Repair Of Oxidative Base Damage Research Articles

A review on protein-protein interaction network of APE1/Ref-1 and its associated biological functions.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a classic example of functionally variable protein. Besides its well-known role in (i) DNA repair of oxidative base damage, APE1 also plays a critical role in (ii) redox regulation of transcription factors controlling gene expression for cell survival pathways, for which it is also known as redox effector factor 1 (Ref-1), and recent evidences advocates for (iii) coordinated control of other non-canonical protein-protein interaction(s) responsible for significant biological functions in mammalian cells. The diverse functions of APE1 can be ascribed to its ability to interact with different protein partners, owing to the attainment of unfolded domains during evolution. Association of dysregulation of APE1 with various human pathologies, such as cancer, cardiovascular diseases and neurodegeneration, is attributable to its multifunctional nature, and this makes APE1 a potential therapeutic target. This review covers the important aspects of APE1 in terms of its significant protein-protein interaction(s), and this knowledge is required to understand the onset and development of human pathologies and to design or improve the strategies to target such interactions for treatment and management of various human diseases.

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K(d) = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.

Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer's disease

Brain aging is associated with synaptic decline and synaptic function is highly dependent on mitochondria. Increased levels of oxidative DNA base damage and accumulation of mitochondrial DNA (mtDNA) mutations or deletions lead to mitochondrial dysfunction, playing an important role in the aging process and the pathogenesis of several neurodegenerative diseases. Here we have investigated the repair of oxidative base damage, in synaptosomes of mouse brain during normal aging and in an AD model. During normal aging, a reduction in the base excision repair (BER) capacity was observed in the synaptosomal fraction, which was associated with a decrease in the level of BER proteins. However, we did not observe changes between the synaptosomal BER activities of presymptomatic and symptomatic AD mice harboring mutated amyolid precursor protein (APP), Tau, and presinilin-1 (PS1) (3xTgAD). Our findings suggest that the age-related reduction in BER capacity in the synaptosomal fraction might contribute to mitochondrial and synaptic dysfunction during aging. The development of AD-like pathology in the 3xTgAD mouse model was, however, not associated with deficiencies of the BER mechanisms in the synaptosomal fraction when the whole brain was analyzed.

Apoptosis and the DNA Damage Response: The Role of the RB Tumor Suppressor Pathway in Oxidative Stress Responses in the Hematopoietic System.

Abstract SCI-15Exposure to pro-oxidants and defects in repair of oxidative base damage is associated with disease and aging and also contributes to the development of anemia, bone marrow failure and hematopoietic malignancies. Our work examines the role of the RB tumor suppressor pathway in the response of the hematopoietic system to oxidative stress and DNA damage. Evidence from mouse models has identified a role for the Rb protein (pRb) in the regulation of hematopoiesis through cell intrinsic functions in blood cell types but also through effects on the bone marrow microenvironment (Spike et al, 2004; Walkley et al, 2007; Daria et al, 2008). Such models have also demonstrated that pRb is required under stress conditions but not under conditions of steady state hematopoiesis (Spike et al, 2004; Spike et al, 2007; Daria et al, 2008). In particular, pRb was required to modulate the response of the hematopoietic system to replicative stress and hypoxia (Spike et al, 2007; Daria et al, 2008). To explain the mechanisms underlying these unique properties of pRb in hematopoiesis, we hypothesized that pRb protein levels are regulated by oxidative stress, including hypoxia and ROS generated as a consequence of stem cell location in the bone marrow niche or in response to replicative stress induced by agents such as 5-fluorouracil. Notably, hypoxia within the bone marrow niche has been reported to promote stem cell expansion and we postulated that this may be due to reduced pRb protein levels in response to hypoxia. We present evidence that pRb protein levels are regulated in wild-type bone marrow in response to replicative stress and that this in turn modulates expansion of stem cells and myeloid progenitors and also impacts end-stage differentiation in the erythroid lineage. Acetylation of pRb stabilized the protein in an active conformation while de-acetylation de-stabilized the protein and promoted pRb protein turnover and increased progenitor cell proliferation. We will present on-going studies that examine how hypoxia and/or ROS affects hematopoietic stem cell proliferation, self-renewal and differentiation in vivo as a function of pRb protein levels using conditional mouse models. The significance of our findings for bone marrow failure in human patients will be discussed. References Spike, B.T. et al. The Rb tumor suppressor is required for stress erythropoiesis. The EMBO J. 2004: 23, 4319-29. Spike, B.T., Dibling, B.C. & Macleod, K.F. Hypoxic stress underlies defects in erythroblast island formation in the Rb null mouse. Blood 2007; 110, 2173-81. Walkley, C.R., Shea, J.M., Sims, N.A., Purton, L.E. & Orkin, S.H. Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell 2007; 129, 1081-95. Daria, D. et al. The retinoblastoma tumor suppressor is a critical intrinsic regulator for hematopoietic stem and progenitor cells under stress. Blood 2008; 111, 1894-902. Funding: The author is grateful to the J.P. McCarthy Foundation, the Aplastic Anemia and MDS International Foundation and the National Heart Lung & Blood Institute (RO1 HL080262) for funding of work in her laboratory relating to oxidative stress, erythropoiesis and hematopoietic diseases. DisclosuresNo relevant conflicts of interest to declare.

Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma

The human hMYH and NEIL1 genes encode DNA glycosylases involved in repair of oxidative base damage and mutations in these genes are associated with certain cancers. Primary sclerosing cholangitis (PSC), a chronic cholestatic liver disease characterized by inflammatory destruction of the biliary tree, is often complicated by the development of cholangiocarcinoma (CCA). Here, we aimed to investigate the influence of genetic variations in the hMYH and NEIL1 genes on risk of CCA in PSC patients. The hMYH and NEIL1 gene loci in addition to the DNA repair genes hOGG1, NTHL1 and NUDT1 were analyzed in 66 PSC patients (37 with CCA and 29 without cancer) by complete genomic sequencing of exons and adjacent intronic regions. Several single-nucleotide polymorphisms and mutations were identified and severe impairment of protein function was observed for three non-synonymous variants. The NEIL1 G83D mutant was dysfunctional for the major oxidation products 7,8-dihydro-8-oxoguanine (8oxoG), thymine glycol and dihydrothymine in duplex DNA, and the ability to perform δ-elimination at abasic sites was significantly reduced. The hMYH R260Q mutant had severe defect in adenine DNA glycosylase activity, whereas hMYH H434D could excise adenines from A:8oxoG pairs but not from A:G mispairs. We found no overall associations between the 18 identified variants and susceptibility to CCA in PSC patients; however, the impaired variants may be of significance for carcinogenesis in general. Our findings demonstrate the importance of complete resequencing of selected candidate genes in order to identify rare genetic variants and their possible contribution to individual susceptibility to cancer development.

Regulatory Role of Human AP-Endonuclease (APE1/Ref-1) in YB-1-Mediated Activation of the Multidrug Resistance Gene MDR1

Human AP-endonuclease (APE1/Ref-1), a central enzyme involved in the repair of oxidative base damage and DNA strand breaks, has a second activity as a transcriptional regulator that binds to several trans-acting factors. APE1 overexpression is often observed in tumor cells and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to such agents. Because the involvement of APE1 in repairing the DNA damage induced by many of these drugs is unlikely, drug resistance may be linked to APE1's transcriptional regulatory function. Here, we show that APE1, preferably in the acetylated form, stably interacts with Y-box-binding protein 1 (YB-1) and enhances its binding to the Y-box element, leading to the activation of the multidrug resistance gene MDR1. The enhanced MDR1 level due to the ectopic expression of wild-type APE1 but not of its nonacetylable mutant underscores the importance of APE1's acetylation in its coactivator function. APE1 downregulation sensitizes MDR1-overexpressing tumor cells to cisplatin or doxorubicin, showing APE1's critical role in YB-1-mediated gene expression and, thus, drug resistance in tumor cells. A systematic increase in both APE1 and MDR1 expression was observed in non-small-cell lung cancer tissue samples. Thus, our study has established the novel role of the acetylation-mediated transcriptional regulatory function of APE1, making it a potential target for the drug sensitization of tumor cells.

The role of the RB tumour suppressor pathway in oxidative stress responses in the haematopoietic system

Exposure to pro-oxidants and defects in the repair of oxidative base damage are associated with disease and ageing and also contribute to the development of anaemia, bone marrow failure and haematopoietic malignancies. This Review assesses emerging data indicative of a specific role for the RB tumour suppressor pathway in the response of the haematopoietic system to oxidative stress. This is mediated through signalling pathways that involve DNA damage sensors, forkhead box O (Foxo) transcription factors and p38 mitogen-activated protein kinases and has downstream consequences for cell cycle progression, antioxidant capacity, mitochondrial mass and cellular metabolism.

