Adenine DNA Glycosylase Research Articles

Crystal structure of MutYX: A novel clusterless adenine DNA glycosylase with a distinct C-terminal domain and 8-Oxoguanine recognition sphere.

The [4Fe-4S] cluster is an important cofactor of the base excision repair (BER) adenine DNA glycosylase MutY to prevent mutations associated with 8-oxoguanine (OG). Several MutYs lacking the [4Fe-4S] cofactor have been identified. Phylogenetic analysis shows that clusterless MutYs are distributed in two clades suggesting cofactor loss in two independent evolutionary events. Herein, we determined the first crystal structure of a clusterless MutY complexed with DNA. On the basis of the dramatic structural divergence from canonical MutYs, we refer to this as representative of a clusterless MutY subgroup "MutYX". Interestingly, MutYX compensates for the missing [4Fe-4S] cofactor to maintain positioning of catalytic residues by expanding a pre-existing α-helix and acquisition of the new α-helix. Surprisingly, MutYX also acquired a new C-terminal domain that uniquely recognizes OG using residue Gln201 and Arg209. Adenine glycosylase assays and binding affinity measurements indicate that Arg209 is the primary residue responsible to specificity for OG:A lesions, while Gln201 bridges OG and Arg209. Surprisingly, replacement of Arg209 and Gln201 with Ala increases activity toward G:A mismatches. The MutYX structure serves as an example of devolution, capturing structural features required to retain function in the absence of a metal cofactor considered indispensable.

Ultrasensitive Detection of FEN1 Activity for Cancer Diagnosis Using a CRISPR/Cas13a‐Based Triple Cascade Amplification System

AbstractFlap endonuclease 1 (FEN1) is closely associated with tumor progression and proliferation, making it a promising biomarker for cancer diagnosis. However, developing a sensitive, reliable, and user‐friendly method for quantitative FEN1 detection remains technically challenging. In this study, an ultrasensitive FEN1 biosensor is established using a target‐induced cleavage‐ligation‐transcription‐activation cascade strategy (LTACas13a) to enhance the cleavage ability of CRISPR/Cas13a. The LTACas13a method has shown excellent performance in screening FEN1 inhibitors and detecting endogenous FEN1 activity in living cells, as well as in clinical biological samples such as human serum and tissue samples. Additionally, using a universal dumbbell probe derived from FEN1, a multiplex LTACas13a strategy is developed for detecting various DNA glycosylases, including formamidopyrimidine DNA glycosylase, uracil DNA glycosylase, and human alkyl adenine DNA glycosylase. This straightforward approach provides a reliable and effective diagnostic tool for early‐stage cancer detection and offers significant opportunities for FEN1 biosensing and related drug discovery.

Preorganized Internal Electric Field Promotes a Double-Displacement Mechanism for the Adenine Excision Reaction by Adenine DNA Glycosylase.

Adenine DNA glycosylase (MutY) is a monofunctional glycosylase, removing adenines (A) misinserted opposite 8-oxo-7,8-dihydroguanine (OG), a common product of oxidative damage to DNA. Through multiscale calculations, we decipher a detailed adenine excision mechanism of MutY that is consistent with all available experimental data, involving an initial protonation step and two nucleophilic displacement steps. During the first displacement step, N-glycosidic bond cleavage is accompanied by the attack of the carboxylate group of residue Asp144 at the anomeric carbon (C1'), forming a covalent glycosyl-enzyme intermediate to stabilize the fleeting oxocarbenium ion. After departure of the excised base, water nucleophiles can be recruited to displace Asp144, completing the catalytic cycle with retention of stereochemistry at the C1' position. The two displacement reactions are found to mostly involve the movement of the oxocarbenium ion, occurring with large charge reorganization and thus sensitive to the internal electric field (IEF) exerted by the polar protein environment. Intriguingly, we find that the negatively charged carboxylate group is a good nucleophile for the oxocarbenium ion, yet an unactivated water molecule is not, and that the electric field catalysis strategy is used by the enzyme to enable its unique double-displacement reaction mechanism. A strong IEF, pointing toward 5' direction of the substrate sugar ring, greatly facilitates the second displacement reaction at the expense of elevating the barrier of the first one, thereby allowing both reactions to occur. These findings not only increase our understanding of the strategies used by DNA glycosylases to repair DNA lesions, but also have important implications for how internal/external electric field can be applied to modulate chemical reactions.

