Base Excision Repair: Mechanisms and Impact in Biology, Disease, and Medicine.

Base excision repair (BER) corrects forms of oxidative, deamination, alkylation, and abasic single-base damage that appear to have minimal effects on the helix. Since its discovery in 1974, the field has grown in several facets: mechanisms, biology and physiology, understanding deficiencies and human disease, and using BER genes as potential inhibitory targets to develop therapeutics. Within its segregation of short nucleotide (SN-) and long patch (LP-), there are currently six known global mechanisms, with emerging work in transcription- and replication-associated BER. Knockouts (KOs) of BER genes in mouse models showed that single glycosylase knockout had minimal phenotypic impact, but the effects were clearly seen in double knockouts. However, KOs of downstream enzymes showed critical impact on the health and survival of mice. BER gene deficiency contributes to cancer, inflammation, aging, and neurodegenerative disorders. Medicinal targets are being developed for single or combinatorial therapies, but only PARP and APE1 have yet to reach the clinical stage.

DNA Glycosylases Define the Outcome of Endogenous Base Modifications.

Chemically modified nucleic acid bases are sources of genomic instability and mutations but may also regulate gene expression as epigenetic or epitranscriptomic modifications. Depending on the cellular context, they can have vastly diverse impacts on cells, from mutagenesis or cytotoxicity to changing cell fate by regulating chromatin organisation and gene expression. Identical chemical modifications exerting different functions pose a challenge for the cell's DNA repair machinery, as it needs to accurately distinguish between epigenetic marks and DNA damage to ensure proper repair and maintenance of (epi)genomic integrity. The specificity and selectivity of the recognition of these modified bases relies on DNA glycosylases, which acts as DNA damage, or more correctly, as modified bases sensors for the base excision repair (BER) pathway. Here, we will illustrate this duality by summarizing the role of uracil-DNA glycosylases, with particular attention to SMUG1, in the regulation of the epigenetic landscape as active regulators of gene expression and chromatin remodelling. We will also describe how epigenetic marks, with a special focus on 5-hydroxymethyluracil, can affect the damage susceptibility of nucleic acids and conversely how DNA damage can induce changes in the epigenetic landscape by altering the pattern of DNA methylation and chromatin structure.

Recognition of 1,N2-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

The adenine, cytosine, and guanine bases of DNA are susceptible to alkylation by the aldehyde products of lipid peroxidation and by the metabolic byproducts of vinyl chloride pollutants. The resulting adducts spontaneously cyclize to form harmful etheno lesions. Cells employ a variety of DNA repair pathways to protect themselves from these pro-mutagenic modifications. Human alkyladenine DNA glycosylase (AAG) is thought to initiate base excision repair of both 1,N6-ethenoadenine (ϵA) and 1,N2-ethenoguanine (ϵG). However, it is not clear how AAG might accommodate ϵG in an active site that is complementary to ϵA. This prompted a thorough investigation of AAG-catalyzed excision of ϵG from several relevant contexts. Using single-turnover and multiple-turnover kinetic analyses, we found that ϵG in its natural ϵG·C context is very poorly recognized relative to ϵA·T. Bulged and mispaired ϵG contexts, which can form during DNA replication, were similarly poor substrates for AAG. Furthermore, AAG could not recognize an ϵG site in competition with excess undamaged DNA sites. Guided by previous structural studies, we hypothesized that Asn-169, a conserved residue in the AAG active-site pocket, contributes to discrimination against ϵG. Consistent with this model, the N169S variant of AAG was 7-fold more active for excision of ϵG compared with the wildtype (WT) enzyme. Taken together, these findings suggest that ϵG is not a primary substrate of AAG, and that current models for etheno lesion repair in humans should be revised. We propose that other repair and tolerance mechanisms operate in the case of ϵG lesions.

Lysines in the lyase active site of DNA polymerase ß destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition.

DNA polymerase (pol) ß catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol ß has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol ß for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol ß, pol λ, and pol µ, and several variants of pol ß, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol ß, 35, 68, and 72, to alanine (pol ß KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol µ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol ß KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol ß KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol ß. These data suggest that the three lysines in the lyase active site destabilize pol ß when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

The DNA repair enzyme MUTYH potentiates cytotoxicity of the alkylating agent MNNG by interacting with abasic sites.

