Base Excision Repair Glycosylases Research Articles

Cellular Repair of Synthetic Analogs of Oxidative DNA Damage Reveals a Key Structure-Activity Relationship of the Cancer-Associated MUTYH DNA Repair Glycosylase.

The base excision repair glycosylase MUTYH prevents mutations associated with the oxidatively damaged base, 8-oxo-7,8-dihydroguanine (OG), by removing undamaged misincorporated adenines from OG:A mispairs. Defects in OG:A repair in individuals with inherited MUTYH variants are correlated with the colorectal cancer predisposition syndrome known as MUTYH-associated polyposis (MAP). Herein, we reveal key structural features of OG required for efficient repair by human MUTYH using structure-activity relationships (SAR). We developed a GFP-based plasmid reporter assay to define SAR with synthetically generated OG analogs in human cell lines. Cellular repair results were compared with kinetic parameters measured by adenine glycosylase assays in vitro. Our results show substrates lacking the 2-amino group of OG, such as 8OI:A (8OI = 8-oxoinosine), are not repaired in cells, despite being excellent substrates in in vitro adenine glycosylase assays, new evidence that the search and detection steps are critical factors in cellular MUTYH repair functionality. Surprisingly, modification of the O8/N7H of OG, which is the distinguishing feature of OG relative to G, was tolerated in both MUTYH-mediated cellular repair and in vitro adenine glycosylase activity. The lack of sensitivity to alterations at the O8/N7H in the SAR of MUTYH substrates is distinct from previous work with bacterial MutY, indicating that the human enzyme is much less stringent in its lesion verification. Our results imply that the human protein relies almost exclusively on detection of the unique major groove position of the 2-amino group of OG within OGsyn:Aanti mispairs to select contextually incorrect adenines for excision and thereby thwart mutagenesis. These results predict that MUTYH variants that exhibit deficiencies in OG:A detection will be severely compromised in a cellular setting. Moreover, the reliance of MUTYH on the interaction with the OG 2-amino group suggests that disrupting this interaction with small molecules may provide a strategy to develop potent and selective MUTYH inhibitors.

Novel Processes Associated with the Repair of Interstrand Cross-Links Derived from Abasic Sites in Duplex DNA: Roles for the Base Excision Repair Glycosylase NEIL3 and the SRAP Protein HMCES.

Recent studies have defined a novel pathway for the repair of interstrand cross-links derived from the reaction of an adenine residue with an apurinic/apyrimidinic (AP) site on the opposing strand of DNA (dA-AP ICL). Stalling of a replication fork at the dA-AP ICL triggers TRAIP-dependent ubiquitylation of the CMG helicase that recruits the base excision repair glycosylase NEIL3 to the lesion. NEIL3 unhooks the dA-AP ICL to regenerate the native adenine residue on one strand and an AP site on the other strand. Covalent capture of the abasic site by the SRAP protein HMCES protects against genomic instability that would result from cleavage of the abasic site in the context of single-stranded DNA at the replication fork. After repair synthesis moves the HMCES-AP adduct into the context of double-stranded DNA, the DNA-protein cross-link is resolved by a nonproteolytic mechanism involving dissociation of thiazolidine attachment. The AP site in duplex DNA is then repaired by the base excision repair pathway.

Construction of bidirectional strand displacement-driven three-dimensional DNA walkers for single-molecule monitoring of multiple DNA glycosylases