Distinctive Formation of a DNA-Protein Cross-Link during the Repair of DNA Oxidative Damage: Insights into Human Disease from MD Simulations and QM/MM Calculations.

Reactive oxygen species damage DNA and result in health issues. The major damage product, 8-oxo-7,8-dihydroguanine (8oG), is repaired by human adenine DNA glycosylase homologue (MUTYH). Although MUTYH misfunction is associated with a genetic disorder called MUTYH-associated polyposis (MAP) and MUTYH is a potential target for cancer drugs, the catalytic mechanism required to develop disease treatments is debated in the literature. This study uses molecular dynamics simulations and quantum mechanics/molecular mechanics techniques initiated from DNA-protein complexes that represent different stages of the repair pathway to map the catalytic mechanism of the wild-type MUTYH bacterial homologue (MutY). This multipronged computational approach characterizes a DNA-protein cross-linking mechanism that is consistent with all previous experimental data and is a distinct pathway across the broad class of monofunctional glycosylase repair enzymes. In addition to clarifying how the cross-link is formed, accommodated by the enzyme, and hydrolyzed for product release, our calculations rationalize why cross-link formation is favored over immediate glycosidic bond hydrolysis, the accepted mechanism for all other monofunctional DNA glycosylases to date. Calculations on the Y126F mutant MutY highlight critical roles for active site residues throughout the reaction, while investigation of the N146S mutant rationalizes the connection between the analogous N224S MUTYH mutation and MAP. In addition to furthering our knowledge of the chemistry associated with a devastating disorder, the structural information gained about the distinctive MutY mechanism compared to other repair enzymes represents an important step for the development of specific and potent small-molecule inhibitors as cancer therapeutics.

Functionalization of Mesoporous Silica with a G-A-Mismatched dsDNA Chain for Efficient Identification and Selective Capturing of the MutY Protein.

MUTYH adenine DNA glycosylase and its homologous protein (collectively MutY) are typical DNA glycosylases with a [4Fe4S] cluster and a helix-hairpin-helix (HhH) motif in its structure. In the present work, the binding behaviors of the MutY protein to dsDNA containing different base mismatches were investigated. The type and distribution of base mismatch in the dsDNA chain were found to influence the DNA-protein binding interaction greatly. The [4Fe4S] cluster of the MutY protein is able to identify a G-A mismatch in the dsDNA chain specifically by monitoring the anomalies of charge transport in the dsDNA chain, allowing the entrance of the identified dsDNA chain into the internal cavity of the MutY protein and the strong DNA-protein binding at the HhH motif of the protein through multiple H-bonds. The dsDNA chain with a centrally located G-A mismatch is thus functionalized on mesoporous silica (MSN) via amination reaction, and the obtained dsDNA(G-A)@MSN is used as a powerful sorbent for the selective capturing of the MutY protein from complex samples. By using 0.5% NH3·H2O (m/v) as a stripping reagent, efficient isolation of the MutY protein from different cell lines and bacteria is achieved and the recovered MutY protein is demonstrated to maintain favorable DNA adenine glycosylase activity.

Cannabidiol and Nano-Selenium Increase Microvascularization and Reduce Degenerative Changes in Superficial Breast Muscle in C. perfringens-Infected Chickens