Higher expression of the human DNA repair enzyme MUTYH has previously been shown to be strongly associated with reduced survival in a panel of 24 human lymphoblastoid cell lines exposed to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The molecular mechanism of MUTYH-enhanced MNNG cytotoxicity is unclear, because MUTYH has a well-established role in the repair of oxidative DNA lesions. Here, we show in mouse embryonic fibroblasts (MEFs) that this MNNG-dependent phenotype does not involve oxidative DNA damage and occurs independently of both O6-methyl guanine adduct cytotoxicity and MUTYH-dependent glycosylase activity. We found that blocking of abasic (AP) sites abolishes higher survival of Mutyh-deficient (Mutyh-/-) MEFs, but this blockade had no additive cytotoxicity in WT MEFs, suggesting the cytotoxicity is due to MUTYH interactions with MNNG-induced AP sites. We found that recombinant mouse MUTYH tightly binds AP sites opposite all four canonical undamaged bases and stimulated apurinic/apyrimidinic endonuclease 1 (APE1)-mediated DNA incision. Consistent with these observations, we found that stable expression of WT, but not catalytically-inactive MUTYH, enhances MNNG cytotoxicity in Mutyh-/- MEFs and that MUTYH expression enhances MNNG-induced genomic strand breaks. Taken together, these results suggest that MUTYH enhances the rapid accumulation of AP-site intermediates by interacting with APE1, implicating MUTYH as a factor that modulates the delicate process of base-excision repair independently of its glycosylase activity.

An autoinhibitory role for the GRF zinc finger domain of DNA glycosylase NEIL3.

The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows that the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA-processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.

Aging and Protein Kinases.

Recently, aging has been tried to be explained with large numbers of theories, but none of them can elucidate the changes occurring in the aging process alone. A unified theory encompassing the mechanisms of genetic factors and repair systems in aging is becoming increasingly required. Almost 37 protein kinases contribute to all processes of aging and senescence. Furthermore, these kinases not only regulate the large number of metabolic pathways related to aging processes, but also control these pathways through 12 checkpoints. Thus, in this chapter, the metabolic targets of protein kinases signal transduction pathways were discussed in terms of the aging perspective under five headings, which are the indispensable stages of the aging process. Although the most popular classical aging theories have been stated as DNA damage theory, mitochondrial theory, free radical theory, and telomere theory, it was concluded that the aging process is controlled by protein kinases regardless of the different theories.

Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis-Element in Response to 5-Formylcytosine and 5-Carboxycytosine.

Stepwise oxidation of the epigenetic mark 5-methylcytosine and base excision repair (BER) of the resulting 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) may provide a mechanism for reactivation of epigenetically silenced genes; however, the functions of 5-fC and 5-caC at defined gene elements are scarcely explored. We analyzed the expression of reporter constructs containing either 2'-deoxy-(5-fC/5-caC) or their BER-resistant 2'-fluorinated analogs, asymmetrically incorporated into CG-dinucleotide of the GC box cis-element (5'-TGGGCGGAGC) upstream from the RNA polymerase II core promoter. In the absence of BER, 5-caC caused a strong inhibition of the promoter activity, whereas 5-fC had almost no effect, similar to 5-methylcytosine or 5-hydroxymethylcytosine. BER of 5-caC caused a transient but significant promoter reactivation, succeeded by silencing during the following hours. Both responses strictly required thymine DNA glycosylase (TDG); however, the silencing phase additionally demanded a 5'-endonuclease (likely APE1) activity and was also induced by 5-fC or an apurinic/apyrimidinic site. We propose that 5-caC may act as a repressory mark to prevent premature activation of promoters undergoing the final stages of DNA demethylation, when the symmetric CpG methylation has already been lost. Remarkably, the downstream promoter activation or repression responses are regulated by two separate BER steps, where TDG and APE1 act as potential switches.

The Influence of 5'R and 5'S cdA and cdG on the Activity of BsmAI and SspI Restriction Enzymes.

