Nucleotide Excision Repair Machinery Research Articles

Ubiquitin signaling and the proteasome drive human DNA-protein crosslink repair.

DNA-protein crosslinks (DPCs) are large cytotoxic DNA lesions that form following exposure to chemotherapeutic drugs and environmental chemicals. Nucleotide excision repair (NER) and homologous recombination (HR) promote survival following exposure to DPC-inducing agents. However, it is not known how cells recognize DPC lesions, or what mechanisms selectively target DPC lesions to these respective repair pathways. To address these questions, we examined DPC recognition and repair by transfecting a synthetic DPC lesion comprised of the human oxoguanine glycosylase (OGG1) protein crosslinked to double-stranded M13MP18 into human cells. In wild-type cells, this lesion is efficiently repaired, whereas cells deficient in NER can only repair this lesion if an un-damaged homologous donor is co-transfected. Transfected DPC is subject to rapid K63 polyubiquitination. In NER proficient cells, the DPC is subject to K48 polyubiquitination, and is removed via a proteasome-dependent mechanism. In NER-deficient cells, the DNA-conjugated protein is not subject to K48 polyubiquitination. Instead, the K63 tag remains attached, and is only lost when a homologous donor molecule is present. Taken together, these results support a model in which selective addition of polyubiquitin chains to DNA-crosslinked protein leads to selective recruitment of the proteasome and the cellular NER and recombinational DNA repair machinery.

ASH1L-MRG15 methyltransferase deposits H3K4me3 and FACT for damage verification in nucleotide excision repair

To recognize DNA adducts, nucleotide excision repair (NER) deploys the XPC sensor, which detects damage-induced helical distortions, followed by engagement of TFIIH for lesion verification. Accessory players ensure that this factor handover takes place in chromatin where DNA is tightly wrapped around histones. Here, we describe how the histone methyltransferase ASH1L, once activated by MRG15, helps XPC and TFIIH to navigate through chromatin and induce global-genome NER hotspots. Upon UV irradiation, ASH1L adds H3K4me3 all over the genome (except in active gene promoters), thus priming chromatin for XPC relocations from native to damaged DNA. The ASH1L-MRG15 complex further recruits the histone chaperone FACT to DNA lesions. In the absence of ASH1L, MRG15 or FACT, XPC is misplaced and persists on damaged DNA without being able to deliver the lesions to TFIIH. We conclude that ASH1L-MRG15 makes damage verifiable by the NER machinery through the sequential deposition of H3K4me3 and FACT.

TFIIH moonlighting at telomeres.

Although telomeres are essential for chromosome stability, they represent fragile structures in our genome. Telomere shortening occurs during aging in cells lacking telomerase due to the end replication problem. In addition, recent work uncovered that the bulk of telomeric DNA poses severe hurdles for the semiconservative DNA replication machinery, requiring the assistance of an increasing number of specialized factors that prevent accidental telomere loss or damage events. In this issue of Genes & Development, Yang and colleagues (pp. 956-969) discover that TFIIH, a basic component of the PolII transcription initiation and nucleotide excision repair machinery, facilitates telomere replication. TFIIH is recruited to telomeres by the shelterin component TRF1, taking on at telomeres a moonlighting function.

TREX1 degrades the 3' end of the small DNA oligonucleotide products of nucleotide excision repair in human cells.

The nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small, excised damage-containing DNA oligonucleotides (sedDNAs) ∼30 nt in length. How cells process and degrade these byproducts of DNA repair is not known. Using a small scale RNA interference screen in UV-irradiated human cells, we identified TREX1 as a major regulator of sedDNA abundance. Knockdown of TREX1 increased the level of sedDNAs containing the two major UV photoproducts and their association with the NER proteins TFIIH and RPA. Overexpression of wild-type but not nuclease-inactive TREX1 significantly diminished sedDNA levels, and studies with purified recombinant TREX1 showed that the enzyme efficiently degrades DNA located 3′ of the UV photoproduct in the sedDNA. Knockdown or overexpression of TREX1 did not impact the overall rate of UV photoproduct removal from genomic DNA or cell survival, which indicates that TREX1 function in sedDNA degradation does not impact NER efficiency. Taken together, these results indicate a previously unknown role for TREX1 in promoting the degradation of the sedDNA products of the repair reaction. Because TREX1 mutations and inefficient DNA degradation impact inflammatory and immune signaling pathways, the regulation of sedDNA degradation by TREX1 may contribute to photosensitive skin disorders.