Here, we demonstrated the potential of Cannabis-derived cannabidiol (CBD) and nanosized selenium (nano-Se) for the modulation of microvascularization and muscle fiber lesions in superficial breast muscle in C. perfringens-challenged chickens. The administration of CBD resulted in a decreased number of atrophic fibers (3.13 vs. 1.13/1.5 mm2) compared with the control, whereas nano-Se or both substances resulted in a decreased split fiber number (4.13 vs. 1.55/1.5 mm2) and in a lower number of necrotic myofibers (2.38 vs. 0.69/1.5 mm2) in breast muscle than the positive control. There was a significantly higher number of capillary vessels in chickens in the CBD+Nano-Se group than in the control and positive control groups (1.31 vs. 0.97 and 0.98, respectively). Feeding birds experimental diets lowered the activity of DNA damage repair enzymes, including 3,N4-ethenodeoxycytosine (by 39.6%), 1,N6-ethenodeoxyadenosine (by 37.5%), 8-oxo-guanine (by 36.2%), formamidopyrimidine (fapy)-DNA glycosylase (by 56.2%) and human alkyl adenine DNA glycosylase (by 40.2%) in the ileal mucosa, but it did not compromise the blood mitochondrial oxygen consumption rate (-2.67 OD/min on average). These findings indicate a potential link between gut mucosa condition and histopathological changes in superficial pectoral muscle under induced inflammation and show the ameliorative effect of CBD and nano-Se in this cross-talk due to their protection from mucosal DNA damage.

Structure of the mammalian adenine DNA glycosylase MUTYH: insights into the base excision repair pathway and cancer.

Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9–Rad1–Hus1 complex (9–1–1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9–1–1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.

Human MettL3-MettL14 RNA adenine methyltransferase complex is active on double-stranded DNA containing lesions.

MettL3-MettL14 methyltransferase complex has been studied widely for its role in RNA adenine methylation. This complex is also recruited to UV- and X-ray exposed DNA damaged sites, and its methyltransfer activity is required for subsequent DNA repair, though in theory this could result from RNA methylation of short transcripts made at the site of damage. We report here that MettL3-MettL14 is active in vitro on double-stranded DNA containing a cyclopyrimidine dimer – a major lesion of UV radiation-induced products – or an abasic site or mismatches. Furthermore, N6-methyladenine (N6mA) decreases misincorporation of 8-oxo-guanine (8-oxoG) opposite to N6mA by repair DNA polymerases. When 8-oxoG is nevertheless incorporated opposite N6mA, the methylation inhibits N6mA excision from the template (correct) strand by the adenine DNA glycosylase (MYH), implying that the methylation decreases inappropriate misrepair. Finally, we observed that the N6mA reader domain of YTHDC1, which is also recruited to sites of DNA damage, binds N6mA that is located across from a single-base gap between two canonical DNA helices. This YTHDC1 complex with a gapped duplex is structurally similar to DNA complexes with FEN1 and GEN1 – two members of the nuclease family that act in nucleotide excision repair, mismatch repair and homologous recombination, and which incise distinct non-B DNA structures. Together, the parts of our study provide a plausible mechanism for N6mA writer and reader proteins acting directly on lesion-containing DNA, and suggest in vivo experiments to test the mechanisms involving methylation of adenine.

GO System, a DNA Repair Pathway to Cope with Oxidative Damage

The GO system is part of the DNA base excision repair pathway and is required for the error-free repair of 8-oxoguanine (oxoG), one of the most common oxidative DNA lesions. Due to the ability of oxoG to form oxoG:A mispairs, this base is highly mutagenic. Its repair requires the action of two enzymes: 8-oxoguanine DNA glycosylase (Fpg or MutM in bacteria and OGG1 in eukaryotes), which removes oxoG from oxoG:C pairs, and adenine DNA glycosylase (MutY in bacteria and MUTYH in eukaryotes), which removes A from oxoG:A mispairs to prevent mutations. The third enzyme of the system (MutT in bacteria and MTH1 or NUDT1 in eukaryotes) hydrolyzes 8-oxo-2'-deoxyguanosine triphosphate, thus preventing its incorporation into DNA. Recent data point to the proteins of the GO system as promising targets for the therapy of cancer, autoimmune diseases, and bacterial infections. This review highlights the structure and specificity of the GO system in bacteria and eukaryotes and its unique role in mutation avoidance.

MUTYH Actively Contributes to Microglial Activation and Impaired Neurogenesis in the Pathogenesis of Alzheimer's Disease.