Human single-stranded-selective monofunctional uracil glycosylase (hSMUG1) and alkyladenine glycosylase (hAAG) are important base-excision repair (BER) glycosylases responsible for deamination and alkylation repair. Their dysfunctional activities have strong association with various multifactorial diseases and cancers. Herein, by integrating three-dimensional (3D) DNA walker with single-molecule technique, we construct bidirectional strand displacement-driven 3D DNA walkers for single-molecule monitoring of multiple DNA glycosylases. In the presence of hSMUG1 and hAAG, the damaged U and I in bifunctional dumbbell probe are removed by APE1, activating bidirectional self-priming strand displacement amplification (SP-SDA) to produce two trigger DNAs. Trigger DNAs hybridize with Cy5/Alexa Fluor 488 signal probes and subsequently serve as the walker DNAs to induce cyclic APE1-powered cleavage of two signal probes, liberating abundant Cy5 and Alexa Fluor 488 from AuNPs. Due to high precision of intracellular BER mechanisms, high efficiency of bidirectional SP-SDA-directed 3D DNA walkers and ultrahigh signal-to-noise ratio of single-molecule imaging, this nanosensor exhibits a detection limit of 8.14 × 10−10 U/μL for hSMUG1 and 4.50 × 10−9 U/μL for hAAG. Importantly, it can measure kinetic parameters, identify potential inhibitors, discriminate cancer from normal cells, and quantify glycosylases activities in various cancer cells at single-cell level, with promising potentials in biomedical and clinical applications.

Direct hOGG1-Myc interactions inhibit hOGG1 catalytic activity and recruit Myc to its promoters under oxidative stress

The base excision repair (BER) glycosylase hOGG1 (human oxoguanine glycosylase 1) is responsible for repairing oxidative lesions in the genome, in particular oxidised guanine bases (oxoG). In addition, a role of hOGG1 in transcription regulation by recruitment of various transcription factors has been reported. Here, we demonstrate direct interactions between hOGG1 and the medically important oncogene transcription factor Myc that is involved in transcription initiation of a large number of genes including inflammatory genes. Using single molecule atomic force microscopy (AFM), we reveal recruitment of Myc to its E-box promoter recognition sequence by hOGG1 specifically under oxidative stress conditions, and conformational changes in hOGG1-Myc complexes at oxoG lesions that suggest loading of Myc at oxoG lesions by hOGG1. Importantly, our data show suppression of hOGG1 catalytic activity in oxoG repair by Myc. Furthermore, mutational analyses implicate the C28 residue in hOGG1 in oxidation induced protein dimerisation and suggest a role of hOGG1 dimerisation under oxidising conditions in hOGG1-Myc interactions. From our data we develop a mechanistic model for Myc recruitment by hOGG1 under oxidising, inflammatory conditions, which may be responsible for the observed enhanced gene expression of Myc target genes.

Histone variants H3.3 and H2A.Z/H3.3 facilitate excision of uracil from nucleosome core particles

At the most fundamental level of chromatin organization, DNA is packaged as nucleosome core particles (NCPs) where DNA is wound around a core of histone proteins. This ubiquitous sequestration of DNA within NCPs presents a significant barrier to many biological processes, including DNA repair. We previously demonstrated that histone variants from the H2A family facilitate excision of uracil (U) lesions by DNA base excision repair (BER) glycosylases. Here, we consider how the histone variant H3.3 and double-variant H2A.Z/H3.3 modulate the BER enzymes uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1). Using an NCP model system with U:G base pairs at a wide variety of geometric positions we generate the global repair profile for both glycosylases. Enhanced excision of U by UDG and SMUG1 is observed with the H3.3 variant. We demonstrate that these H3.3-containing NCPs form two species: (1) octasomes, which contain the full complement of eight histone proteins and (2) hexasomes which are sub-nucleosomal particles that contain six histones. Both the octasome and hexasome species facilitate excision activity of UDG and SMUG1, with the largest impacts observed at sterically-occluded lesion sites and in terminal regions of DNA of the hexasome that do not closely interact with histones. For the double-variant H2A.Z/H3.3 NCPs, which exist as octasomes, the global repair profile reveals that UDG but not SMUG1 has increased U excision activity. The enhanced glycosylase activity reveals potential functions for these histone variants to facilitate BER in packaged DNA and contributes to our understanding of DNA repair in chromatin and its significance regarding mutagenesis and genomic integrity.

Oxidative Damage in Sporadic Colorectal Cancer: Molecular Mapping of Base Excision Repair Glycosylases MUTYH and hOGG1 in Colorectal Cancer Patients.