Mechanism of action of nucleotide excision repair machinery.

Nucleotide excision repair (NER) is a versatile DNA repair pathway essential for the removal of a broad spectrum of structurally diverse DNA lesions arising from a variety of sources, including UV irradiation and environmental toxins. Although the core factors and basic stages involved in NER have been identified, the mechanisms of the NER machinery are not well understood. This review summarizes our current understanding of the mechanisms and order of assembly in the core global genome (GG-NER) pathway.

XPA is susceptible to proteolytic cleavage by cathepsin L during lysis of quiescent cells

The xeroderma pigmentosum group A (XPA) protein plays an essential role in the removal of UV photoproducts and other bulky lesions from DNA as a component of the nucleotide excision repair (NER) machinery. Using cell lysates prepared from confluent cultures of human cells and from human skin epidermis, we observed an additional XPA antibody-reactive band on immunoblots that was approximately 3–4 kDa smaller than the native, full-length XPA protein. Biochemical studies revealed this smaller molecular weight XPA species to be due to proteolysis at the C-terminus of the protein, which negatively impacted the ability of XPA to interact with the NER protein TFIIH. Further work identified the endopeptidase cathepsin L, which is expressed at higher levels in quiescent cells, as the protease responsible for cleaving XPA during cell lysis. These results suggest that supplementation of lysis buffers with inhibitors of cathepsin L is important to prevent cleavage of XPA during lysis of confluent cells.

The intrinsic ATPase activity of Mycobacteriumtuberculosis UvrC is crucial for its damage-specific DNA incision function.

To ensure genome stability, bacteria have evolved a network of DNA repair mechanisms; among them, the UvrABC-dependent nucleotide excision repair (NER) pathway is essential for the incision of a variety of bulky adducts generated by exogenous chemicals, UV radiation and by-products of cellular metabolism. However, very little is known about the enzymatic properties of Mycobacteriumtuberculosis UvrABC excinuclease complex. Furthermore, the biochemical properties of Escherichiacoli UvrC (EcUvrC) are not well understood (compared to UvrA and UvrB), perhaps due to its limited availability and/or activity instability invitro. In addition, homology modelling of M.tuberculosis UvrC (MtUvrC) revealed the presence of a putative ATP-binding pocket, although its function remains unknown. To elucidate the biochemical properties of UvrC, we constructed and purified wild-type MtUvrC and its eight variants harbouring mutations within the ATP-binding pocket. The data from DNA-binding studies suggest that MtUvrC exhibits high-affinity for duplex DNA containing a bubble or fluorescein-dT moiety, over fluorescein-adducted single-stranded DNA. Most notably, MtUvrC has an intrinsic UvrB-independent ATPase activity, which drives dual incision of the damaged DNA strand. In contrast, EcUvrC is devoid of ATPase activity; however, it retains the ability to bind ATP at levels comparable to that of MtUvrC. The ATPase-deficient variants map to residues lining the MtUvrC ATP-binding pocket. Further analysis of these variants revealed separation of function between ATPase and DNA-binding activities in MtUvrC. Altogether, these findings reveal functional diversity of the bacterial NER machinery and a paradigm for the evolution of a catalytic scaffold in UvrC.

Interplay between BRCA1 and GADD45A and Its Potential for Nucleotide Excision Repair in Breast Cancer Pathogenesis.

A fraction of breast cancer cases are associated with mutations in the BRCA1 (BRCA1 DNA repair associated, breast cancer type 1 susceptibility protein) gene, whose mutated product may disrupt the repair of DNA double-strand breaks as BRCA1 is directly involved in the homologous recombination repair of such DNA damage. However, BRCA1 can stimulate nucleotide excision repair (NER), the most versatile system of DNA repair processing a broad spectrum of substrates and playing an important role in the maintenance of genome stability. NER removes carcinogenic adducts of diol-epoxy derivatives of benzo[α]pyrene that may play a role in breast cancer pathogenesis as their accumulation is observed in breast cancer patients. NER deficiency was postulated to be intrinsic in stage I of sporadic breast cancer. BRCA1 also interacts with GADD45A (growth arrest and DNA damage-inducible protein GADD45 alpha) that may target NER machinery to actively demethylate genome sites in order to change the expression of genes that may be important in breast cancer. Therefore, the interaction between BRCA1 and GADD45 may play a role in breast cancer pathogenesis through the stimulation of NER, increasing the genomic stability, removing carcinogenic adducts, and the local active demethylation of genes important for cancer transformation.