Oxidative stress is a major risk factor for Alzheimer's disease (AD), which is characterized by brain atrophy, amyloid plaques, neurofibrillary tangles, and loss of neurons. 8-Oxoguanine, a major oxidatively generated nucleobase highly accumulated in the AD brain, is known to cause neurodegeneration. In mammalian cells, several enzymes play essential roles in minimizing the 8-oxoguanine accumulation in DNA. MUTYH with adenine DNA glycosylase activity excises adenine inserted opposite 8-oxoguanine in DNA. MUTYH is reported to actively contribute to the neurodegenerative process in Parkinson and Huntington diseases and some mouse models of neurodegenerative diseases by accelerating neuronal dysfunction and microgliosis under oxidative conditions; however, whether or not MUTYH is involved in AD pathogenesis remains unclear. In the present study, we examined the contribution of MUTYH to the AD pathogenesis. Using postmortem human brains, we showed that various types of MUTYH transcripts and proteins are expressed in most hippocampal neurons and glia in both non-AD and AD brains. We further introduced MUTYH deficiency into AppNL-G-F/NL-G-F knock-in AD model mice, which produce humanized toxic amyloid-β without the overexpression of APP protein, and investigated the effects of MUTYH deficiency on the behavior, pathology, gene expression, and neurogenesis. MUTYH deficiency improved memory impairment in AppNL-G-F/NL-G-F mice, accompanied by reduced microgliosis. Gene expression profiling strongly suggested that MUTYH is involved in the microglial response pathways under AD pathology and contributes to the phagocytic activity of disease-associated microglia. We also found that MUTYH deficiency ameliorates impaired neurogenesis in the hippocampus, thus improving memory impairment. In conclusion, we propose that MUTYH, which is expressed in the hippocampus of AD patients as well as non-AD subjects, actively contributes to memory impairment by inducing microgliosis with poor neurogenesis in the preclinical AD phase and that MUTYH is a novel therapeutic target for AD, as its deficiency is highly beneficial for ameliorating AD pathogenesis.

MUTYH: Not just polyposis.

MUTYH is a base excision repair enzyme, it plays a crucial role in the correction of DNA errors from guanine oxidation and may be considered a cell protective factor. In humans it is an adenine DNA glycosylase that removes adenine misincorporated in 7,8-dihydro-8-oxoguanine (8-oxoG) pairs, inducing G:C to T:A transversions. MUTYH functionally cooperates with OGG1 that eliminates 8-oxodG derived from excessive reactive oxygen species production. MUTYH mutations have been linked to MUTYH associated polyposis syndrome (MAP), an autosomal recessive disorder characterized by multiple colorectal adenomas. MAP patients show a greatly increased lifetime risk for gastrointestinal cancers. The cancer risk in mono-allelic carriers associated with one MUTYH mutant allele is controversial and it remains to be clarified whether the altered functions of this protein may have a pathophysiological involvement in other diseases besides familial gastrointestinal diseases. This review evaluates the role of MUTYH, focusing on current studies of human neoplastic and non-neoplastic diseases different to colon polyposis and colorectal cancer. This will provide novel insights into the understanding of the molecular basis underlying MUTYH-related pathogenesis. Furthermore, we describe the association between MUTYH single nucleotide polymorphisms (SNPs) and different cancer and non-cancer diseases. We address the utility to increase our knowledge regarding MUTYH in the light of recent advances in the literature with the aim of a better understanding of the potential for identifying new therapeutic targets. Considering the multiple functions and interactions of MUTYH protein, its involvement in pathologies based on oxidative stress damage could be hypothesized. Although the development of extraintestinal cancer in MUTYH heterozygotes is not completely defined, the risk for malignancies of the duodenum, ovary, and bladder is also increased as well as the onset of benign and malignant endocrine tumors. The presence of MUTYH pathogenic variants is an independent predictor of poor prognosis in sporadic gastric cancer and in salivary gland secretory carcinoma, while its inhibition has been shown to reduce the survival of pancreatic ductal adenocarcinoma cells. Furthermore, some MUTYH SNPs have been associated with lung, hepatocellular and cervical cancer risk. An additional role of MUTYH seems to contribute to the prevention of numerous other disorders with an inflammatory/degenerative basis, including neurological and ocular diseases. Finally, it is interesting to note that MUTYH could be a new therapeutic target and future studies will shed light on its specific functions in the prevention of diseases and in the improvement of the chemo-sensitivity of cancer cells.

