NEIL1 Recoding due to RNA Editing Impacts Lesion-Specific Recognition and Excision.

A-to-I RNA editing is widespread in human cells but is uncommon in the coding regions of proteins outside the nervous system. An unusual target for recoding by the adenosine deaminase ADAR1 is the pre-mRNA of the base excision DNA repair enzyme NEIL1 that results in the conversion of a lysine (K) to arginine (R) within the lesion recognition loop and alters substrate specificity. Differences in base removal by unedited (UE, K242) vs edited (Ed, R242) NEIL1 were evaluated using a series of oxidatively modified DNA bases to provide insight into the chemical and structural features of the lesion base that impact isoform-specific repair. We find that UE NEIL1 exhibits higher activity than Ed NEIL1 toward the removal of oxidized pyrimidines, such as thymine glycol, uracil glycol, 5-hydroxyuracil, and 5-hydroxymethyluracil. Gas-phase calculations indicate that the relative rates in excision track with the more stable lactim tautomer and the proton affinity of N3 of the base lesion. These trends support the contribution of tautomerization and N3 protonation in NEIL1 excision catalysis of these pyrimidine base lesions. Structurally similar but distinct substrate lesions, 5-hydroxycytosine and guanidinohydantoin, are more efficiently removed by the Ed NEIL1 isoform, consistent with the inherent differences in tautomerization, proton affinities, and lability. We also observed biphasic kinetic profiles and lack of complete base removal with specific combinations of the lesion and NEIL1 isoform, suggestive of multiple lesion binding modes. The complexity of NEIL1 isoform activity implies multiple roles for NEIL1 in safeguarding accurate repair and as an epigenetic regulator.

Unique Hydrogen Bonding of Adenine with the Oxidatively Damaged Base 8-Oxoguanine Enables Specific Recognition and Repair by DNA Glycosylase MutY.

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.

Detection of OG:A Lesion Mispairs by MutY Relies on a Single His Residue and the 2-Amino Group of 8-Oxoguanine.

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.

Structure-Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase.

Base excision repair glycosylases locate and remove damaged bases in DNA with remarkable specificity. The MutY glycosylases, unusual for their excision of undamaged adenines mispaired to the oxidized base 8-oxoguanine (OG), must recognize both bases of the mispair in order to prevent promutagenic activity. Moreover, MutY must effectively find OG:A mismatches within the context of highly abundant and structurally similar T:A base pairs. Very little is known about the factors that initiate MutY's interaction with the substrate when it first encounters an intrahelical OG:A mispair, or about the order of recognition checkpoints. Here, we used structure-activity relationships (SAR) to investigate the features that influence the in vitro measured parameters of mismatch affinity and adenine base excision efficiency by E. coli MutY. We also evaluated the impacts of the same substrate alterations on MutY-mediated repair in a cellular context. Our results show that MutY relies strongly on the presence of the OG base and recognizes multiple structural features at different stages of recognition and catalysis to ensure that only inappropriately mispaired adenines are excised. Notably, some OG modifications resulted in more dramatic reductions in cellular repair than in the in vitro kinetic parameters, indicating their importance for initial recognition events needed to locate the mismatch within DNA. Indeed, the initial encounter of MutY with its target base pair may rely on specific interactions with the 2-amino group of OG in the major groove, a feature that distinguishes OG:A from T:A base pairs. These results furthermore suggest that inefficient substrate location in human MutY homologue variants may prove predictive for the early onset colorectal cancer phenotype known as MUTYH-Associated Polyposis, or MAP.

Base Excision Repair of N6-Deoxyadenosine Adducts of 1,3-Butadiene.

The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including N6-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N6-HB-dA), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-HMHP-dA), and N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A → T and A → G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2'-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (S)-N6-HB-dA, (R,S)-1,N6-HMHP-dA, and (R,R)-N6,N6-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (S)-N6-HB-dA > (R,R)-N6,N6-DHB-dA > (R,S)-1,N6-HMHP-dA. The extent of strand cleavage at the adduct site was decreased in the presence of BER inhibitor methoxyamine and by competitor duplexes containing known BER substrates. Similar strand cleavage assays conducted using several eukaryotic DNA glycosylases/lyases (AAG, Mutyh, hNEIL1, and hOGG1) have failed to observe correct incision products at the BD-dA lesion sites, suggesting that a different BER enzyme may be involved in the removal of BD-dA adducts in human cells.

DNMT1 and Cancer: An Electrifying Link.

Aberrant epigenetic methylation is linked to the onset and progression of cancer. In this issue of Chemistry & Biology, Furst and Barton (2015) describe a sensitive electrochemical assay that can detect hyperactive epigenetic methylation in tumor tissue.

Gas-phase studies of substrates for the DNA mismatch repair enzyme MutY.

The gas-phase thermochemical properties (tautomeric energies, acidity, and proton affinity) have been measured and calculated for adenine and six adenine analogues that were designed to test features of the catalytic mechanism used by the adenine glycosylase MutY. The gas-phase intrinsic properties are correlated to possible excision mechanisms and MutY excision rates to gain insight into the MutY mechanism. The data support a mechanism involving protonation at N7 and hydrogen bonding to N3 of adenine. We also explored the acid-catalyzed (non-enzymatic) depurination of these substrates, which appears to follow a different mechanism than that employed by MutY, which we elucidate using calculations.