Restriction endonucleases (REs) are intra-bacterial scissors that are considered tools in the fight against foreign genetic material. SspI and BsmAI, examined in this study, cleave dsDNA at their site of recognition or within a short distance of it. Both enzymes are representatives of type II REs, which have played an extremely important role in research on the genetics of organisms and molecular biology. Therefore, the study of agents affecting their activity has become highly important. Ionizing radiation may damage basic cellular mechanisms by inducing lesions in the genome, with 5',8-cyclo-2'-deoxypurines (cdPus) as a model example. Since cdPus may become components of clustered DNA lesions (CDLs), which are unfavorable for DNA repair pathways, their impact on other cellular mechanisms is worthy of attention. This study investigated the influence of cdPus on the elements of the bacterial restriction-modification system. In this study, it was shown that cdPus present in DNA affect the activity of REs. SspI was blocked by any cdPu lesion present at the enzyme's recognition site. When lesions were placed near the recognition sequence, the SspI was inhibited up to 46%. Moreover, (5'S)-5',8-cyclo-2'-deoxyadenosine (ScdA) present in the oligonucleotide sequence lowered BsmAI activity more than (5'R)-5',8-cyclo-2'-deoxyadenosine (RcdA). Interestingly, in the case of 5',8-cyclo-2'-deoxyguanosine (cdG), both 5'S and 5'R diastereomers inhibited BsmAI activity (up to 55% more than cdA). The inhibition was weaker when cdG was present at the recognition site rather than the cleavage site.

Molecular basis for the faithful replication of 5-methylcytosine and its oxidized forms by DNA polymerase ß.

DNA methylation is an epigenetic mark that regulates gene expression in mammals. One method of methylation removal is through ten-eleven translocation-catalyzed oxidation and the base excision repair pathway. The iterative oxidation of 5-methylcytosine catalyzed by ten-eleven translocation enzymes produces three oxidized forms of cytosine: 5-hydroxmethylcytosine, 5-formylcytosine, and 5-carboxycytosine. The effect these modifications have on the efficiency and fidelity of the base excision repair pathway during the repair of opposing base damage, and in particular DNA polymerization, remains to be elucidated. Using kinetic assays, we show that the catalytic efficiency for the incorporation of dGTP catalyzed by human DNA polymerase ß is not affected when 5-methylcytosine, 5-hydroxmethylcytosine, and 5-formylcytosine are in the DNA template. In contrast, the catalytic efficiency of dGTP insertion decreases ∼20-fold when 5-carboxycytosine is in the templating position, as compared with unmodified cytosine. However, DNA polymerase fidelity is unaltered when these modifications are in the templating position. Structural analysis reveals that the methyl, hydroxymethyl, and formyl modifications are easily accommodated within the polymerase active site. However, to accommodate the carboxyl modification, the phosphate backbone on the templating nucleotide shifts ∼2.5 Å to avoid a potential steric/repulsive clash. This altered conformation is stabilized by lysine 280, which makes a direct interaction with the carboxyl modification and the phosphate backbone of the templating strand. This work provides the molecular basis for the accommodation of epigenetic base modifications in a polymerase active site and suggests that these modifications are not mutagenically copied during base excision repair.

Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast.

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.

Genome-wide mapping of regions preferentially targeted by the human DNA-cytosine deaminase APOBEC3A using uracil-DNA pulldown and sequencing.

Activation-induced deaminase (AID) and apolipoprotein B mRNA-editing enzyme catalytic subunit (APOBEC) enzymes convert cytosines to uracils, creating signature mutations that have been used to predict sites targeted by these enzymes. Mutation-based targeting maps are distorted by the error-prone or error-free repair of these uracils and by selection pressures. To directly map uracils created by AID/APOBEC enzymes, here we used uracil-DNA glycosylase and an alkoxyamine to covalently tag and sequence uracil-containing DNA fragments (UPD-Seq). We applied this technique to the genome of repair-defective, APOBEC3A-expressing bacterial cells and created a uracilation genome map, i.e. uracilome. The peak uracilated regions were in the 5'-ends of genes and operons mainly containing tRNA genes and a few protein-coding genes. We validated these findings through deep sequencing of pulldown regions and whole-genome sequencing of independent clones. The peaks were not correlated with high transcription rates or stable RNA:DNA hybrid formation. We defined the uracilation index (UI) as the frequency of occurrence of TT in UPD-Seq reads at different original TC dinucleotides. Genome-wide UI calculation confirmed that APOBEC3A modifies cytosines in the lagging-strand template during replication and in short hairpin loops. APOBEC3A's preference for tRNA genes was observed previously in yeast, and an analysis of human tumor sequences revealed that in tumors with a high percentage of APOBEC3 signature mutations, the frequency of tRNA gene mutations was much higher than in the rest of the genome. These results identify multiple causes underlying selection of cytosines by APOBEC3A for deamination, and demonstrate the utility of UPD-Seq.

Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination.

The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.

Oxidative DNA damage is concurrently repaired by base excision repair (BER) and apyrimidinic endonuclease 1 (APE1)-initiated nonhomologous end joining (NHEJ) in cortical neurons.