Oxidative stress, oxidative DNA damage and resulting mutations play a role in colorectal carcinogenesis. Impaired equilibrium between DNA damage formation, antioxidant status, and DNA repair capacity is responsible for the accumulation of genetic mutations and genomic instability. The lesion-specific DNA glycosylases, e.g., hOGG1 and MUTYH, initiate the repair of oxidative DNA damage. Hereditary syndromes (MUTYH-associated polyposis, NTHL1-associated tumor syndrome) with germline mutations causing a loss-of-function in base excision repair glycosylases, serve as straight forward evidence on the role of oxidative DNA damage and its repair. Altered or inhibited function of above glycosylases result in an accumulation of oxidative DNA damage and contribute to the adenoma-adenocarcinoma transition. Oxidative DNA damage, unless repaired, often gives rise G:C > T:A mutations in tumor suppressor genes and proto-oncogenes with subsequent occurrence of chromosomal copy-neutral loss of heterozygosity. For instance, G>T transversions in position c.34 of a KRAS gene serves as a pre-screening tool for MUTYH-associated polyposis diagnosis. Since sporadic colorectal cancer represents more complex and heterogenous disease, the situation is more complicated. In the present study we focused on the roles of base excision repair glycosylases (hOGG1, MUTYH) in colorectal cancer patients by investigating tumor and adjacent mucosa tissues. Although we found downregulation of both glycosylases and significantly lower expression of hOGG1 in tumor tissues, accompanied with G>T mutations in KRAS gene, oxidative DNA damage and its repair cannot solely explain the onset of sporadic colorectal cancer. In this respect, other factors (especially microenvironment) per se or in combination with oxidative DNA damage warrant further attention. Base excision repair characteristics determined in colorectal cancer tissues and their association with disease prognosis have been discussed as well.

Breakage of cytoplasmic chromosomes by pathological DNA base excision repair.

Chromothripsis is a catastrophic mutational process that promotes tumorigenesis and causes congenital disease1-4. Chromothripsis originates from aberrations of nuclei called micronuclei or chromosome bridges5-8. These structures are associated with fragile nuclear envelopes that spontaneously rupture9,10, leading to DNA damage when chromatin is exposed to the interphase cytoplasm. Here we identify a mechanism explaining a major fraction of this DNA damage. Micronuclei accumulate large amounts of RNA-DNA hybrids, which are edited by adenine deaminases acting on RNA (ADAR enzymes) to generate deoxyinosine. Deoxyinosine is then converted into abasic sites by a DNA base excision repair (BER) glycosylase, N-methyl-purine DNA glycosylase11,12 (MPG). These abasic sites are cleaved by the BER endonuclease, apurinic/apyrimidinic endonuclease12 (APE1), creating single-stranded DNA nicks that can be converted to DNA double strand breaks by DNA replication or when closely spaced nicks occur on opposite strands13,14. This model predicts that MPG should be able to remove the deoxyinosine base from the DNA strand of RNA-DNA hybrids, which we demonstrate using purified proteins and oligonucleotide substrates. These findings identify a mechanism for fragmentation of micronuclear chromosomes, an important step in generating chromothripsis. Rather than breaking any normal chromosome, we propose that the eukaryotic cytoplasm only damages chromosomes with pre-existing defects such as the DNA base abnormality described here.

Unexpected Complexity in the Products Arising from NaOH-, Heat-, Amine-, and Glycosylase-Induced Strand Cleavage at an Abasic Site in DNA.

Hydrolytic loss of nucleobases from the deoxyribose backbone of DNA is one of the most common unavoidable types of damage in synthetic and cellular DNA. The reaction generates abasic sites in DNA, and it is important to understand the properties of these lesions. The acidic nature of the α-protons of the ring-opened abasic aldehyde residue facilitates the β-elimination of the 3'-phosphoryl group. This reaction is expected to generate a DNA strand break with a phosphoryl group on the 5'-terminus and a trans-α,β-unsaturated aldehyde residue on the 3'-terminus; however, a handful of studies have identified noncanonical sugar remnants on the 3'-terminus, suggesting that the products arising from strand cleavage at apurinic/apyrimidinic sites in DNA may be more complex than commonly thought. We characterized the strand cleavage induced by the treatment of an abasic site-containing DNA oligonucleotide with heat, NaOH, piperidine, spermine, and the base excision repair glycosylases Fpg and Endo III. The results showed that under multiple conditions, cleavage at an abasic site in a DNA oligomer generated noncanonical sugar remnants including cis-α,β-unsaturated aldehyde, 2-deoxyribose, and 3-thio-2,3-dideoxyribose products on the 3'-terminus of the strand break.