Global transcriptomic and proteomics analysis of Lactobacillus plantarum Y44 response to 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) stress

Our previous study demonstrated that Lactobacillus plantarum Y44 exhibited antioxidant activity. However, the physiological characteristics of L. plantarum Y44 exposure to oxidative stress was not clear. In this research, the differentially expressed proteins and genes in L. plantarum Y44 under 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) stress at different concentrations were studied by using integrated transcriptomic and proteomic methods. Under 100 mM AAPH stress condition, 1139 differentially expressed genes (DEGs, 546 up-regulated and 593 down-regulated) and 329 differentially expressed proteins (DEPs, 127 up-regulated and 202 down-regulated) were observed. Under 200 mM AAPH stress condition, 1526 DEGs (751 up-regulated and 775 down-regulated) and 382 DEPs (139 up-regulated and 243 down-regulated) were observed. Overall, we found that L. plantarum Y44 fought against AAPH induced oxidative stress by up-regulating antioxidant enzymes and DNA repair proteins, such as ATP-dependent DNA helicase RuvA, adenine DNA glycosylase, single-strand DNA-binding protein SSB, DNA-binding ferritin-like protein DPS, thioredoxin reductase, protein-methionine-S-oxide reductase and glutathione peroxidase. Additionally, cell envelope composition of L. plantarum Y44 was highly remodeled by accelerating peptidoglycan and teichoic-acid (LTA) biosynthesis and modulating the fatty acids (FA) composition to achieve a higher ratio of unsaturated/saturated fatty acids (UFAs/SFAs) against AAPH stress. Moreover, metabolism processes including carbohydrate metabolism, amino acid biosynthesis, and nucleotide metabolism altered to respond to AAPH-induced damage. Altogether, our findings allow us to facilitate a better understanding of L. plantarum Y44 against oxidative stress. SignificanceThis study represents an integrated proteomic and transcriptomic analysis of Lactobacillus plantarum Y44 response to 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) stress. Differentially expressed proteins and genes were identified between the proteome and transcriptome of L. plantarum Y44 under different AAPH stress. AAPH-induced response of L. plantarum Y44 appears to be primarily based on ROS scavenging, DNA repair, highly remodeled cell surface and specific metabolic processes. The knowledge about these proteomes and transcriptomes provides significant insights into the oxidative stress response of Lactobacillus plantarum.

Aberrant repair initiated by the adenine-DNA glycosylase does not play a role in UV-induced mutagenesis in Escherichia coli.

BackgroundDNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2′-deoxyguanosine-5′-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage.MethodsHere, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproduct (6–4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured.ResultsWe demonstrated that MutY does not excise Adenine when it is paired with CPD and 6–4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains.DiscussionThese findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

Structural Basis for the Lesion-scanning Mechanism of the MutY DNA Glycosylase

The highly mutagenic A:8-oxoguanine (oxoG) base pair is generated mainly by misreplication of the C:oxoG base pair, the oxidation product of the C:G base pair. The A:oxoG base pair is particularly insidious because neither base in it carries faithful information to direct the repair of the other. The bacterial MutY (MUTYH in humans) adenine DNA glycosylase is able to initiate the repair of A:oxoG by selectively cleaving the A base from the A:oxoG base pair. The difference between faithful repair and wreaking mutagenic havoc on the genome lies in the accurate discrimination between two structurally similar base pairs: A:oxoG and A:T. Here we present two crystal structures of the MutY N-terminal domain in complex with either undamaged DNA or DNA containing an intrahelical lesion. These structures have captured for the first time a DNA glycosylase scanning the genome for a damaged base in the very first stage of lesion recognition and the base extrusion pathway. The mode of interaction observed here has suggested a common lesion-scanning mechanism across the entire helix-hairpin-helix superfamily to which MutY belongs. In addition, small angle X-ray scattering studies together with accompanying biochemical assays have suggested a possible role played by the C-terminal oxoG-recognition domain of MutY in lesion scanning.