AIMS: Accumulating studies have suggested that base excision repair (BER) is the major repair pathway of oxidative DNA damage in neurons, and neurons are deficient in other DNA repair pathways, including nucleotide excision repair and homologous recombination repair. However, some studies have demonstrated that neurons could efficiently repair glutamate- and menadione-induced double-strand breaks (DSBs), suggesting that the DSB repair mechanisms might be implicated in neuronal health. In this study, we hypothesized that BER and nonhomologous end joining (NHEJ) work together to repair oxidative DNA damage in neurons. METHODS: Immunohistochemistry and confocal microscopy were employed to examine the colocalization of apyrimidinic endonuclease 1 (APE1), histone variant 2AX (γH2AX) and phosphorylated p53-binding protein (53BP1). APE1 inhibitor and shRNA were respectively applied to suppress APE1 activity and protein expression to determine the correlation of APE1 and DSB formation. The neutral comet assay was used to determine and quantitate the formation of DSB. RESULTS: Both γH2AX and 53BP1 were upregulated and colocalized with APE1 in the nuclei of rat cortical neurons subjected to menadione-induced oxidative insults. Phospho53BP1 foci were efficiently abolished, but γH2AX foci persisted following the suppression of APE1 activity. Comet assays demonstrated that the inhibition of APE1 decreased the DSB formation. CONCLUSIONS: Our results indicate that APE1 can engage the NHEJ mechanism in the repair of oxidative DNA damage in neurons. These findings provide insights into the mechanisms underlying the efficient repair of oxidative DNA damage in neurons despite the high oxidative burden.

Oxidative Damage in Sporadic Colorectal Cancer: Molecular Mapping of Base Excision Repair Glycosylases in Colorectal Cancer Patients.

Oxidative stress with subsequent premutagenic oxidative DNA damage has been implicated in colorectal carcinogenesis. The repair of oxidative DNA damage is initiated by lesion-specific DNA glycosylases (hOGG1, NTH1, MUTYH). The direct evidence of the role of oxidative DNA damage and its repair is proven by hereditary syndromes (MUTYH-associated polyposis, NTHL1-associated tumor syndrome), where germline mutations cause loss-of-function in glycosylases of base excision repair, thus enabling the accumulation of oxidative DNA damage and leading to the adenoma-colorectal cancer transition. Unrepaired oxidative DNA damage often results in G:C>T:A mutations in tumor suppressor genes and proto-oncogenes and widespread occurrence of chromosomal copy-neutral loss of heterozygosity. However, the situation is more complicated in complex and heterogeneous disease, such as sporadic colorectal cancer. Here we summarized our current knowledge of the role of oxidative DNA damage and its repair on the onset, prognosis and treatment of sporadic colorectal cancer. Molecular and histological tumor heterogeneity was considered. Our study has also suggested an additional important source of oxidative DNA damage due to intestinal dysbiosis. The roles of base excision repair glycosylases (hOGG1, MUTYH) in tumor and adjacent mucosa tissues of colorectal cancer patients, particularly in the interplay with other factors (especially microenvironment), deserve further attention. Base excision repair characteristics determined in colorectal cancer tissues reflect, rather, a disease prognosis. Finally, we discuss the role of DNA repair in the treatment of colon cancer, since acquired or inherited defects in DNA repair pathways can be effectively used in therapy.

Analysis of MUTYH alternative transcript expression, promoter function, and the effect of human genetic variants.

The human DNA repair gene MUTYH, whose mutational loss causes a colorectal polyposis and cancer predisposition, contains three alternative first exons. In order to analyze alternative transcription and the effect of genetic alterations found in humans, we established a cell-based minigene experimental model supporting transcription and splicing and thoroughly verified its functionality. We identified highly conserved promoter areas and inactivated them in the minigene, and also introduced six human variants. Moreover, the potential contribution of CpG island methylation and specific transcription factors on MUTYH transcription was addressed. The findings allowed to attribute regulatory roles to three conserved motifs in the promoter: an M4 motif, a transcription factor IIB recognition element, and a GC box. Moreover, the data showed that three patient variants compromised MUTYH expression and therefore have the potential to cause pathogenic effects. We did not find evidence for a biologically relevant contribution of CpG island methylation or a direct transcriptional activation by DNA damage. Besides insight into the regulation of MUTYH transcription, the work therefore provides a functional MUTYH minigene experimental system suitable as a diagnostic tool for analyzing patient variants, and a functional map of the promotor that also can facilitate pathogenicity classifications of human variants.