A key interaction with RPA orients XPA in NER complexes.

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD–RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

Detection of the small oligonucleotide products of nucleotide excision repair in UVB-irradiated human skin

UVB radiation results in the formation of potentially mutagenic photoproducts in the DNA of epidermal skin cells. In vitro approaches have demonstrated that the nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small (∼30-nt-long), excised, damage-containing DNA oligonucleotides (sedDNAs). Though this process presumably takes place in human skin exposed to UVB radiation, sedDNAs have not previously been detected in human skin. Using surgically discarded human skin, we have optimized the detection of the sedDNA products of NER from small amounts of human epidermal tissue ex vivo within minutes of UVB exposure and after UVB doses that normally lead to minimal erythema. Moreover, sedDNA generation was inhibited by treatment of skin explants with spironolactone, which depletes the epidermis of the essential NER protein XPB to mimic the skin of xeroderma pigmentosum patients. Time course experiments revealed that a partially degraded form of the sedDNAs could be readily detected even 12 hours following UVB exposure, which indicates that these repair products are relatively stable in human skin epidermis. Together, these data suggest that sedDNA detection may be a useful assay for determining how genetic, environmental, and other factors influence NER activity in human skin epidermis and whether abnormal sedDNA processing contributes to photosensitive skin disorders.

Isolation of a Novel Complex Between Human NER Proteins XPC and XPA

Nucleotide excision repair (NER) is the primary mechanism to remove large covalent DNA lesions resulting from exposure to a wide variety of genotoxic agents. The human NER mechanism is dependent on the coordinated activity of over thirty proteins that build a series of protein machines to repair the damaged DNA. Two proteins, XPC and XPA, play critical roles in NER despite lacking any enzymatic activity. XPC serves as a generic sensor of damaged DNA, marking a site for repair and initiating assembly of the NER machinery. XPA is generally believed to act as a molecular scaffold upon which the NER machines are built. Previous studies have suggested a weak but direct physical interaction between XPC and XPA, and genetic evidence suggests this interaction may aid in recruitment of XPA to potential damage sites in cells. In this study, we identified a novel protein complex between human XPA and the globular transglutaminase-like domain of XPC (XPC-TGD). We employed a chemical cross-linking strategy to isolate protein complexes formed from highly purified recombinant proteins. Complexes were purified by SDS-PAGE and subjected to trypsin proteolysis. MALDI-TOF mass spectrometry analysis identified polypeptide fragments from both the XPA and XPC proteins in tryptic digests of isolated complex samples. These results suggest the XPA may interact with XPC by binding to the XPC-TGD during assembly of the NER machinery. Support or Funding Information Supported by The University of Virginia's College at Wise Fellowship in Natural Sciences (FINS) Undergraduate Research Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantification of DNA Lesions Induced by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol in Mammalian Cells.

Quantitative measurement of DNA adducts in carcinogen-exposed cells provides the information about the frequency of formation and the rate of removal of DNA lesions in vivo, which yields insights into the initial events of mutagenesis. Metabolic activation of tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), leads to pyridyloxobutylation and pyridylhydroxybutylation of DNA. In this study, we employed a highly robust nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS) coupled with the isotope-dilution method for simultaneous quantification of O6-[4-(3-pyridyl)-4-hydroxylbut-1-yl]-2'-deoxyguanosine ( O6-PHBdG) and O2- and O4-[4-(3-pyridyl)-4-hydroxylbut-1-yl]-thymidine ( O2-PHBdT and O4-PHBdT). Cultured mammalian cells were exposed to a model pyridylhydroxybutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALOAc), followed by DNA extraction, enzymatic digestion, and sample enrichment prior to nLC-nESI-MS/MS quantification. Our results demonstrate, for the first time, that O4-PHBdT is quantifiable in cellular DNA and naked DNA upon NNALOAc exposure. We also show that nucleotide excision repair (NER) machinery may counteract the formation of O2-PHBdT and O4-PHBdT, and O6-alkylguanine DNA alkyltransferase (AGT) may be responsible for the repair of O6-PHBdG and O4-PHBdT in mammalian cells. Together, our study provides new knowledge about the occurrence and repair of NNAL-induced DNA lesions in mammalian cells.