Interaction features of adenine DNA glycosylase MutY from E. coli with DNA substrates

Kinetic characteristics of specific recognition of damaged base by the DNA glycosylase MutY in model DNA substrates, containing oxoG/A-, G/A-, oxoG/C- and F/G pairs in the central position, were investigated. Conformational changes of the MutY enzyme during the recognition of the damaged base in DNA have been recorded by the change in the fluorescence intensity of tryptophan residues using the stopped-flow technique in real time. DNA duplexes containing a fluorescein residue were used for the registration of DNA conformational changes. Analysis of the kinetic curves allowed us to determine the values of rate constants for the kinetic stages of the interaction. It was shown that nonspecific contacts between the DNA-binding site of the enzyme and the DNA duplex are formed at the first stage of the interaction. It was found that the discrimination of Gua and oxoGua bases occurs at the second stage of the MutY interaction with the DNA duplex. The data obtained for the oxoG/C-substrate showed that the recognition of the base located opposite oxoGua also occurs at this stage.

Molecular pathophysiology of impaired glucose metabolism, mitochondrial dysfunction, and oxidative DNA damage in Alzheimer's disease brain

In normal brain, neurons in the cortex and hippocampus produce insulin, which modulates glucose metabolism and cognitive functions. It has been shown that insulin resistance impairs glucose metabolism and mitochondrial function, thus increasing production of reactive oxygen species. Recent progress in Alzheimer’s disease (AD) research revealed that insulin production and signaling are severely impaired in AD brain, thereby resulting in mitochondrial dysfunction and increased oxidative stress. Among possible oxidative DNA lesions, 8-oxoguanine (8-oxoG) is highly accumulated in the brain of AD patients. Previously we have shown that incorporating 8-oxoG in nuclear and mitochondrial DNA promotes MUTYH (adenine DNA glycosylase) dependent neurodegeneration. Moreover, cortical neurons prepared from MTH1 (8-oxo-dGTPase)/OGG1 (8-oxoG DNA glycosylase)-double deficient adult mouse brains is shown to exhibit significantly poor neuritogenesis in vitro with increased 8-oxoG accumulation in mitochondrial DNA in the absence of antioxidants. Therefore, 8-oxoG can be considered involved in the neurodegenerative process in AD brain. In mild cognitive impairment, mitochondrial dysfunction and oxidative damage may induce synaptic dysfunction due to energy failures in neurons thus resulting in impaired cognitive function. If such abnormality lasts long, it can lead to vicious cycles of oxidative damage, which may then trigger the neurodegenerative process seen in Alzheimer type dementia.

Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase

The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site.

MUTYH, an adenine DNA glycosylase, mediates p53 tumor suppression via PARP-dependent cell death.

p53-regulated caspase-independent cell death has been implicated in suppression of tumorigenesis, however, the regulating mechanisms are poorly understood. We previously reported that 8-oxoguanine (8-oxoG) accumulation in nuclear DNA (nDNA) and mitochondrial DNA triggers two distinct caspase-independent cell death through buildup of single-strand DNA breaks by MutY homolog (MUTYH), an adenine DNA glycosylase. One pathway depends on poly-ADP-ribose polymerase (PARP) and the other depends on calpains. Deficiency of MUTYH causes MUTYH-associated familial adenomatous polyposis. MUTYH thereby suppresses tumorigenesis not only by avoiding mutagenesis, but also by inducing cell death. Here, we identified the functional p53-binding site in the human MUTYH gene and demonstrated that MUTYH is transcriptionally regulated by p53, especially in the p53/DNA mismatch repair enzyme, MLH1-proficient colorectal cancer-derived HCT116+Chr3 cells. MUTYH-small interfering RNA, an inhibitor for p53 or PARP suppressed cell death without an additive effect, thus revealing that MUTYH is a potential mediator of p53 tumor suppression, which is known to be upregulated by MLH1. Moreover, we found that the p53-proficient, mismatch repair protein, MLH1-proficient colorectal cancer cell line express substantial levels of MUTYH in nuclei but not in mitochondria, suggesting that 8-oxoG accumulation in nDNA triggers MLH1/PARP-dependent cell death. These results provide new insights on the molecular mechanism of tumorigenesis and potential new strategies for cancer therapies.