Oxidative stress-mediated epigenetic regulation by G-quadruplexes.

Many cancer-associated genes are regulated by guanine (G)-rich sequences that are capable of refolding from the canonical duplex structure to an intrastrand G-quadruplex. These same sequences are sensitive to oxidative damage that is repaired by the base excision repair glycosylases OGG1 and NEIL1–3. We describe studies indicating that oxidation of a guanosine base in a gene promoter G-quadruplex can lead to up- and downregulation of gene expression that is location dependent and involves the base excision repair pathway in which the first intermediate, an apurinic (AP) site, plays a key role mediated by AP endonuclease 1 (APE1/REF1). The nuclease activity of APE1 is paused at a G-quadruplex, while the REF1 capacity of this protein engages activating transcription factors such as HIF-1α, AP-1 and p53. The mechanism has been probed by in vitro biophysical studies, whole-genome approaches and reporter plasmids in cellulo. Replacement of promoter elements by a G-quadruplex sequence usually led to upregulation, but depending on the strand and precise location, examples of downregulation were also found. The impact of oxidative stress-mediated lesions in the G-rich sequence enhanced the effect, whether it was positive or negative.

Genetic variations in 3'UTRs of SMUG1 and NEIL2 genes modulate breast cancer risk, survival and therapy response.

Breast cancer (BC) is the most frequent malignancy in women accounting for approximately 2 million new cases worldwide annually. Several genetic, epigenetic and environmental factors are known to be involved in BC development and progression, including alterations in post-transcriptional gene regulation mediated by microRNAs (miRNAs). Single nucleotide polymorphisms (SNPs) located in miRNA binding sites (miRSNPs) in 3'-untranslated regions of target genes may affect miRNA-binding affinity and consequently modulate gene expression. We have previously reported a significant association of miRSNPs in the SMUG1 and NEIL2 genes with overall survival in colorectal cancer patients. SMUG1 and NEIL2 are DNA glycosylases involved in base excision DNA repair. Assuming that certain genetic traits are common for solid tumours, we have investigated wherever variations in SMUG1 and NEIL2 genes display an association with BC risk, prognosis, and therapy response in a group of 673 BC patients and 675 healthy female controls. Patients with TC genotype of NEIL2 rs6997097 and receiving only hormonal therapy displayed markedly shorter overall survival (HR = 4.15, 95% CI = 1.7-10.16, P = 0.002) and disease-free survival (HR = 2.56, 95% CI = 1.5-5.7, P = 0.02). Our results suggest that regulation of base excision repair glycosylases operated by miRNAs may modulate the prognosis of hormonally treated BC.

A Novel Role for the DNA Repair Enzyme 8-Oxoguanine DNA Glycosylase in Adipogenesis.

Cells sustain constant oxidative stress from both exogenous and endogenous sources. When unmitigated by antioxidant defenses, reactive oxygen species damage cellular macromolecules, including DNA. Oxidative lesions in both nuclear and mitochondrial DNA are repaired via the base excision repair (BER) pathway, initiated by DNA glycosylases. We have previously demonstrated that the BER glycosylase 8-oxoguanine DNA glycosylase (OGG1) plays a novel role in body weight maintenance and regulation of adiposity. Specifically, mice lacking OGG1 (Ogg1−/−) are prone to increased fat accumulation with age and consumption of hypercaloric diets. Conversely, transgenic animals with mitochondrially-targeted overexpression of OGG1 (Ogg1Tg) are resistant to age- and diet-induced obesity. Given these phenotypes of altered adiposity in the context of OGG1 genotype, we sought to determine if OGG1 plays a cell-intrinsic role in adipocyte maturation and lipid accumulation. Here, we report that preadipocytes from Ogg1−/− mice differentiate more efficiently and accumulate more lipids than those from wild-type animals. Conversely, OGG1 overexpression significantly blunts adipogenic differentiation and lipid accretion in both pre-adipocytes from Ogg1Tg mice, as well as in 3T3-L1 cells with adenovirus-mediated OGG1 overexpression. Mechanistically, changes in adipogenesis are accompanied by significant alterations in cellular PARylation, corresponding with OGG1 genotype. Specifically, deletion of OGG1 reduces protein PARylation, concomitant with increased adipogenic differentiation, while OGG1 overexpression significantly increases PARylation and blunts adipogenesis. Collectively, these data indicate a novel role for OGG1 in modulating adipocyte differentiation and lipid accretion. These findings have important implications to our knowledge of the fundamental process of adipocyte differentiation, as well as to our understanding of lipid-related diseases such as obesity.

