neuroblastoma susceptibility in eastern Chinese children
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Dear Editor, Neuroblastoma is the most common non-central nerve system (CNS) solid tumor in pediatrics [1]. Neuroblastoma accounts for approximately 8% of all pediatric cancers but disproportionally causes a high cancer mortality (15%) in children [2]. Pediatric patients with low-risk neuroblastoma witness a 5-year overall survival rate > 90%, whereas the 5-year overall survival rate in high-risk neuroblastoma pediatric patients is < 40% [3]. Genetic susceptibility to neuroblastoma is a promising area of research and needs to be fully investigated. For sporadic neuroblastoma, genome-wide association studies (GWASs) have identified over a dozen causal genetic loci. Studies of candidate genes also reported a decent number of variants predisposing to neuroblastoma. However, the known genetic alternations still could not unveil the full genetic underpinnings of neuroblastoma. The base excision repair (BER) pathway, one of the DNA repair systems, is responsible for repairing numerous oxidized and alkylated bases by recognizing and excising damaged bases [4]. Many core proteins are involved in the BER pathway, including poly(ADP)ribose polymerase 1 (PARP1), human 8-oxoguanine DNA glycosylase (OGG1), flap endonuclease 1 (FEN1), apurinic/apyrimidinic endonuclease 1 (APEX1), DNA ligase III (LIG3), and x-ray repair cross-complementing group 1 (XRCC1). OGG1 is a bifunctional enzyme (DNA glycosylase and AP lyase) that incises at abasic sites via an AP lyase activity, leaving a single-strand DNA break intermediate. APEX1 initiates the repair of abasic sites in DNA by cleaving the phosphodiester backbone 5′ to an AP site, creating a nick in the DNA backbone. FEN1 participates in the penultimate steps of Okazaki fragment maturation and 5′-flap removal during long-patch BER. LIG3 catalyzes the last stage of BER by sealing the gap. XRCC1 and PARP1 serve as the scaffold protein. Intensive evidence suggests that aberrant BER pathway proteins result in a variety of diseases, especially cancers [4]. Single nucleotide polymorphisms (SNPs) of the BER pathway genes are associated with the risk of various cancer types. Functional analysis revealed that SNPs in the BER pathway genes may modify the kinetics of BER proteins and the DNA repair capacity of the BER system, ultimately affecting carcinogenesis [4]. However, evidence regarding the role of BER pathway gene SNPs in the risk of neuroblastoma waits to be added. To identify more neuroblastoma susceptibility variations in the BER pathway genes, we performed a case-control study in children at three center hospitals in East China. This study was conducted in Children's Hospital of Nanjing Medical University (Nanjing, Jiangsu), Anhui Provincial Children's Hospital (Hefei, Anhui), and Yuying Children's Hospital of Wenzhou Medical University (Wenzhou, Zhejiang) in East China. A total of 313 neuroblastoma pediatric patients and 762 cancer-free children were recruited in this study. The characteristics of the study subjects are summarized in Supplementary Table S1. Age (P = 0.823) and gender (P = 0.610) were distributed equivalently between the two groups. The study design and participant recruitment were described in our previous work [5]. We successfully genotyped 20 SNPs from 6 BER pathway genes in 313 neuroblastoma pediatric patients and 762 control children (Table 1). Specifically, 3 PARP1, 3 OGG1, 2 FEN1, 3 APEX1, 3 LIG3, and 6 XRCC1 SNPs were genotyped. The genotypic distributions of all candidate SNPs were in Hardy-Weinberg equilibrium (P ≥ 0.05) in the controls. The rs174538 of the FEN1 gene was associated with decreased neuroblastoma risk under the dominant model (adjusted odd ratio [OR] = 0.71, 95% confidence interval [CI] = 0.54-0.93, P = 0.012). However, no significant associations with neuroblastoma risk were found for the remaining SNPs in the single-locus analysis (all P ≥ 0.05; Supplementary Figure S1). We conducted the stratified analyses (Supplementary Table S2) to eliminate potential influences of FEN1 genotypes on neuroblastoma susceptibility by adjusting confounding factors (age, gender, and site of tumor origin). The protective role of rs174538 AG/GG in decreasing neuroblastoma risk was found in subgroups of age ≤18 months (adjusted OR = 0.60, 95% CI = 0.40-0.89, P = 0.011), females (adjusted OR = 0.59, 95% CI = 0.40-0.87, P = 0.009), and tumors arising from the mediastinum (adjusted OR = 0.53, 95% CI = 0.35-0.81, P = 0.003). Combined analysis stated that the 2 protective genotypes (rs174538 AG/GG and rs4246215 TG/GG genotypes) also decreased neuroblastoma risk in the following subgroups: age ≤ 18 months (adjusted OR = 0.62, 95% CI = 0.42-0.93, P = 0.019), females (adjusted OR = 0.61, 95% CI = 0.41-0.91, P = 0.015), and tumors originated from the mediastinum (adjusted OR = 0.54, 95% CI = 0.36-0.83, P = 0.005). We carried out false-positive report probability (FPRP) analysis to validate significant associations (Supplementary Table S3). The threshold for FPRP was preset as 0.2. At the prior probability level of 0.1, significant associations with FEN1 rs174538 A > G (GG/AG vs. AA) remained noteworthy in all subjects (FPRP = 0.121) as well as in the subgroups of females (FPRP = 0.185) and tumors originating from the mediastinum (FPRP = 0.160). In the combined analysis, significant findings for 2 vs. 0-1 protective genotypes (FPRP = 0.166) and its subgroup tumors originated from the mediastinum (FPRP = 0.183) could be called noteworthy. We further explored the biological effects of FEN1 rs174538 A > G on the neighboring gene expression by using released data from Genotype-Tissue Expression (GTEx) Portal (https://www.gtexportal.org/). We observed that rs174538 A allele was significantly associated with increased mRNA expression levels of fatty acid desaturase 2 (FADS2) and transmembrane protein 258 (TMEM258) in the whole blood, nerve-tibial, and cell-cultured fibroblasts (Figure 1A). The rs174538 A allele was also associated with increased expression of fatty acid desaturase 1 (FADS1) mRNA in the whole blood, but with decreased expression of FADS1 mRNA in the nerve-tibial (Figure 1B). eQTL analysis of the neuroblastoma risk factor FEN1 rs174538 A > G. A. FADS2 and TMEM258 levels in the whole blood, nerve-tibial, and cell-cultured fibroblasts; B. FADS1 level in the whole blood and nerve-tibial. Abbreviations: eQTL, expression quantitative loci; FEN1, flap endonuclease 1; FADS2, fatty acid desaturase 2; TMEM258, transmembrane protein 258 The implication of the BER pathway gene SNPs in cancer susceptibility has been highly documented. Plenty of SNPs within the BER pathway genes were found to predispose to various types of cancer. Our group previously carried out a study on BER gene polymorphisms and Wilms tumor susceptibility [6]. Significant associations with Wilms tumor susceptibility were shown for the OGG1 rs1052133, FEN1 rs174538, and FEN1 rs4246215 polymorphisms. Regarding the association of the BER pathway gene SNPs with neuroblastoma risk, only 3 studies were available by far; and all of them were performed by our research group. In these studies, we found that, none of the studied APEX1 polymorphisms were associated with neuroblastoma risk [5]. Such a negative association was also observed between neuroblastoma risk and polymorphisms in the OGG1 [7] and LIG3 genes [8]. However, all these studies were conducted to analyze a single gene in the BER pathway, and the results need to be validated in another independent study. Thus, here we attempted to validate the previous studies by adopting a systematical analysis of potentially functional SNPs in 6 core genes in the BER pathway. In the current study, no significant relationships were detected between neuroblastoma risk and the SNPs in PARP1, OGG1, APEX1, LIG3, and XRCC1 genes. Such results strengthen the previous findings that these variations may be too weak to impact neuroblastoma risk. To be noted, significant conferring roles of the same BER SNPs to the risk of other cancer types have been detected, such as PARP1 rs1136410 and thyroid cancer [9], OGG1 rs1052133 and Wilms tumor [6], FEN1 rs4246215 and Wilms tumor [6], APEX1 rs1130409 and renal cell carcinoma [10], LIG3 rs1052536 and lung cancer [11]. The different roles of these SNPs in specific cancer types indicated that specific cancer types should be set before interpreting the role of SNPs. Excitedly, we demonstrated that the rs174538 of the FEN1 gene could protect from neuroblastoma. FEN1 is a structure-specific nuclease involved in the removal of 5′-flap during long-patch BER and the maturation of Okazaki fragments in DNA replication. Moreover, FEN1 