The human DNA base excision repair enzyme MUTYH (MutY homolog DNA glycosylase) excises undamaged adenine that has been misincorporated opposite the oxidatively damaged 8-oxoG, preventing transversion ...mutations and serving as an important defense against the deleterious effects of this damage. Mutations in the MUTYH gene predispose patients to MUTYH-associated polyposis and colorectal cancer, and MUTYH expression has been documented as a biomarker for pancreatic cancer. Measuring MUTYH activity is therefore critical for evaluating and diagnosing disease states as well as for testing this enzyme as a potential therapeutic target. However, current methods for measuring MUTYH activity rely on indirect electrophoresis and radioactivity assays, which are difficult to implement in biological and clinical settings. Herein, we synthesize and identify novel fluorescent adenine derivatives that can act as direct substrates for excision by MUTYH as well as bacterial MutY. When incorporated into synthetic DNAs, the resulting fluorescently modified adenine-release turn-on (FMART) probes report on enzymatic base excision activity in real time, both in vitro and in mammalian cells and human blood. We also employ the probes to identify several promising small-molecule modulators of MUTYH by employing FMART probes for in vitro screening.
Conspectus Base excision repair (BER) enzymes are genomic superheroes that stealthily and accurately identify and remove chemically modified DNA bases. DNA base modifications erode the informational ...content of DNA and underlie many disease phenotypes, most conspicuously, cancer. The “OG” of oxidative base damage, 8-oxo-7,8-dihydroguanine (OG), is particularly insidious due to its miscoding ability that leads to the formation of rare, pro-mutagenic OG:A mismatches. Thwarting mutagenesis relies on the capture of OG:A mismatches prior to DNA replication and removal of the mis-inserted adenine by MutY glycosylases to initiate BER. The threat of OG and the importance of its repair are underscored by the association between inherited dysfunctional variants of the MutY human homologue (MUTYH) and colorectal cancer, known as MUTYH-associated polyposis (MAP). Our functional studies of the two founder MUTYH variants revealed that both have compromised activity and a reduced affinity for OG:A mismatches. Indeed, these studies underscored the challenge of the recognition of OG:A mismatches that are only subtly structurally different than T:A base pairs. Since the original discovery of MAP, many MUTYH variants have been reported, with most considered to be “variants of uncertain significance.” To reveal features associated with damage recognition and adenine excision by MutY and MUTYH, we have developed a multipronged chemical biology approach combining enzyme kinetics, X-ray crystallography, single-molecule visualization, and cellular repair assays. In this review, we highlight recent work in our laboratory where we defined MutY structure–activity relationship (SAR) studies using synthetic analogs of OG and A in cellular and in vitro assays. Our studies revealed the 2-amino group of OG as the key distinguishing feature of OG:A mismatches. Indeed, the unique position of the 2-amino group in the major groove of OG syn :A anti mismatches provides a means for its rapid detection among a large excess of highly abundant and structurally similar canonical base pairs. Furthermore, site-directed mutagenesis and structural analysis showed that a conserved C-terminal domain β-hairpin “FSH’’ loop is critical for OG recognition with the “His” serving as the lesion detector. Notably, MUTYH variants located within and near the FSH loop have been associated with different forms of cancer. Uncovering the role(s) of this loop in lesion recognition provided a detailed understanding of the search and repair process of MutY. Such insights are also useful to identify mutational hotspots and pathogenic variants, which may improve the ability of physicians to diagnose the likelihood of disease onset and prognosis. The critical importance of the “FSH” loop in lesion detection suggests that it may serve as a unique locus for targeting probes or inhibitors of MutY/MUTYH to provide new chemical biology tools and avenues for therapeutic development.
UV-DDB, a key protein in human global nucleotide excision repair (NER), binds avidly to abasic sites and 8-oxo-guanine (8-oxoG), suggesting a noncanonical role in base excision repair (BER). We ...investigated whether UV-DDB can stimulate BER for these two common forms of DNA damage, 8-oxoG and abasic sites, which are repaired by 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease (APE1), respectively. UV-DDB increased both OGG1 and APE1 strand cleavage and stimulated subsequent DNA polymerase β-gap filling activity by 30-fold. Single-molecule real-time imaging revealed that UV-DDB forms transient complexes with OGG1 or APE1, facilitating their dissociation from DNA. Furthermore, UV-DDB moves to sites of 8-oxoG repair in cells, and UV-DDB depletion sensitizes cells to oxidative DNA damage. We propose that UV-DDB is a general sensor of DNA damage in both NER and BER pathways, facilitating damage recognition in the context of chromatin.
Abstract
The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the ...removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4–5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.
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•DNA glycosylases remove damaged bases, initiating BER, but mechanisms for many are poorly defined.•MBD4, of the HhH superfamily, repairs mutagenic G·T mispairs arising from ...5-methylcytosine deamination.•New structures of enzyme-DNA complexes, at three stages of catalysis, illuminate the MBD4 mechanism.•Our structural and biochemical findings inform the role of a catalytic Asp conserved in HhH glycosylases.•Detailed snapshots of metal-bound HhH motifs inform how these ubiquitous elements mediate DNA binding.
DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.