Structure of a Stable Interstrand DNA Cross-Link Involving a β-N-Glycosyl Linkage Between an N6-dA Amino Group and an Abasic Site.

Abasic (AP) sites are one of the most common forms of DNA damage. The deoxyribose ring of AP sites undergoes anomerization between α and β configurations, via an electrophilic aldehyde intermediate. In sequences where an adenine residue is located on the opposing strand and offset 1 nt to the 3' side of the AP site, the nucleophilic N6-dA amino group can react with the AP aldehyde residue to form an interstrand cross-link (ICL). Here, we present an experimentally determined structure of the dA-AP ICL by NMR spectroscopy. The ICL was constructed in the oligodeoxynucleotide 5'-d(T1A2T3G4T5C6T7A8A9G10T11T12C13A14T15C16T17A18)-3':5'-d(T19A20G21A22T23G24A25A26C27X28T29A30G31A32C33A34T35A36)-3' (X=AP site), with the dA-AP ICL forming between A8 and X28. The NMR spectra indicated an ordered structure for the cross-linked DNA duplex and afforded detailed spectroscopic resonance assignments. Structural refinement, using molecular dynamics calculations restrained by NOE data (rMD), revealed the structure of the ICL. In the dA-AP ICL, the 2'-deoxyribosyl ring of the AP site was ring-closed and in the β configuration. Juxtapositioning the N6-dA amino group and the aldehydic C1 of the AP site within bonding distance while simultaneously maintaining two flanking unpaired A9 and T29 bases stacked within the DNA is accomplished by the unwinding of the DNA at the ICL. The structural data is discussed in the context of recent studies describing the replication-dependent unhooking of the dA-AP ICL by the base excision repair glycosylase NEIL3.

Correlation between Charge Transport and Base Excision Repair in the MutY-DNA Glycosylase.

Experimental evidence suggests that DNA-mediated redox signaling between high-potential [Fe4S4] proteins is relevant to DNA replication and repair processes, and protein-mediated charge transfer (CT) between [Fe4S4] clusters and nucleic acids is a fundamental process of the signaling and repair mechanisms. We analyzed the dominant CT pathways in the base excision repair glycosylase MutY using molecular dynamics simulations and hole hopping pathway analysis. We find that the adenine nucleobase of the mismatched A·oxoG DNA base pair facilitates [Fe4S4]-DNA CT prior to adenine excision by MutY. We also find that the R153L mutation in MutY (linked to colorectal adenomatous polyposis) influences the dominant [Fe4S4]-DNA CT pathways and appreciably decreases their effective CT rates.

Relationship between MUTYH, OGG1 and BRCA1 mutations and mRNA expression in breast and ovarian cancer predisposition