is also characterized as a 5′ exonuclease and a gap-dependent endonuclease, which mediates apoptotic DNA degradation during apoptosis. The FEN1 gene is mapped to chromosome 11 (11q12.2). Yang et al. [12] identified that the rs174538 A allele of the FEN1 gene decreased risk for lung cancer by decreasing FEN1 expression. Moreover, they detected that coke oven workers who carried the AA genotype have significantly lower DNA damage level than those with GG or GA genotypes. In a meta-analysis conducted for the overall cancer, the results suggested that the subjects with FEN1 rs174538 A allele have a decreased susceptibility to cancer in Chinese populations [13]. We further performed online expression quantitative trait loci (eQTL) analysis to interpret the possible mechanism of how rs174538 impacts neuroblastoma risk. eQTL evidence suggested that the A allele in rs174538 was significantly associated with the increased mRNA expression levels of FADS2 and TMEM258. Further functional experiments conducted in neuroblastoma cells are needed to show how the FEN1 rs174538 A allele can be associated with altered expressions of these genes. FADS2 was found to function as a potential oncogene in some types of cancer [14]. TMEM258 is a central mediator of endoplasmic reticulum quality control and intestinal homeostasis, yet its role in cancer remains unknown [15]. The exact relationship of FADS2 and TMEM258 with neuroblastoma risk waits to be elucidated. Taken together, the significant role of rs174538 A allele in cancer deserves more attention for further exploration. Although at the preliminary stage, our findings represent a novel mechanism by which rs174538 may modulate the expression of multiple nearby genes, thereby impacting the risk of neuroblastoma. Our study has several limitations. First, the sample sizes were small in some stratification analyses. Second, the number of analyzed SNPs was limited. Another limitation was the lack of incorporating analysis on environment factors and genetic-environmental factors. The fourth limitation was that the current study only focused on the subjects of the Han population. Replication of these findings in additional individuals of non-Chinese descent should be helpful to validate our findings. In conclusion, we showed a robust association of genetic variants in the FEN1 gene with neuroblastoma risk in a relatively large sample size of pediatric patients in East China. Intensive future research is warranted to extend the role of FEN1 gene loci in neuroblastoma susceptibility in individuals of non-Chinese ancestries. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Ethics Committees of Children's Hospital of Nanjing Medical University, Anhui Provincial Children's Hospital, and the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University. Each participant signed an informed consent before participating to this study. Not applicable. All data generated or analyzed during this study are included in this published article and its additional files. The authors declare that they have no competing interests. This work was supported by the grants from Natural Science Foundation of Guangdong Province (2019A1515010360) and Guangdong Provincial Key Laboratory of Research in Structural Birth Defect Disease (2019B030301004). Z.Z., J.Z., H.L., J.H., and Y.W. designed the study, performed the experiments and wrote the manuscript. C.Z., Y.F., H.Z., H.W., and Y.W. collected the clinical samples and information. Z.Z. and J.H. analyzed the data and prepared all the tables and figures. Z.Z., J.H., and Y.W. coordinated the study. All authors reviewed and approved the final manuscript. Not applicable. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
- Research Article
- 62
- 10.1074/jbc.m109.000505
- May 1, 2009
- Journal of Biological Chemistry
Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase beta (pol beta), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol beta is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol beta or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol beta with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a "hit and run" mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process.
- Research Article
- 807
- 10.1093/emboj/16.11.3341
- Jun 1, 1997
- The EMBO Journal
Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.
- Research Article
- 64
- 10.1074/jbc.m409200200
- Feb 1, 2005
- Journal of Biological Chemistry
In the present investigation, we report a previously unsuspected function of the tumor suppressor protein, APC (adenomatous polyposis coli), in the regulation of base excision repair (BER). We identified a proliferating cell nuclear antigen-interacting protein-like box sequence in APC that binds DNA polymerase beta and blocks DNA polymerase beta-mediated strand-displacement synthesis in long patch BER without affecting short patch BER. We further showed that the colon cancer cell line expressing the wild-type APC gene was more sensitive to a DNA-methylating agent due to decreased DNA repair by long patch BER than the cell line expressing the mutant APC gene lacking the proliferating cell nuclear antigen-interacting protein-like box. Experiments based on RNA interference showed that the wild-type APC gene expression is required for DNA methylation-induced sensitivity of colon cancer cells. Thus, APC may play a critical role in determining utilization of long versus short patch BER pathways and affect the susceptibility of colon cancer cells to carcinogenic and chemotherapeutic agents.
- Research Article
- 123
- 10.1016/j.molcel.2011.02.016
- Mar 1, 2011
- Molecular Cell
USP47 Is a Deubiquitylating Enzyme that Regulates Base Excision Repair by Controlling Steady-State Levels of DNA Polymerase β
- Research Article
- 138
- 10.1016/j.molcel.2006.01.036
- Mar 1, 2006
- Molecular Cell
Condensin I Interacts with the PARP-1-XRCC1 Complex and Functions in DNA Single-Strand Break Repair
- Research Article
- 22
- 10.3390/genes14010098
- Dec 29, 2022
- Genes
Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3′-5′ exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.
- Research Article
- 10.1007/s12291-015-0476-2
- Jan 20, 2015
- Indian Journal of Clinical Biochemistry
Genes in the base excision repair (BER) pathway influence the generation and repair of oxidative lesions. The mammalian short-patch BER is primarily attributed to the human 8-oxoguanine DNA glycosylase (hOGG1), apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β (pol β), and X-ray repair cross-complementing group 1 (XRCC1) pathways. There have been many reports of differences among individuals and populations regarding polymorphisms of hOGG1, APE1, and XRCC1 genes and metabolism. However, there are only a limited number of reports available concerning pol β polymorphisms. Therefore, the aim of the present study was to evaluate the frequencies of a common single nucleotide polymorphism (SNP) of the polymerase β rs3136794 gene in the worldwide population. Our sample consisted of 1,540 healthy individuals from Japan, Korea, Tibet, Nepal, Sri Lanka, Vietnam, Mexico, Namibia, Ghana, and South Africa. We observed that the A allele was the most common allele in Asia and Mexico, ranging from 58.5 to 94.7 %, respectively. On the other hand, the G allele was the most common allele in Africa, ranging from 23.4 to 25.3 %. In conclusion, the distribution of the rs3136794 gene polymorphism distinguishes the African population studied from other populations; however, it is necessary to increase the size of the samples to acquire more conclusive results. This study is the first to demonstrate the existence of genetic heterogeneity in a worldwide distribution of SNPs in a pol β gene (rs3136794) in the BER genes.
- Research Article
- 120
- 10.1074/jbc.m703343200
- Sep 1, 2007
- The Journal of biological chemistry
The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.
- Research Article
- 90
- 10.1158/1055-9965.epi-06-0712
- Jan 1, 2007
- Cancer Epidemiology, Biomarkers & Prevention
Cancer is a common multifactor human disease resulting from complex interactions between many genetic and environmental factors. In this study, we used a multifaceted analytic approach to explore the relationship between eight single nucleotide polymorphisms in base excision repair (BER) pathway genes, smoking, and bladder cancer susceptibility in a hospital-based case-control study. Overall, we did not find an association between any single BER gene single nucleotide polymorphism and bladder cancer risk. However, in stratified analysis, the OGG1 S326C variant genotypes in ever smokers (odds ratio, 0.74; 95% confidence interval, 0.56-0.99) and ADP-ribosyltransferase (ADPRT) V762A variant genotypes in never smokers (odds ratio, 0.58; 95% confidence interval, 0.37-0.91) conferred a significantly reduced risk. Using logistic regression, we observed that there was a two-way interaction between ADPRT V762A and smoking status. We next used classification and regression tree analysis to explore high-order gene-gene and gene-environment interactions. We found that smoking is the most important influential factor for bladder cancer risk. Consistent with the above findings, we found that the ADPRT V762A was only significantly involved in bladder cancer risk in never smokers and the OGG1 S326C was only significantly involved in ever smokers. We also observed gene-gene interactions among OGG1 S326C, XRCC1 R194W, and MUTYH H335Q in ever smokers. Using multifactor dimensionality reduction approach, the four-factor model, including smoking status, OGG1 S326C (rs1052133), APEX1 D148E (rs3136820), and ADPRT762 (rs1136410), had the best ability to predict bladder cancer risk with the highest cross-validation consistency (100%) and the lowest prediction error (37.02%; P < 0.001). These results support the hypothesis that genetic variants in BER genes contribute to bladder cancer risk through gene-gene and gene-environmental interactions.
- Research Article
- 79
- 10.1016/j.mrfmmm.2009.08.009
- Aug 22, 2009
- Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Poly(ADP-ribose) polymerase 1 regulates activity of DNA polymerase β in long patch base excision repair
- Research Article
- 1
- 10.1158/1538-7445.am10-4656
- Apr 15, 2010
- Cancer Research
Graft versus host disease (GVHD) remains a major source of morbidity and mortality among patients undergoing hematopoietic cell transplants (HCT). Tissue damage as a result of treatment is the initiating event in the pathogenesis of GVHD and variations in DNA repair can influence the amount of tissue damage in response to treatment modalities used in HCT. Since DNA damage caused by therapeutic agents such as alkylating agents and ionizing radiation used frequently in pre- HCT conditioning regimens is commonly repaired by the base excision repair (BER) pathway we hypothesized that genetic polymorphisms in the BER pathway will be associated with GVHD after HCT. We evaluated the association between single nucleotide polymorphisms (SNPs) (n= 179) in the BER pathway with acute GVHD and chronic GVHD in a cohort of 470 recipients who underwent allogeneic HCT for treatment of hematologic malignancies at the University of Minnesota. After adjustment for donor type, diagnosis, disease status at transplant and gender mismatch, two SNPs in RFC1 (Replication Factor C) gene (rs1057807 and rs4975003) were associated with a decreased risk of grade II-IV acute GVHD (HR:0.74-0.77; p≤0.01) and one SNP, rs6844176, in RFC1 gene was associated with increased risk of grade II-IV acute GVHD (HR:1.39, p=0.001). The rs3730914 SNP in the LIG1 (Ligase I) gene was also associated with a decreased risk of grade III-IV acute GVHD (HR: 0.38; p=0.005). One SNP, rs1805410, in the PARP1 (poly ADP-ribose polymerase 1) gene was associated with increased risk of chronic GVHD (HR: 1.44; p=0.01) post HCT. These findings suggest that SNPs in the BER pathway can be used as genetic biomarkers to predict individuals at high risk for GVHD. If these findings are confirmed, modulation of pre-transplant conditioning may alter risk in these patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4656.
- Research Article
- 87
- 10.1038/sj.embor.embor796
- Mar 7, 2003
- EMBO reports
The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.
- Research Article
- 157
- 10.1074/jbc.m803491200
- Sep 1, 2008
- Journal of Biological Chemistry
The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5'-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase gamma (pol gamma) and polymerase beta in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would require 5'-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5'-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5'-exo/endonuclease and pol gamma with 3'-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol gamma, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5'-exo/endonuclease in mitochondrial LP-BER.
- Research Article
- 96
- 10.1016/s0079-6603(01)68086-3
- Jan 1, 2001
- Progress in Nucleic Acid Research and Molecular Biology
The mechanism of switching among multiple BER pathways
- Research Article
- 10
- 10.1593/neo.12680
- Jun 1, 2012
- Neoplasia
Adenomatous Polyposis Coli Interacts with Flap Endonuclease 1 to Block Its Nuclear Entry and Function
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