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.

Vertebrate POLQ and POLβ Cooperate in Base Excision Repair of Oxidative DNA Damage

Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage induced by H(2)O(2), but not to UV or cisplatin. Surprisingly, this phenotype was synergistically increased by concomitant deletion of the major BER polymerase, POLbeta. Moreover, extracts from a polq null mutant cell line show reduced BER activity, and POLQ, like POLbeta, accumulated rapidly at sites of base damage. Accordingly, POLQ and POLbeta share an overlapping function in the repair of oxidative base damage. Taken together, these results suggest a role for vertebrate POLQ in BER.

Host cell reactivation of plasmids containing oxidative DNA lesions is defective in Cockayne syndrome but normal in UV-sensitive syndrome fibroblasts

UV-sensitive syndrome (UV SS) is a human DNA repair-deficient disease with mild clinical manifestations. No neurological or developmental abnormalities or predisposition to cancer have been reported. In contrast, Cockayne syndrome (CS) patients exhibit severe developmental and neurological defects, in addition to photosensitivity. The cellular and biochemical responses of UV SS and CS cells to UV are indistinguishable, and result from defective transcription-coupled repair (TCR) of photoproducts in expressed genes. We propose that UV SS patients develop normally because they are proficient in repair of oxidative base damage. Consistent with our model, we show that Cockayne syndrome cells from complementation groups A and B (CS-A, CS-B) are more sensitive to treatment with hydrogen peroxide than wild type or UV SS cells. Using a host cell reactivation assay with plasmids containing UV-induced photoproducts, we find that expression of the plasmid-encoded lacZ gene is reduced in the TCR-deficient CS-B and UV SS cells. When the plasmids contain the oxidative base lesion thymine glycol, CS-B cells are defective in recovery of expression, whereas UV SS cells show levels of expression similar to those in wild type cells. 8-oxoguanine in the plasmids result in similarly defective host cell reactivation in CS-A and CS-B cells; abasic sites or single strand breaks in the plasmids cause similar decreases in expression in all the cell lines examined. Repair of thymine glycols in the lacZ gene was measured in plasmids extracted from transfected cells; removal of the lesions is efficient and without strand bias in all the cell lines tested.

UV-sensitive syndrome

UV-sensitive syndrome (UV SS) is a human DNA repair-deficiency disorder with mild clinical manifestations. In contrast to other disorders with photosensitivity, no neurological or developmental abnormalities and no predisposition to cancer have been reported. The cellular and biochemical responses of UV SS and Cockayne syndrome (CS) cells to UV light are indistinguishable, and result from defective transcription-coupled repair of photoproducts in expressed genes [G. Spivak, T. Itoh, T. Matsunaga, O. Nikaido, P. Hanawalt, M. Yamaizumi, Ultra violet-sensitive syndrome cells are defective in transcription-coupled repair of cyclobutane pyrimidine dimers, DNA Repair, 1, 2002, 629–643]. The severe neurological and developmental deficiency characteristic of CS may arise from unresolved blockage of transcription at oxidative DNA lesions, which could result in excessive cell death and/or attenuated transcription. We have proposed that individuals with UV SS develop normally because they are proficient in repair of oxidative base damage or in transcriptional bypass of these lesions; consistent with this hypothesis, CS-B cells, but not UV SS cells, are deficient in host cell reactivation of plasmids containing oxidative base lesions [G. Spivak, P. Hanawalt, Host cell reactivation of plasmids containing oxidative DNA lesions is defective in Cockayne syndrome but normal in UV-sensitive syndrome, 2005, submitted for publication]. In this review, I will summarize the current understanding of the UV-sensitive syndrome and compare it with the Cockayne syndrome.

How heterologously expressed Escherichia coli genes contribute to understanding DNA repair processes in Saccharomyces cerevisiae