The aetiology of breast and ovarian cancer (BC/OC) is multi-factorial. At present, the involvement of base excision repair (BER) glycosylases (MUTYH and OGG1) in BC/OC predisposition is controversial. The present study investigated whether germline mutation status and mRNA expression of two BER genes, MUTHY and OGG1, were correlated with BRCA1 in 59 patients with BC/OC and 50 matched population controls. In addition, to evaluate the relationship between MUTYH, OGG1 and BRCA1, their possible mutual modulation and correlation among mutational spectrum, gene expression and demographic characteristics were evaluated. The results identified 18 MUTYH and OGG1 variants, of which 4 were novel (2 MUTYH and 2 OGG1) in 44 of the 59 patients. In addition, two pathogenic mutations were identified: OGG1 p.Arg46Gln, detected in a patient with BC and a family history of cancer, and MUTYH p.Val234Gly in a patient with OC, also with a family history of cancer. A significant reduced transcript expression in MUTYH was observed (P=0.033) in cases, and in association with the presence of rare variants in the same gene (P=0.030). A significant correlation in the expression of the two BER genes was observed in cases (P=0.004), whereas OGG1 and BRCA1 was significantly correlated in cases (P=0.001) compared with controls (P=0.010). The results of the present study indicated that the relationship among mutational spectrum, gene expression and demographic characteristics may improve the genetic diagnosis and primary prevention of at-risk individuals belonging to families with reduced mRNA expression, regardless of mutation presence.

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.

2'-Fluorinated Hydantoins as Chemical Biology Tools for Base Excision Repair Glycosylases.

The guanine oxidation products, 5-guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), are mutagenic and toxic base lesions that are removed by Fpg, Nei, and the Nei-like (NEIL) glycosylases as the first step in base excision repair (BER). The hydantoins are excellent substrates for the NEIL glycosylases in a variety of DNA contexts beyond canonical duplex DNA, implicating the potential impact of repair activity on a multitude of cellular processes. In order to prepare stable derivatives as chemical biology tools, oligonucleotides containing fluorine at the 2'-position of the sugar of 8-oxo-7,8-dihydro-2'-deoxyguanosine2'-F-OG) were synthesized in ribo and arabino configuration. Selective oxidation of 2'-F-OG within a DNA oligonucleotide provided the corresponding 2'-F-Gh or 2'-F-Sp containing DNA. The 2'-F-hydantoins in duplex DNA were found to be highly resistant to the glycosylase activity of Fpg and NEIL1 compared to the unmodified lesion substrates. Surprisingly, however, some glycosylase-mediated base removal from both the 2'-F-ribo- and 2'-F-arabinohydantoin duplex DNA was observed. Notably, the associated β-lyase strand scission reaction of the 2'-F-arabinohydantoins was inhibited such that the glycosylases were "stalled" at the Schiff-base intermediate. Fpg and NEIL1 showed high affinity for the 2'-F-Gh duplexes in both ribo and arabino configurations. However, binding affinity assessed using catalytically inactive variants of Fpg and NEIL1 indicated higher affinity for the 2'-F-riboGh-containing duplexes. The distinct features of glycosylase processing of 2'-F-ribohydantoins and 2'-F-arabinohydantoins illustrate their utility to reveal structural insight into damage recognition and excision by NEIL and related glycosylases and provide opportunities for delineating the impact of lesion formation and repair in cells.

Evolution of Base Excision Repair in Entamoeba histolytica is shaped by gene loss, gene duplication, and lateral gene transfer

During its life cycle, the protist parasite Entamoeba histolytica encounters reactive oxygen and nitrogen species that alter its genome. Base excision repair (BER) is one of the most important pathways for the repair of DNA base lesions. Analysis of the E. histolytica genome revealed the presence of most of the BER components. Surprisingly, this included a gene encoding an apurinic/apyrimidinic (AP) endonuclease that previous studies had assumed was absent. Indeed, our analysis showed that the genome of E. histolytica harbors the necessary genes needed for both short and long-patch BER sub-pathways. These genes include DNA polymerases with predicted 5′-dRP lyase and strand-displacement activities and a sole DNA ligase. A distinct feature of the E. histolytica genome is the lack of several key damage-specific BER glycosylases, such as OGG1/MutM, MDB4, Mag1, MPG, SMUG, and TDG. Our evolutionary analysis indicates that several E. histolytica DNA glycosylases were acquired by lateral gene transfer (LGT). The genes that encode for MutY, AlkD, and UDG (Family VI) are included among these cases. Endonuclease III and UNG (family I) are the only DNA glycosylases with a eukaryotic origin in E. histolytica. A gene encoding a MutT 8-oxodGTPase was also identified that was acquired by LGT. The mixed composition of BER genes as a DNA metabolic pathway shaped by LGT in E. histolytica indicates that LGT plays a major role in the evolution of this eukaryote. Sequence and structural prediction of E. histolytica DNA glycosylases, as well as MutT, suggest that the E. histolytica DNA repair proteins evolved to harbor structural modifications that may confer unique biochemical features needed for the biology of this parasite.