The reactivity of DNA nucleobases towards oxidants and alkylators lends itself to the probability of introducing errors and mutations into the genetic code. Damaged DNA bases are removed from the ...genome by an arsenal of DNA glycosylases that initiate the base excision repair (BER) process. The mechanism by which glycosylases locate their target damaged base is one of the key questions in the field since it is estimated that a single glycosylase must interrogate between 10,000 – 100,000 normal bases before it finds its target.MutY is an unusual DNA glycosylase that excises undamaged but miscoding adenine bases that are misincorporated opposite 8-oxo-7,8-dihydroguanine (OG), arising from the oxidation of guanine. The indispensable role of MutY in maintaining genomic integrity is evidenced by its conservation across all domains of life. In humans, inheritance of functionally deficient variants of MUTYH are associated with a colorectal cancer predisposition syndrome, known as MUTYH associated polyposis or MAP, that is characterized by an accumulation of G:C to T:A mutations in the tumor suppressor adenomatous polyposis coli (APC) gene. Indeed, among the ca. 300 variants of MUTYH that have been reported in humans, many are associated with cancers and diseases.In this work, I present a structure activity relationship (SAR) study using substrate analogs to probe the structural requirements from the adenine and OG bases required for recognition and repair. I correlate the in vitro parameters of binding affinity and base excision to the experimental and calculated gas phase acidities of the analogs to evaluate their impact on the enzymatic reaction. These data have shown that within the active site, the enzyme modulates the intrinsic lability of the bases through specific interactions to effect cleavage. A cell-based repair assay revealed that deficiencies in recognition rather than catalysis affect the ability of cellular MutY to repair the modified lesions. The SAR also highlight the possible modifications to the bases that are tolerated by the enzyme and may guide the development of MutY specific probes or inhibitors.Uncovering the process by which OG recognition occurs also involves the identification of the amino acids in MutY that are responsible for the lesion recognition. Using a combination of in vitro and bacterial cell repair assays with single molecule fluorescence microscopy I studied amino acid variants of a conserved C-terminal domain histidine residue to probe a possible recognition role. These studies showed that recognition-deficient variants are incapable of successful repair and are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. Overall, these data suggest that defects in lesion detection of human MutY variants affect overall repair and may prove predictive of early onset MAP. Additionally, characterization of “damage-sensor” residues can lead to the identification of an allosteric site that may be targeted for MutY specific inhibition.Lastly, MutY and its homologs are associated with iron sulfur (Fe-S) clusters that are essential for catalytic activity. Fe-S clusters are fascinating cofactors implicated in the DNA damage location process of glycosylases. Since Fe-S clusters are known to be oxygen sensitive, the presence of ambient oxygen in typical recombinant protein purification strategies leads to less than optimal yields of cluster carrying protein. Therefore, I also discuss the design and development of a novel oxygen-free purification scheme to isolate high yields of Fe-S cluster loaded MutY to enable the complete characterization of their role in DNA damage location.Taken together, the work presented in this dissertation provides new insights into the nuances of lesion recognition and adenine excision by the MutY glycosylase. The results show that damage location and excision is exquisitely specific to the chemical structure of OG:A and occurs through a multi-step process that is tightly regulated. These insights not just further our present understanding of the enzyme, but also open new avenues for exploring the repair and recognition process by MutY/Mutyh.
The DNA glycosylase MutY prevents deleterious mutations resulting from guanine oxidation by recognition and removal of adenine (A) misincorporated opposite 8-oxo-7,8-dihydroguanine (OG). Correct ...identification of OG:A is crucial to prevent improper and detrimental MutY-mediatedadenine excision from G:A or T:A base pairs. Here we present a structure–activity relationship (SAR) study using analogues of A to probe the basis for OG:A specificity of MutY. We correlate observed in vitro MutY activity on A analogue substrates with their experimental and calculated acidities to provide mechanistic insight into the factors influencing MutY base excision efficiency. These data show that H-bonding and electrostatic interactions of the base within the MutY active site modulate the lability of the N-glycosidic bond. A analogues that were not excised from duplex DNA as efficiently as predicted by calculations provided insight into other required structural features, such as steric fit and H-bonding within the active site for proper alignment with MutY catalytic residues. We also determined MutY-mediated repair of A analogues paired with OG within the context of a DNA plasmid in bacteria. Remarkably, the magnitudes of decreased in vitro MutY excision rates with different A analogue duplexes do not correlate with the impact on overall MutY-mediated repair. The feature that most strongly correlated with facile cellular repair was the ability of the A analogues to H-bond with the Hoogsteen face of OG. Notably, base pairing of A with OG uniquely positions the 2-amino group of OG in the major groove and provides a means to indirectly select only these inappropriately placed adenines for excision. This highlights the importance of OG lesion detection for efficient MutY-mediated cellular repair. The A analogue SARs also highlight the types of modifications tolerated by MutY and will guide the development of specific probes and inhibitors of MutY.
MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8-dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. ...Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of in vitro and bacterial cell repair assays with single-molecule fluorescence microscopy to demonstrate that both a C-terminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY (MUTYH) variants may prove predictive of early-onset colorectal cancer known an MUTYH-associated polyposis. Furthermore, unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.
MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8-dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. ...Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of
in vitro
and bacterial cell repair assays with single molecule fluorescence microscopy to demonstrate that both a C-terminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY variants may prove predictive of early onset colorectal cancer known an MUTYH-associated polyposis. Furthermore unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.
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