Sequence-specific DNA glycosylase found in a restriction-modification system

Restriction-modification systems consist of genes that encode a restriction enzyme and a cognate modification methyltransferase. It was believed that restriction enzymes are sequence-specific endonucleases that introduce double-strand breaks at specific sites by catalyzing the cleavages of phosphodiester bonds. R.PabI is a type II restriction enzyme from a hyperthermophilic archaea Pyrococcus abyssi that recognizes 5'-GTAC-3' sequence and cleaves DNA duplexes without the addition of a divalent cation. The structural and mutational analyses of R.PabI in our previous work showed that R.PabI forms a homodimer and has a novel DNA-binding fold called a "half-pipe," which consists of a highly curved anti-parallel β-sheet. Because the structure of R.PabI shares no structural similarity to any other protein, the structural basis of the sequence-recognition and DNA-cleavage mechanisms remained unclear. In this study, we report the crystal structure of R.PabI in complex with a DNA duplex containing the R.PabI recognition sequence. The structure of the R.PabI-DNA complex shows that R.PabI unwinds a DNA duplex at a 5'-GTAC-3' site and flips the guanine and adenine bases out of the DNA helix to recognize the sequence. The electron-density map of the R.PabI-DNA complex shows that R.PabI releases adenine bases from the R.PabI recognition sequence. Biochemical assays using HPLC and MALDI-TOF MS spectrometry also support the observation that R.PabI releases adenine bases by hydrolysis. These results show that R.PabI is not an endonuclease but a sequence-specific adenine DNA glycosylase. R.PabI is the first example of a restriction enzyme that shows DNA glycosylase activity. Mutational analysis reveals the active site of the adenine DNA glycosylase activity of R.PabI. The two opposing apurinic/apyrimidinic (AP) sites generated by R.PabI are cleaved by heat promoted β elimination and/or by endogenous AP endonucleases of host cells to introduce a double-strand break.

Cellular levels of 8-oxoguanine in either DNA or the nucleotide pool play pivotal roles in carcinogenesis and survival of cancer cells.

8-Oxoguanine, a major oxidized base lesion formed by reactive oxygen species, causes G to T transversion mutations or leads to cell death in mammals if it accumulates in DNA. 8-Oxoguanine can originate as 8-oxo-dGTP, formed in the nucleotide pool, or by direct oxidation of the DNA guanine base. MTH1, also known as NUDT1, with 8-oxo-dGTP hydrolyzing activity, 8-oxoguanine DNA glycosylase (OGG1) an 8-oxoG DNA glycosylase, and MutY homolog (MUTYH) with adenine DNA glycosylase activity, minimize the accumulation of 8-oxoG in DNA; deficiencies in these enzymes increase spontaneous and induced tumorigenesis susceptibility. However, different tissue types have different tumorigenesis susceptibilities. These can be reversed by combined deficiencies in the defense systems, because cell death induced by accumulation of 8-oxoG in DNA is dependent on MUTYH, which can be suppressed by MTH1 and OGG1. In cancer cells encountering high oxidative stress levels, a high level of 8-oxo-dGTP accumulates in the nucleotide pool, and cells therefore express increased levels of MTH1 in order to eliminate 8-oxo-dGTP. Suppression of MTH1 may be an efficient strategy for killing cancer cells; however, because MTH1 and OGG1 protect normal tissues from oxidative-stress-induced cell death, it is important that MTH1 inhibition does not increase the risk of healthy tissue degeneration.