Targeting Base Excision Repair Glycosylases with DNA Containing Transition State Mimics Prepared via Click Chemistry.

DNA glycosylases of the base excision repair (BER) pathway are front-line defenders in removing compromising modifications of the DNA nucleobases. Aberrantly modified nucleobases mediate genomic mutations and inhibit DNA replication leading to adverse health consequences such as cancer, neurological diseases, and aging. In an effort to develop high-affinity transition state (TS) analogues as chemical biology probes for DNA glycosylases, oligonucleotides containing a propargyl-modified pyrrolidine TS mimic nucleotide were synthesized. A small library of TS mimic-containing oligonucleotides was generated using a structurally diverse set of five azides via copper(I)-catalyzed azide-alkyne cycloaddition "click" chemistry. The relative affinity ( Kd) was evaluated for BER glycosylases Escherichia coli MutY, bacterial formamidopyrimidine glycosylase (Fpg), and human OG glycosylase 1 (hOGG1) with the library of TS mimic DNA duplexes. All of the BER glycosylases were found to exhibit extremely high affinities (approximately picomolar Kd values) for the TS mimics. However, binding preferences, distinct for each glycosylase, for the TS mimic library members were observed, suggesting different modes of binding and transition state stabilization among the three glycosylases. Fpg bound all of the TS mimics with exceptionally high affinities, while the MutY binding affinity correlated inversely with the size of the appended moiety. Of note, we identified one member of the small TS mimic library that exhibited a particularly high affinity for hOGG1. These results strongly support the use of the propargyl-TS mimic oligonucleotides and elaboration via click chemistry in screening and identification of high-affinity ligands for BER glycosylases of interest.

The Zinc Linchpin Motif in the DNA Repair Glycosylase MUTYH: Identifying the Zn2+ Ligands and Roles in Damage Recognition and Repair.

The DNA base excision repair (BER) glycosylase MUTYH prevents DNA mutations by catalyzing adenine (A) excision from inappropriately formed 8-oxoguanine (8-oxoG):A mismatches. The importance of this mutation suppression activity in tumor suppressor genes is underscored by the association of inherited variants of MUTYH with colorectal polyposis in a hereditary colorectal cancer syndrome known as MUTYH-associated polyposis, or MAP. Many of the MAP variants encompass amino acid changes that occur at positions surrounding the two-metal cofactor-binding sites of MUTYH. One of these cofactors, found in nearly all MUTYH orthologs, is a [4Fe-4S]2+ cluster coordinated by four Cys residues located in the N-terminal catalytic domain. We recently uncovered a second functionally relevant metal cofactor site present only in higher eukaryotic MUTYH orthologs: a Zn2+ ion coordinated by three Cys residues located within the extended interdomain connector (IDC) region of MUTYH that connects the N-terminal adenine excision and C-terminal 8-oxoG recognition domains. In this work, we identified a candidate for the fourth Zn2+ coordinating ligand using a combination of bioinformatics and computational modeling. In addition, using in vitro enzyme activity assays, fluorescence polarization DNA binding assays, circular dichroism spectroscopy, and cell-based rifampicin resistance assays, the functional impact of reduced Zn2+ chelation was evaluated. Taken together, these results illustrate the critical role that the "Zn2+ linchpin motif" plays in MUTYH repair activity by providing for proper engagement of the functional domains on the 8-oxoG:A mismatch required for base excision catalysis. The functional importance of the Zn2+ linchpin also suggests that adjacent MAP variants or exposure to environmental chemicals may compromise Zn2+ coordination, and ability of MUTYH to prevent disease.

Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements.

A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S]2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease.