Human Nucleotide Excision Repair Research Articles

Nucleotide excision repair in humans.

The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process.

Genome Instability in Development and Aging: Insights from Nucleotide Excision Repair in Humans, Mice, and Worms.

DNA damage causally contributes to aging and cancer. Congenital defects in nucleotide excision repair (NER) lead to distinct cancer-prone and premature aging syndromes. The genetics of NER mutations have provided important insights into the distinct consequences of genome instability. Recent work in mice and C. elegans has shed new light on the mechanisms through which developing and aging animals respond to persistent DNA damage. The various NER mouse mutants have served as important disease models for Xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD), while the traceable genetics of C. elegans have allowed the mechanistic delineation of the distinct outcomes of genome instability in metazoan development and aging. Intriguingly, highly conserved longevity assurance mechanisms respond to transcription-blocking DNA lesions in mammals as well as in worms and counteract the detrimental consequences of persistent DNA damage. The insulin-like growth factor signaling (IIS) effector transcription factor DAF-16 could indeed overcome DNA damage-driven developmental growth delay and functional deterioration even when DNA damage persists. Longevity assurance mechanisms might thus delay DNA damage-driven aging by raising the threshold when accumulating DNA damage becomes detrimental for physiological tissue functioning.

Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution.

We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells.

DNA Repair Synthesis and Ligation Affect the Processing of Excised Oligonucleotides Generated by Human Nucleotide Excision Repair

Ultraviolet (UV) photoproducts are removed from genomic DNA by dual incisions in humans in the form of 24- to 32-nucleotide-long oligomers (canonical 30-mers) by the nucleotide excision repair system. How the small, excised, damage-containing DNA oligonucleotides (sedDNAs) are processed in cells following the dual incision event is not known. Here, we demonstrate that sedDNAs are localized to the nucleus in two biochemically distinct forms, which include chromatin-associated, transcription factor II H-bound complexes and more readily solubilized, RPA-bound complexes. Because the nuclear mobility and repair functions of transcription factor II H and RPA are influenced by post-incision gap-filling events, we examined how DNA repair synthesis and DNA ligation affect sedDNA processing. We found that although these gap filling activities are not essential for the dual incision/sedDNA generation event per se, the inhibition of DNA repair synthesis and ligation is associated with a decrease in UV photoproduct removal rate and an accumulation of RPA-sedDNA complexes in the cell. These findings indicate that sedDNA processing and association with repair proteins following the dual incisions may be tightly coordinated with gap filling during nucleotide excision repair in vivo.

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates.

How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.

Nuclear Magnetic Resonance Studies of an N2-Guanine Adduct Derived from the Tumorigen Dibenzo[a,l]pyrene in DNA: Impact of Adduct Stereochemistry, Size, and Local DNA Sequence on Solution Conformations

The dimensions and arrangements of aromatic rings (topology) in adducts derived from the reactions of polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites with DNA influence the distortions and stabilities of double-stranded DNA, and hence their recognition and processing by the human nucleotide excision repair (NER) system. Dibenzo[a,l]pyrene (DB[a,l]P) is a highly tumorigenic six-ring PAH, which contains a nonplanar and aromatic fjord region that is absent in the structurally related bay region five-ring PAH benzo[a]pyrene (B[a]P). The PAH diol epoxide–DNA adducts formed include the stereoisomeric 14S and 14Rtrans-anti-DB[a,l]P-N2-dG and the stereochemically analogous 10S- and 10R-B[a]P-N2-dG (B[a]P-dG) guanine adducts. However, nuclear magnetic resonance (NMR) solution studies of the 14S-DB[a,l]P-N2-dG adduct in DNA have not yet been presented. Here we have investigated the 14S-DB[a,l]P-N2-dG adduct in two different sequence contexts using NMR methods with distance-restrained molecular dynamics simulations. In duplexes with dC opposite the adduct deleted, a well-resolved base-displaced intercalative adduct conformation can be observed. In full duplexes, in contrast to the intercalated 14R stereoisomeric adduct, the bulky DB[a,l]P residue in the 14S adduct is positioned in a greatly widened and distorted minor groove, with significant disruptions and distortions of base pairing at the lesion site and two 5′-side adjacent base pairs. These unique structural features are significantly different from those of the stereochemically analogous but smaller B[a]P-dG adduct. The greater size and different topology of the DB[a,l]P aromatic ring system lead to greater structurally destabilizing DNA distortions that are partially compensated by stabilizing DB[a,l]P-DNA van der Waals interactions, whose combined effects impact the NER response to the adduct. These structural results broaden our understanding of the structure–function relationship in NER.