A cassette of basic amino acids in histone H2B regulates nucleosome dynamics and access to DNA damage.

Nucleosome dynamics, such as spontaneous DNA unwrapping, are postulated to have a critical role in regulating the access of DNA repair machinery to DNA lesions within nucleosomes. However, the specific histone domains that regulate nucleosome dynamics and the impact of such changes in intrinsic nucleosome dynamics on DNA repair are not well understood. Previous studies identified a highly conserved region in the N-terminal tail of histone H2B known as the histone H2Brepression (or HBR) domain, which has a significant influence on gene expression, chromatin assembly, and DNA damage formation and repair. However, the molecular mechanism(s) that may account for these observations are limited. In this study, we characterized the stability and dynamics of ΔHBR mutant nucleosome core particles (NCPs) in vitro by restriction enzyme accessibility (REA), FRET, and temperature-induced sliding of histone octamers. Our results indicate that ΔHBR-NCPs are more dynamic, with a larger steady-state fraction of the NCP population occupying the unwrapped state than for WT-NCPs. Additionally, ΔHBR-histone octamers are more susceptible to temperature-induced sliding on DNA than WT histone octamers. Furthermore, we show that the activity of base excision repair enzymes at uracil lesions and single nucleotide gaps is enhanced in a site-specific manner in ΔHBR-NCPs. This enhanced activity correlates well with regions exhibiting increased DNA unwrapping. Finally, removal of the HBR domain is not sufficient to completely alleviate the structural constraints imposed by histone octamers on the activity of base excision repair enzymes.

Poly(ADP-ribose) polymerase 1 (PARP1) promotes oxidative stress-induced association of Cockayne syndrome group B protein with chromatin.

Cockayne syndrome protein B (CSB) is an ATP-dependent chromatin remodeler that relieves oxidative stress by regulating DNA repair and transcription. CSB is proposed to participate in base-excision repair (BER), the primary pathway for repairing oxidative DNA damage, but exactly how CSB participates in this process is unknown. It is also unclear whether CSB contributes to other repair pathways during oxidative stress. Here, using a patient-derived CS1AN-sv cell line, we examined how CSB is targeted to chromatin in response to menadione-induced oxidative stress, both globally and locus-specifically. We found that menadione-induced, global CSB-chromatin association does not require CSB's ATPase activity and is, therefore, mechanistically distinct from UV-induced CSB-chromatin association. Importantly, poly(ADP-ribose) polymerase 1 (PARP1) enhanced the kinetics of global menadione-induced CSB-chromatin association. We found that the major BER enzymes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), do not influence this association. Additionally, the level of γ-H2A histone family member X (γ-H2AX), a marker for dsDNA breaks, was not increased in menadione-treated cells. Therefore, our results support a model whereby PARP1 localizes to ssDNA breaks and recruits CSB to participate in DNA repair. Furthermore, this global CSB-chromatin association occurred independently of RNA polymerase II-mediated transcription elongation. However, unlike global CSB-chromatin association, both PARP1 knockdown and inhibition of transcription elongation interfered with menadione-induced CSB recruitment to specific genomic regions. This observation supports the hypothesis that CSB is also targeted to specific genomic loci to participate in transcriptional regulation in response to oxidative stress.

DNA Oxidation and Excision Repair Pathways.

The physiological impact of the aberrant oxidation products on genomic DNA were demonstrated by embryonic lethality or the cancer susceptibility and/or neurological symptoms of animal impaired in the base excision repair (BER); the major pathway to maintain genomic integrity against non-bulky DNA oxidation. However, growing evidence suggests that other DNA repair pathways or factors that are not primarily associated with the classical BER pathway are also actively involved in the mitigation of oxidative assaults on the genomic DNA, according to the corresponding types of DNA oxidation. Among others, factors dedicated to lesion recognition in the nucleotide excision repair (NER) pathway have been shown to play eminent roles in the process of lesion recognition and stimulation of the enzyme activity of some sets of BER factors. Besides, substantial bulky DNA oxidation can be preferentially removed by a canonical NER mechanism; therefore, loss of function in the NER pathway shares common features arising from BER defects, including cancer predisposition and neurological disorders, although NER defects generally are nonlethal. Here we discuss recent achievements for delineating newly arising roles of NER lesion recognition factors to facilitate the BER process, and cooperative works of BER and NER pathways in response to the genotoxic oxidative stress.

The Human Ligase IIIα-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA.

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin.