Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis.

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and ß-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

Cooperation of the NEIL3 and Fanconi anemia/BRCA pathways in interstrand crosslink repair.

The NEIL3 DNA glycosylase is a base excision repair enzyme that excises bulky base lesions from DNA. Although NEIL3 has been shown to unhook interstrand crosslinks (ICL) in Xenopus extracts, how NEIL3 participants in ICL repair in human cells and its corporation with the canonical Fanconi anemia (FA)/BRCA pathway remain unclear. Here we show that the NEIL3 and the FA/BRCA pathways are non-epistatic in psoralen-ICL repair. The NEIL3 pathway is the major pathway for repairing psoralen-ICL, and the FA/BRCA pathway is only activated when NEIL3 is not present. Mechanistically, NEIL3 is recruited to psoralen-ICL in a rapid, PARP-dependent manner. Importantly, the NEIL3 pathway repairs psoralen-ICLs without generating double-strand breaks (DSBs), unlike the FA/BRCA pathway. In addition, we found that the RUVBL1/2 complex physically interact with NEIL3 and function within the NEIL3 pathway in psoralen-ICL repair. Moreover, TRAIP is important for the recruitment of NEIL3 but not FANCD2, and knockdown of TRAIP promotes FA/BRCA pathway activation. Interestingly, TRAIP is non-epistatic with both NEIL3 and FA pathways in psoralen-ICL repair, suggesting that TRAIP may function upstream of the two pathways. Taken together, the NEIL3 pathway is the major pathway to repair psoralen-ICL through a unique DSB-free mechanism in human cells.

The lyase activity of bifunctional DNA glycosylases and the 3'-diesterase activity of APE1 contribute to the repair of oxidized bases in nucleosomes.

The vast majority of oxidized bases that form in DNA are subject to base excision repair (BER). The DNA intermediates generated during successive steps in BER may prove mutagenic or lethal, making it critical that they be 'handed' from one BER enzyme to the next in a coordinated fashion. Here, we report that the handoff of BER intermediates that occurs during the repair of naked DNA substrates differs significantly from that in nucleosomes. During BER of oxidized bases in naked DNA, products generated by the DNA glycosylase NTHL1 were efficiently processed by the downstream enzyme, AP-endonuclease (APE1). In nucleosomes, however, NTHL1-generated products accumulated to significant levels and persisted for some time. During BER of naked DNA substrates, APE1 completely bypasses the inefficient lyase activity of NTHL1. In nucleosomes, the NTHL1-associated lyase contributes to BER, even in the presence of APE1. Moreover, in nucleosomes but not in naked DNA, APE1 was able to process NTHL1 lyase-generated substrates just as efficiently as it processed abasic sites. Thus, the lyase activity of hNTHL1, and the 3' diesterase activity of APE1, which had been seen as relatively dispensable, may have been preserved during evolution to enhance BER in chromatin.

Single molecule glycosylase studies with engineered 8-oxoguanine DNA damage sites show functional defects of a MUTYH polyposis variant.

Proper repair of oxidatively damaged DNA bases is essential to maintain genome stability. 8-Oxoguanine (7,8-dihydro-8-oxoguanine, 8-oxoG) is a dangerous DNA lesion because it can mispair with adenine (A) during replication resulting in guanine to thymine transversion mutations. MUTYH DNA glycosylase is responsible for recognizing and removing the adenine from 8-oxoG:adenine (8-oxoG:A) sites. Biallelic mutations in the MUTYH gene predispose individuals to MUTYH-associated polyposis (MAP), and the most commonly observed mutation in some MAP populations is Y165C. Tyr165 is a 'wedge' residue that intercalates into the DNA duplex in the lesion bound state. Here, we utilize single molecule fluorescence microscopy to visualize the real-time search behavior of Escherichia coli and Mus musculus MUTYH WT and wedge variant orthologs on DNA tightropes that contain 8-oxoG:A, 8-oxoG:cytosine, or apurinic product analog sites. We observe that MUTYH WT is able to efficiently find 8-oxoG:A damage and form highly stable bound complexes. In contrast, MUTYH Y150C shows decreased binding lifetimes on undamaged DNA and fails to form a stable lesion recognition complex at damage sites. These findings suggest that MUTYH does not rely upon the wedge residue for damage site recognition, but this residue stabilizes the lesion recognition complex.

A functional assay-based procedure to classify mismatch repair gene variants in Lynch syndrome.

PURPOSE: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data. METHODS: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories. RESULTS: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible. CONCLUSION: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants.

Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase.

The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis and mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.

The Tumor-Associated Variant RAD51 G151D Induces a Hyper-Recombination Phenotype.

The RAD51 protein plays a key role in the homology-directed repair of DNA double-strand breaks and is important for maintaining genome stability. Here we report on a novel human RAD51 variant found in an aggressive and therapy-refractive breast carcinoma. Expression of the RAD51 G151D variant in human breast epithelial cells increases the levels of homology-directed repair. Expression of RAD51 G151D in cells also promotes high levels of chromosomal aberrations and sister chromatid exchanges. In vitro, the purified RAD51 G151D protein directly and significantly enhances DNA strand exchange activity in the presence of RPA. In concordance with this result, co-incubation of G151D with BRCA2 resulted in a much higher level of strand-exchange activity compared to WT RAD51. Strikingly, the RAD51 G151D variant confers resistance to multiple DNA damaging agents, including ionizing radiation, mitomycin C, and doxorubicin. Our findings demonstrate that the RAD51 G151D somatic variant has a novel hyper-recombination phenotype and suggest that this property of the protein is important for the repair of DNA damage, leading to drug resistance.

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.

The NEIL glycosylases remove oxidized guanine lesions from telomeric and promoter quadruplex DNA structures.

G-quadruplex is a four-stranded G-rich DNA structure that is highly susceptible to oxidation. Despite the important roles that G-quadruplexes play in telomere biology and gene transcription, neither the impact of guanine lesions on the stability of quadruplexes nor their repair are well understood. Here, we show that the oxidized guanine lesions 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) reduce the thermostability and alter the folding of telomeric quadruplexes in a location-dependent manner. Also, the NEIL1 and NEIL3 DNA glycosylases can remove hydantoin lesions but none of the glycosylases, including OGG1, are able to remove 8-oxoG from telomeric quadruplexes. Interestingly, a hydantoin lesion at the site most prone to oxidation in quadruplex DNA is not efficiently removed by NEIL1 or NEIL3. However, NEIL1, NEIL2 and NEIL3 remove hydantoins from telomeric quadruplexes formed by five TTAGGG repeats much more rapidly than the commonly studied four-repeat quadruplex structures. We also show that APE1 cleaves furan in selected positions in Na(+)-coordinated telomeric quadruplexes. In promoter G-quadruplex DNA, the NEIL glycosylases primarily remove Gh from Na(+)-coordinated antiparallel quadruplexes but not K(+)-coordinated parallel quadruplexes containing VEGF or c-MYC promoter sequences. Thus, the NEIL DNA glycosylases may be involved in both telomere maintenance and in gene regulation.

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases.

DNA glycosylases are enzymes that perform the initial steps of base excision repair, the principal repair mechanism that identifies and removes endogenous damages that occur in an organism's DNA. We characterized the motion of single molecules of three bacterial glycosylases that recognize oxidized bases, Fpg, Nei, and Nth, as they scan for damages on tightropes of λ DNA. We find that all three enzymes use a key "wedge residue" to scan for damage because mutation of this residue to an alanine results in faster diffusion. Moreover, all three enzymes bind longer and diffuse more slowly on DNA that contains the damages they recognize and remove. Using a sliding window approach to measure diffusion constants and a simple chemomechanical simulation, we demonstrate that these enzymes diffuse along DNA, pausing momentarily to interrogate random bases, and when a damaged base is recognized, they stop to evert and excise it.

Germ-line variant of human NTH1 DNA glycosylase induces genomic instability and cellular transformation.

Base excision repair (BER) removes at least 20,000 DNA lesions per human cell per day and is critical for the maintenance of genomic stability. We hypothesize that aberrant BER, resulting from mutations in BER genes, can lead to genomic instability and cancer. The first step in BER is catalyzed by DNA N-glycosylases. One of these, n(th) endonuclease III-like (NTH1), removes oxidized pyrimidines from DNA, including thymine glycol. The rs3087468 single nucleotide polymorphism of the NTH1 gene is a G-to-T base substitution that results in the NTH1 D239Y variant protein that occurs in ∼6.2% of the global population and is found in Europeans, Asians, and sub-Saharan Africans. In this study, we functionally characterize the effect of the D239Y variant expressed in immortal but nontransformed human and mouse mammary epithelial cells. We demonstrate that expression of the D239Y variant in cells also expressing wild-type NTH1 leads to genomic instability and cellular transformation as assessed by anchorage-independent growth, focus formation, invasion, and chromosomal aberrations. We also show that cells expressing the D239Y variant are sensitive to ionizing radiation and hydrogen peroxide and accumulate double strand breaks after treatment with these agents. The DNA damage response is also activated in D239Y-expressing cells. In combination, our data suggest that individuals possessing the D239Y variant are at risk for genomic instability and cancer.

Visualizing the Search for Radiation-damaged DNA Bases in Real Time.

The Base Excision Repair (BER) pathway removes the vast majority of damages produced by ionizing radiation, including the plethora of radiation-damaged purines and pyrimidines. The first enzymes in the BER pathway are DNA glycosylases, which are responsible for finding and removing the damaged base. Although much is known about the biochemistry of DNA glycosylases, how these enzymes locate their specific damage substrates among an excess of undamaged bases has long remained a mystery. Here we describe the use of single molecule fluorescence to observe the bacterial DNA glycosylases, Nth, Fpg and Nei, scanning along undamaged and damaged DNA. We show that all three enzymes randomly diffuse on the DNA molecule and employ a wedge residue to search for and locate damage. The search behavior of the Escherichia coli DNA glycosylases likely provides a paradigm for their homologous mammalian counterparts.

Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages.

Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.

Neil3 and NEIL1 DNA glycosylases remove oxidative damages from quadruplex DNA and exhibit preferences for lesions in the telomeric sequence context.

The telomeric DNA of vertebrates consists of d(TTAGGG)n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.

Structural characterization of viral ortholog of human DNA glycosylase NEIL1 bound to thymine glycol or 5-hydroxyuracil-containing DNA.

Thymine glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublié, S. (2009) J. Biol. Chem. 284, 26174-26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen-bonding contact with either damaged base, and mutating these residues did not markedly affect the glycosylase activity. This finding suggests that lesion recognition by Nei occurs before the damaged base flips into the glycosylase active site.

Rules of engagement for base excision repair in chromatin.

Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA- and nucleosome-associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology-directed repair (HDR), and non-homologous end-joining (NHEJ) double strand break repair pathways have led to an "access-repair-restore" paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ~20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value.

Neil3, the final frontier for the DNA glycosylases that recognize oxidative damage.

DNA glycosylases are the enzymes that initiate the Base Excision Repair (BER) process that protects all organisms from the mutagenic and/or cytotoxic effects of DNA base lesions. Endonuclease VIII like proteins (Neil1, Neil2 and Neil3) are found in vertebrate genomes and are homologous to the well-characterized bacterial DNA glycosylases, Formamidopyrimidine DNA glycosylase (Fpg) and Endonuclease VIII (Nei). Since the initial discovery of the Neil proteins, much progress has been made on characterizing Neil1 and Neil2. It was not until recently, however, that Neil3 was shown to be a functional DNA glycosylase having a different substrate specificity and unusual structural features compared with other Fpg/Nei homologs. Although the biological functions of Neil3 still remain an enigma, this review highlights recent biochemical and structural data that may ultimately shed light on its biological role.

Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA.

Within the base excision repair (BER) pathway, the DNA N-glycosylases are responsible for locating and removing the majority of oxidative base damages. Endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families: the helix-hairpin-helix (HhH) superfamily and the Fpg/Nei family. The search mechanisms employed by these two families of glycosylases were examined using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with YOYO-1 stained λ-DNA molecules suspended between 5 µm silica beads. The HhH and Fpg/Nei families were found to have a similar diffusive search mechanism described as a continuum of motion, in keeping with rotational diffusion along the DNA molecule ranging from slow, sub-diffusive to faster, unrestricted diffusion. The search mechanism for an Fpg variant, F111A, lacking a phenylalanine wedge residue no longer displayed slow, sub-diffusive motion compared to wild type, suggesting that Fpg base interrogation may be accomplished by Phe(111) insertion.

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo.

To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino- 5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Delta324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.

Molecular radiobiology and the origins of the base excision repair pathway: an historical perspective.

PURPOSE: To demonstrate how the search by the Molecular Radiobiologists for enzymes that could recognize and remove DNA damage produced by ionizing radiation was intertwined with the development of the Base Excision Repair pathway. CONCLUSION: The Base Excision Repair pathway repairs the vast majority of radiation-induced DNA damages including base damages, alkali labile lesions, and single strand breaks. It turns out that Base Excision Repair actually evolved to repair some thirty to forty thousand endogenous lesions formed in each of our cells every day. Thus, this process is extremely efficient and accordingly, at relatively low doses of radiation, the single lesions repaired by base excision repair result in few lethal or mutagenic events. This efficiency is a double-edged sword since ionizing radiation-induced hydroxyl radicals produced along the radiation track form both bistranded and tandem clustered lesions in DNA. These damages are recognized by the efficient Base Excision Repair enzymes, which, during attempted repair, lead to double strand breaks and/or multiple lesions that can collapse replication forks. Double strand breaks and other complex or clustered lesions formed by ionizing radiation present distinct challenges to DNA repair systems compared to the relative ease and efficiency by which isolated lesions are repaired.