P.4.08 Metabolic syndrome, inflammation and oxidative stress as potential risk factors for affective disorders – a twin study

UVB radiation results in the formation of potentially mutagenic photoproducts in the DNA of epidermal skin cells. In vitro approaches have demonstrated that the nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small (∼30-nt-long), excised, damage-containing DNA oligonucleotides (sedDNAs). Though this process presumably takes place in human skin exposed to UVB radiation, sedDNAs have not previously been detected in human skin. Using surgically discarded human skin, we have optimized the detection of the sedDNA products of NER from small amounts of human epidermal tissue ex vivo within minutes of UVB exposure and after UVB doses that normally lead to minimal erythema. Moreover, sedDNA generation was inhibited by treatment of skin explants with spironolactone, which depletes the epidermis of the essential NER protein XPB to mimic the skin of xeroderma pigmentosum patients. Time course experiments revealed that a partially degraded form of the sedDNAs could be readily detected even 12 hours following UVB exposure, which indicates that these repair products are relatively stable in human skin epidermis. Together, these data suggest that sedDNA detection may be a useful assay for determining how genetic, environmental, and other factors influence NER activity in human skin epidermis and whether abnormal sedDNA processing contributes to photosensitive skin disorders.

Alternative excision repair of topoisomerase inhibitor-induced DNA damage

Topoisomerase 1 (Top1) and 2 (Top2) are common targets of anti-cancer drugs, which induce specific DNA lesions that are thought to lead to cell death in tumour cells. Camptothecin blocks DNA religation in the Top1–DNA complex and induces Top1-attached nick DNA lesions (Top1–DNA damage). We propose that nucleotide excision repair (NER) factors, excision repair cross complementing 1 protein-xeroderma pigmentosum group F (ERCC1–XPF) endonuclease, and replication protein A (RPA), participate in the repair of Top1-attached nick DNA lesions. ERCC1–XPF exhibits nuclease activity in the presence of RPA on a 3′-phosphotyrosyl bond nick-containing DNA (3′-Tyr-nick DNA) substrate, which mimics a Top1-attached nick DNA lesion. Moreover, repair of the 3′-Tyr-nick DNA occurred in the presence of NER factors, including ERCC1–XPF, RPA, DNA polymerase delta, flap endonuclease 1 (FEN1) and DNA ligase 1 (LIG1). We also illustrate a repair model for DNA lesions induced by Top2 inhibitor etoposide (VP-16), Top2-attached nick DNA (Top2-DNA damage). FEN1 participates in the cleavage of the 5′-phosphotyrosyl bond nick-containing DNA (5′-Tyr-nick DNA) substrates, which mimic Top2-attached nick DNA lesions. Under DNA repair synthesis conditions, FEN1 efficiently repaired the 5′-Tyr-nick DNA substrates in the presence of other base excision repair (BER) factors, LIG1 and DNA polymerase beta (POLB). Taken together, some of the NER and BER machinery might represent an alternative repair pathway for Top1- and Top2-DNA damage.

Repair protein persistence at DNA lesions characterizes XPF defect with Cockayne syndrome features.

The structure-specific ERCC1-XPF endonuclease plays a key role in DNA damage excision by nucleotide excision repair (NER) and interstrand crosslink repair. Mutations in this complex can either cause xeroderma pigmentosum (XP) or XP combined with Cockayne syndrome (XPCS-complex) or Fanconi anemia. However, most patients carry compound heterozygous mutations, which confounds the dissection of the phenotypic consequences for each of the identified XPF alleles. Here, we analyzed the functional impact of individual pathogenic XPF alleles on NER. We show that XP-causing mutations diminish XPF recruitment to DNA damage and only mildly affect global genome NER. In contrast, an XPCS-complex-specific mutation causes persistent recruitment of XPF and the upstream core NER machinery to DNA damage and severely impairs both global genome and transcription-coupled NER. Remarkably, persistence of NER factors at DNA damage appears to be a common feature of XPCS-complex cells, suggesting that this could be a determining factor contributing to the development of additional developmental and/or neurodegenerative features in XP patients.