DNA-damaging agents constantly challenge cellular DNA; and efficient DNA repair is therefore essential to maintain genome stability and cell viability. Several DNA repair mechanisms have evolved and these have been shown to be highly conserved from bacteria to man. DNA repair studies were originally initiated in very simple organisms such as Escherichia coli and Saccharomyces cerevisiae, bacteria being the best understood organism to date. As a consequence, bacterial DNA repair genes encoding proteins with well characterized functions have been transferred into higher organisms in order to increase repair capacity, or to complement repair defects, in heterologous cells. While indicating the contribution of these repair functions to protection against the genotoxic effects of DNA-damaging agents, heterologous expression studies also highlighted the role of the DNA lesions that are substrates for such processes. In addition, bacterial DNA repair-like functions could be identified in higher organisms using this approach. We heterologously expressed three well characterized E. coli repair genes in S. cerevisiae cells of different genetic backgrounds: (1) the ada gene encoding O(6)-methylguanine DNA-methyltransferase, a protein involved in the repair of alkylation damage to DNA, (2) the recA gene encoding the main recombinase in E. coli and (3) the nth gene, the product of which (endonuclease III) is responsible for the repair of oxidative base damage. Here, we summarize our results and indicate the possible implications they have for a better understanding of particular DNA repair processes in S. cerevisiae.

Compromised Incision of Oxidized Pyrimidines in Liver Mitochondria of Mice Deficient in NTH1 and OGG1 Glycosylases

Mitochondrial DNA is constantly exposed to high levels of endogenously produced reactive oxygen species, resulting in elevated levels of oxidative damaged DNA bases. A large spectrum of DNA base alterations can be detected after oxidative stress, and many of these are highly mutagenic. Thus, an efficient repair of these is necessary for survival. Some of the DNA repair pathways involved have been characterized, but others are not yet determined. A DNA repair activity for thymine glycol and other oxidized pyrimidines has been described in mammalian mitochondria, but the nature of the glycosylases involved in this pathway remains unclear. The generation of mouse strains lacking murine thymine glycol-DNA glycosylase (mNTH1) and/or murine 8-oxoguanine-DNA glycosylase (mOGG1), the two major DNA N-glycosylase/apurinic/apyrimidinic (AP) lyases involved in the repair of oxidative base damage in the nucleus, has provided very useful biological model systems for the study of the function of these and other glycosylases in mitochondrial DNA repair. In this study, mouse liver mitochondrial extracts were generated from mNTH1-, mOGG1-, and [mNTH1, mOGG1]-deficient mice to ascertain the role of each of these glycosylases in the repair of oxidized pyrimidine base damage. We also characterized for the first time the incision of various modified bases in mitochondrial extracts from a double-knock-out [mNTH1, mOGG1]-deficient mouse. We show that mNTH1 is responsible for the repair of thymine glycols in mitochondrial DNA, whereas other glycosylase/AP lyases also participate in removing other oxidized pyrimidines, such as 5-hydroxycytosine and 5-hydroxyuracil. We did not detect a backup glycosylase or glycosylase/AP lyase activity for thymine glycol in the mitochondrial mouse extracts.

Long-patch DNA repair synthesis during base excision repair in mammalian cells.

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.

Defective transcription-coupled repair of oxidative base damage in Cockayne syndrome patients from XP group G.

In normal human cells, damage due to ultraviolet light is preferentially removed from active genes by nucleotide excision repair (NER) in a transcription-coupled repair (TCR) process that requires the gene products defective in Cockayne syndrome (CS). Oxidative damage, including thymine glycols, is shown to be removed by TCR in cells from normal individuals and from xeroderma pigmentosum (XP)-A, XP-F, and XP-G patients who have NER defects but not from XP-G patients who have severe CS. Thus, TCR of oxidative damage requires an XPG function distinct from its NER endonuclease activity. These results raise the possibility that defective TCR of oxidative damage contributes to the developmental defects associated with CS.

Gene PSO5 of Saccharomyces cerevisiae, involved in repair of oxidative DNA damage, is allelic to RAD16.

The pos5-1 mutation renders Saccharomyces cerevisiae cells sensitive to DNA-damaging agents. We have isolated plasmids from a S. cerevisiae genomic library capable of restoring wild-type levels of 254-nm ultraviolet light sensitivity of the pso5-1 mutant. DNA sequence analysis revealed that the complementing activity resides in RAD16, a gene involved in excision repair. Tetrad analysis showed that PSO5, like RAD16, is tightly linked to LYS2 on chromosome II. Moreover, allelism between the pso5-1 and rad16 mutants was demonstrated by the comparison of mutagen sensitivity phenotypes, complementation tests, and by meiotic analysis. The cloned RAD16 gene was capable of restoring wild-type resistance of the pso5-1 mutant to H2O2 and photoactivated 3-carbethoxypsoralen, both treatments generating oxidative stress-related DNA damage. This indicates that RAD16/PSO5 might also participate in the repair of oxidative base damage.