Structural basis for the recognition of diastereomeric 5′,8-cyclo-2′-deoxypurine lesions by the human nucleotide excision repair system

The hydroxyl radical is a powerful oxidant that generates DNA lesions including the stereoisomeric R and S 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) pairs that have been detected in cellular DNA. Unlike some other oxidatively generated DNA lesions, cdG and cdA are repaired by the human nucleotide excision repair (NER) apparatus. The relative NER efficiencies of all four cyclopurines were measured and compared in identical human HeLa cell extracts for the first time under identical conditions, using identical sequence contexts. The cdA and cdG lesions were excised with similar efficiencies, but the efficiencies for both 5′R cyclopurines were greater by a factor of ∼2 than for the 5′S lesions. Molecular modeling and dynamics simulations have revealed structural and energetic origins of this difference in NER-incision efficiencies. These lesions cause greater DNA backbone distortions and dynamics relative to unmodified DNA in 5′R than in 5′S stereoisomers, producing greater impairment in van der Waals stacking interaction energies in the 5′R cases. The locally impaired stacking interaction energies correlate with relative NER incision efficiencies, and explain these results on a structural basis in terms of differences in dynamic perturbations of the DNA backbone imposed by the R and S covalent 5′,8 bonds.

DNA Repair Synthesis and Ligation Affect the Processing of Excised Oligonucleotides Generated by Human Nucleotide Excision Repair

DNA Repair Synthesis and Ligation Affect the Processing of Excised Oligonucleotides Generated by Human Nucleotide Excision Repair

Highly specific and sensitive method for measuring nucleotide excision repair kinetics of ultraviolet photoproducts in human cells

The nucleotide excision repair pathway removes ultraviolet (UV) photoproducts from the human genome in the form of short oligonucleotides ∼30 nt in length. Because there are limitations to many of the currently available methods for investigating UV photoproduct repair in vivo, we developed a convenient non-radioisotopic method to directly detect DNA excision repair events in human cells. The approach involves extraction of oligonucleotides from UV-irradiated cells, DNA end-labeling with biotin and streptavidin-mediated chemiluminescent detection of the excised UV photoproduct-containing oligonucleotides that are released from the genome during excision repair. Our novel approach is robust, with essentially no signal in the absence of UV or a functional excision repair system. Furthermore, our non-radioisotopic methodology allows for the sensitive detection of excision products within minutes following UV irradiation and does not require additional enrichment steps such as immunoprecipitation. Finally, this technique allows for quantitative measurements of excision repair in human cells. We suggest that the new techniques presented here will be a useful and powerful approach for studying the mechanism of human nucleotide excision repair in vivo.

Free Energy Profiles of Base Flipping in Intercalative Polycyclic Aromatic Hydrocarbon-Damaged DNA Duplexes: Energetic and Structural Relationships to Nucleotide Excision Repair Susceptibility

The crystal structure of Rad4/Rad23, the yeast homolog of the human nucleotide excision repair (NER) lesion recognition factor XPC-RAD23B ( Min , J. H. and Pavletich , N. P. ( 2007 ) Nature 449 , 570 - 575 ) reveals that the lesion-partner base is flipped out of the helix and binds to amino acids of the protein. This suggests the hypothesis that the flipping of this partner base must overcome a free energy barrier, which constitutes one element contributing to changes in the thermodynamic properties induced by the DNA damage and sensed by the recognition protein. We explored this hypothesis by computing complete flipping free energy profiles for two lesions derived from the procarcinogenic polycyclic aromatic hydrocarbons (PAHs), dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P), R-trans-anti-DB[a,l]P-N(6)-dA (R-DB[a,l]P-dA) and R-trans-anti-B[a]P-N(6)-dA (R-B[a]P-dA), and the corresponding unmodified duplex. The DB[a,l]P and B[a]P adducts differ in number and organization of their aromatic rings. We integrate these results with prior profiles for the R-trans-anti-DB[a,l]P-dG adduct ( Zheng , H. et al. ( 2010 ) Chem. Res. Toxicol. 23 , 1868 - 1870 ). All adopt conformational themes involving intercalation of the PAH aromatic ring system into the DNA duplex; however, R-DB[a,l]P-dA and R-B[a]P-dA intercalate from the major groove, while R-DB[a,l]P-dG intercalates from the minor groove. These structural differences produce different computed van der Waals stacking interaction energies between the flipping partner base with the lesion aromatic ring system and adjacent bases; we find that the better the stacking, the higher the relative flipping free energy barrier and hence lower flipping probability. The better relative NER susceptibilities correlate with greater ease of flipping in these three differently intercalated lesions. In addition to partner base flipping, the Rad4/Rad23 crystal structure shows that a protein-β-hairpin, BHD3, intrudes from the major groove side between the DNA strands at the lesion site. We present a molecular modeling study for the R-DB[a,l]P-dG lesion in Rad4/Rad23 showing BHD3 β-hairpin intrusion with lesion eviction, and we hypothesize that lesion steric effects play a role in the recognition of intercalated adducts.

Nucleotide Excision Repair in Human Cells: FATE OF THE EXCISED OLIGONUCLEOTIDE CARRYING DNA DAMAGE IN VIVO

Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.

WT UV-DDB Performs a 3D Search on DNA whereas the XP-E Mutant (K244E DDB2) Mutant Slides

The DNA damage binding protein complex (UV-DDB) recognizes ultraviolet light (UV) induced lesions such as 6-4 photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs) in DNA and initiates human nucleotide excision repair in chromatin. Crystallographic studies have revealed that UV-DDB binds to damaged DNA as a heterodimer of DDB1 and DDB2 on short DNA substrates; however, its oligomeric state on longer, physiologically relevant substrates and on nucleosomes remains undetermined. Additionally, the question of how UV-DDB searches a sea of undamaged chromatin for UV-induced lesions remains unresolved. We assayed purified UV-DDB for binding to a 517 bp UV-irradiated, PCR fragment using atomic force microscopy (AFM). Volume analysis revealed that UV-DDB binds primarily as a dimer of heterodimers to UV damaged DNA, with 19% of these UV-DDB dimers binding simultaneously to two DNA molecules In order to study damage recognition in real time, we used a His-tag on UV-DDB (either DDB1 or DDB2) to conjugate quantum dots(QDs). QD-UV-DDB retained DNA damage binding activity, as assayed by electrophoretic mobility shift assays. To identify the search mode, we have employed an oblique angle fluorescence microscopy setup to track single molecules of QD tagged UV-DDB on UV-damaged DNA tightropes. We have identified that WT UV-DDB employs a 3D search to identify DNA damage, with a long residence time when bound to sites of damage. Consistent with several salt-bridges observed in the co-crystal structure, we have found that the mobility of UV-DDB on DNA is salt dependent. Further, we have assayed the disease causing K244E mutant of DDB2 and observed that the mutant retains DNA binding activity, but slides on DNA compared to the WT. Our results reveal the stoichiometry and search mechanism of UV-DDB in damage surveillance.

Nuclear Magnetic Resonance Solution Structure of an N2-Guanine DNA Adduct Derived from the Potent Tumorigen Dibenzo[a,l]pyrene: Intercalation from the Minor Groove with Ruptured Watson–Crick Base Pairing

The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

The Efficiencies of Damage Recognition and Excision Correlate with Duplex Destabilization Induced by Acetylaminofluorene Adducts in Human Nucleotide Excision Repair

Nucleotide excision repair (NER) removes lesions caused by environmental mutagens or UV light from DNA. A hallmark of NER is the extraordinarily wide substrate specificity, raising the question of how one set of proteins is able to recognize structurally diverse lesions. Two key features of good NER substrates are that they are bulky and thermodynamically destabilize DNA duplexes. To understand what the limiting step in damage recognition in NER is, we set out to test the hypothesis that there is a correlation of the degree of thermodynamic destabilization induced by a lesion, binding affinity to the damage recognition protein XPC-RAD23B, and overall NER efficiency. We chose to use acetylaminofluorene (AAF) and aminofluorene (AF) adducts at the C8 position of guanine in different positions within the NarI (GGCGCC) sequence, as it is known that the structures of the duplexes depend on the position of the lesion in this context. We found that the efficiency of NER and the binding affinity of the damage recognition factor XPC-RAD23B correlated with the thermodynamic destabilization induced by the lesion. Our study is the first systematic analysis correlating these three parameters and supports the idea that initial damage recognition by XPC-RAD23B is a key rate-limiting step in NER.

Repair efficiency of (5′S)-8,5′-cyclo-2′-deoxyguanosine and (5′S)-8,5′-cyclo-2′-deoxyadenosine depends on the complementary base

5′-R and 5′-S diastereoisomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24–32bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N2-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N2-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.

XPF-ERCC1: On the Bubble

In this issue of Structure, Das et al. report the structure of the helix-hairpin-helix dimerization domain of XPF bound to ssDNA. These results provide insight into the architecture of nucleotide excision repair machinery and how it interacts with damaged DNA substrates.

Oxidative Damage Targets Complexes Containing DNA Methyltransferases, SIRT1, and Polycomb Members to Promoter CpG Islands

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an invivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Probing for DNA damage with β-hairpins: Similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[ a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend toward similar relative NER incision efficiencies for ∼65% of these substrates; the other cases deviate mostly by ∼30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER.

Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein

The XPA (Xeroderma pigmentosum A) protein is one of the six core factors of the human nucleotide excision repair system. In this study we show that XPA is a rate-limiting factor in all human cell lines tested, including a normal human fibroblast cell line. The level of XPA is controlled at the transcriptional level by the molecular circadian clock and at the post-translational level by a HECT domain family E3 ubiquitin ligase called HERC2. Stabilization of XPA by downregulation of HERC2 moderately enhances excision repair activity. Conversely, downregulation of XPA by siRNA reduces excision repair activity in proportion to the level of XPA. Ubiquitination and proteolysis of XPA are inhibited by DNA damage that promotes tight association of the protein with chromatin and its dissociation from the HERC2 E3 ligase. Finally, in agreement with a recent report, we find that XPA is post-translationally modified by acetylation. However, contrary to the previous claim, we find that in mouse liver only a small fraction of XPA is acetylated and that downregulation of SIRT1 deacetylase in two human cell lines does not affect the overall repair rate. Collectively, the data reveal that XPA is a limiting factor in excision repair and that its level is coordinately regulated by the circadian clock, the ubiquitin–proteasome system and DNA damage.

Both base excision repair and nucleotide excision repair in humans are influenced by nutritional factors

Lack of reliable assays for DNA repair has largely prevented measurements of DNA repair from being included in human biomonitoring studies. Using newly developed modifications of the comet assay we tested whether a fruit- and antioxidant-rich plant-based intervention could affect base excision repair (BER) and nucleotide excision repair (NER) in a group of 102 male volunteers. BER and NER repair capacities were measured in lymphocytes before and after a dietary intervention lasting 8 weeks. The study had one control group, one group consuming three kiwifruits per day and one group consuming a variety of antioxidant-rich fruits and plant products in addition to their normal diet. DNA strand breaks were reduced following consumption of both kiwifruits (13%, p = 0.05) and antioxidant-rich plant products (20%, p = 0.02). Increased BER (55%, p = 0.01) and reduced NER (-39%, p < 0.01) were observed in the group consuming a wide variety of plant products. Reduced NER was also observed in the kiwifruit group (-38%, p = 0.05), but BER was not affected in this group. Here we have demonstrated that DNA repair is affected by diet and that modified versions of the comet assay can be used to assess activity of different DNA repair pathways in human biomonitoring studies.