A quantitative PCR-based assay reveals that nucleotide excision repair plays a predominant role in the removal of DNA-protein crosslinks from plasmids transfected into mammalian cells

DNA-protein crosslinks (DPCs) are complex DNA lesions that induce mutagenesis and cell death. DPCs are created by common antitumor drugs, reactive oxygen species, and endogenous aldehydes. Since these agents create other types of DNA damage in addition to DPCs, identification of the mechanisms of DPC repair is challenging. In this study, we created plasmid substrates containing site-specific DPC lesions, as well as plasmids harboring lesions that are selectively repaired by the base excision or nucleotide excision repair (NER) pathways. These substrates were transfected into mammalian cells and a quantitative real-time PCR assay employed to study their repair. This assay revealed that DPC lesions were rapidly repaired in wild-type human and Chinese hamster derived cells, as were plasmids harboring an oxoguanine residue (base excision repair substrate) or cholesterol lesion (NER substrate). Interestingly, the DPC substrate was repaired in human cells nearly three times as efficiently as in Chinese hamster cells (>75% vs ∼25% repair at 8 h post-transfection), while there was no significant species-specific difference in the efficiency with which the cholesterol lesion was repaired (∼60% repair). Experiments revealed that both human and hamster cells deficient in NER due to mutations in the xeroderma pigmentosum A or D genes were five to ten-fold less able to repair the cholesterol and DPC lesions than were wild-type control clones, and that both the global genome and transcription-coupled sub-pathways of NER were capable of repairing DPCs. In addition, analyses using this PCR-based assay revealed that a 4 kDa peptide DNA crosslink was repaired nearly twice as efficiently as was a ∼38 kDa DPC, suggesting that proteolytic degradation of crosslinked proteins occurs during DPC repair. These results highlight the utility of this PCR-based assay to study DNA repair and indicate that the NER machinery rapidly and efficiently repairs plasmid DPC lesions in mammalian cells.

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.

Trabectedin Mechanism of Action and Platinum Resistance: Molecular Rationale

Trabectedin presents a complex mode of action affecting key cell biology processes in tumor cells and in the tumor microenvironment. In ovarian cancer patients with a platinum treatment-free interval of 6-12months treated with trabectedin+pegylated liposomal doxorubicin (PLD) or single-agent PLD, and retreated with platinum after relapse, overall survival was significantly prolonged in the trabectedin+PLD group. Mechanisms by which trabectedin restores tumor sensitivity to platinum include its interaction with components of the nucleotide excision repair machinery in tumor cells and inhibition of inflammatory mediators such as IL-6 in the tumor microenvironment. Additionally, BRCA mutations and associated homologous recombination repair deficiency may contribute to enhanced sensitivity to trabectedin observed in BRCA-mutated patients with ovarian cancer.

Synthesis, structure, and biological evaluation of a platinum-carbazole conjugate.

Cisplatin resistance is caused, in part, by the efficient removal of the helix-distorting cisplatin 1,2-intrastrand cross-links by nucleotide excision repair (NER) machinery. To make a platinum-DNA adduct that causes less helical distortion than the cisplatin 1,2-intrastrand adduct, we designed and synthesized a monofunctional platinum-carbazole conjugate (carbazoplatin). The 2.5Å crystal structure of carbazoplatin-DNA adduct revealed both the monoplatination of the N7 of a guanine (G) base and the intercalation into two G:C base pairs, while causing a minor distortion of the DNA helix. A 50-mer dsDNA containing a single carbazoplatin lesion was poorly processed by UvrABC endonuclease, the prokaryotic NER machinery that detects helical distortion and performs dual incision around the lesion. Our cell viability assay indicated that the cytotoxic pathways of carbazoplatin might be different from those of cisplatin; carbazoplatin was 5-8 times more cytotoxic than cisplatin against PANC-1 and MDA-MB-231 cancer cell lines.

Structure of a DNA glycosylase that unhooks interstrand cross-links

DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.