N-methylpurine DNA Glycosylase Research Articles

Developing guanine base editors for G-to-T editing in rice.

Two guanine base editors created using an engineered N-methylpurine DNA glycosylase with CRISPR systems achieved targeted G-to-T editing with 4.94-12.50% efficiency in rice (Oryza sativa). The combined use of the DNA glycosylase and deaminases enabled co-editing of target guanines with adenines or cytosines.

Effect of kynurenic acid on enzymatic activity of the DNA base excision repair pathway in specific areas of the sheep brain

Relatively low levels of antioxidant enzymes coupled with high oxygen metabolism result in the formation of numerous oxidative DNA damages in the tissues of the central nervous system. Recently, kynurenic acid (KYNA), knowns for its neuroprotective properties, has gained increasing attention in this context. Therefore, our hypothesis assumed that increased KYNA levels in the brain would positively influence mRNA expression of selected enzymes of the base excision repair pathway as well as enhance their efficiency in excising damaged nucleobases in specific areas of the sheep brain. The study was conducted on adult anestrous sheep (n = 18), in which two different doses of KYNA (20 and 100 μg/day) were infused into the third brain ventricle for three days. Molecular and biochemical analysis included the hypothalamus (preoptic and mediol-basal areas), hippocampus (CA3 field) and amygdala (central amygdaloid nucleus), dissected from the brain of sheep euthanized immediately after the last infusion. The results revealed a significant increase P < 0.001) in the relative mRNA abundance of N-methylpurine DNA glycosylase (MPG) following administration of both dose of KYNA across all examined tissues. The transcription of thymine-DNA glycosylase (TDG) increased significantly (P < 0.001) in all tissues in response to the lower KYNA dose compared to the control group. Moreover, 8-oxoguanine (8-oxoG) DNA glycosylase (OGG1) mRNA levels were also higher in both animal groups (P < 0.001). In addition, in the hypothalamus, hippocampus and amygdala, AP endonuclease 1 (APE1) mRNA expression increased under both doses of KYNA. Moreover, the both dose of KYNA significantly stimulated the efficiency of 8-oxoG excision in hypothalamus and amygdala (P < 0.05–0.001). The lower and higher doses of KYNA significantly influenced the effectiveness of εA and εC in all structures (P < 0.01–0.001). In conclusion, the favorable effect of KYNA in the brain may include the protection of genetic material in nerve and glial cells by stimulating the expression and efficiency of BER pathway enzymes.

Fusion of a rice endogenous N-methylpurine DNA glycosylase to a plant adenine base transition editor ABE8e enables A-to-K base editing in rice plants

Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement. Here, we isolated a rice endogenous hypoxanthine excision protein, N-methylpurine DNA glycosylase (OsMPG), and engineered two plant A-to-K (K = G or T) base editors, rAKBE01 and rAKBE02, for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor, ABE8e. We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04, respectively. Testing these four rAKBEs, at five endogenous loci in rice protoplasts, indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64. Furthermore, whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus, in comparison with rAKBE02 and rAKBE03, rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57- and 1.75-fold in the rice protoplasts, respectively. Moreover, although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04, rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing, at all the five target loci, with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31% and 1.67 to 4.84% in rice stable lines, respectively. Together, these rAKBEs enable different portfolios of editing products and, thus, now expands the potential of base editing in diverse application scenario for crop improvement.

Epigenetic silencing of HTATIP2 in glioblastoma contributes to treatment resistance by enhancing nuclear translocation of the DNA repair protein MPG.

Glioblastoma, the most malignant brain tumor in adults, exhibits characteristic patterns of epigenetic alterations that await elucidation. The DNA methylome of glioblastoma revealed recurrent epigenetic silencing of HTATIP2, which encodes a negative regulator of importin β-mediated cytoplasmic-nuclear protein translocation. Its deregulation may thus alter the functionality of cancer-relevant nuclear proteins, such as the base excision repair (BER) enzyme N-methylpurine DNA glycosylase (MPG), which has been associated with treatment resistance in GBM. We found that induction of HTATIP2 expression in GBM cells leads to a significant shift of predominantly nuclear to cytoplasmic MPG, whereas depletion of endogenous HTATIP2 results in enhanced nuclear MPG localization. Reduced nuclear MPG localization, prompted by HTATIP2 expression or by depletion of MPG, yielded less phosphorylated-H2AX-positive cells upon treatment with an alkylating agent. This suggested reduced MPG-mediated formation of apurinic/apyrimidinic sites, leaving behind unrepaired DNA lesions, reflecting a reduced capacity of BER in response to the alkylating agent. Epigenetic silencing of HTATIP2 may thus increase nuclear localization of MPG, thereby enhancing the capacity of the glioblastoma cells to repair treatment-related lesions and contributing to treatment resistance.

Adenine base editor incorporating the N-methylpurine DNA glycosylase MPGv3 enables efficient A-to-K base editing in rice

Adenine base editor incorporating the N-methylpurine DNA glycosylase MPGv3 enables efficient A-to-K base editing in rice

Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase.

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.

Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase.

Here we developed an adenine transversion base editor, AYBE, for A-to-C and A-to-T transversion editing in mammalian cells by fusing an adenine base editor (ABE) with hypoxanthine excision protein N-methylpurine DNA glycosylase (MPG). We also engineered AYBE variants enabling targeted editing at genomic loci with higher transversion editing activity (up to 72% for A-to-C or A-to-T editing).

Central Stimulatory Effect of Kynurenic Acid on BDNF-TrkB Signaling and BER Enzymatic Activity in the Hippocampal CA1 Field in Sheep

Deficiency of neurotrophic factors and oxidative DNA damage are common causes of many neurodegenerative diseases. Recently, the importance of kynurenic acid (KYNA), an active metabolite of tryptophan, has increased as a neuroprotective molecule in the brain. Therefore, the present study tested the hypothesis that centrally acting KYNA would positively affect: (1) brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signaling and (2) selected base excision repair (BER) pathway enzymes activities in the hippocampal CA1 field in sheep. Both lower (20 μg in total) and higher (100 μg in total) doses of KYNA infused into the third brain ventricle differentially increased the abundance of BDNF and TrkB mRNA in the CA1 field; additionally, the higher dose increased BDNF tissue concentration. The lower dose of KYNA increased mRNA expression for 8-oxoguanine glycosylase (OGG1), N-methylpurine DNA glycosylase (MPG), and thymine DNA glycosylase and stimulated the repair of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxy-cytosine as determined by the excision efficiency of lesioned nucleobases. The higher dose increased the abundance of OGG1 and MPG transcripts, however, its stimulatory effect on repair activity was less pronounced in all cases compared to the lower dose. The increased level of AP-endonuclease mRNA expression was dose-dependent. In conclusion, the potential neurotrophic and neuroprotective effects of KYNA in brain cells may involve stimulation of the BDNF-TrkB and BER pathways.

Cooperative interaction between AAG and UV-DDB in the removal of modified bases.

UV-DDB is a DNA damage recognition protein recently discovered to participate in the removal of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG) by stimulating multiple steps of base excision repair (BER). In this study, we examined whether UV-DDB has a wider role in BER besides oxidized bases and found it has specificity for two known DNA substrates of alkyladenine glycosylase (AAG)/N-methylpurine DNA glycosylase (MPG): 1, N6-ethenoadenine (ϵA) and hypoxanthine. Gel mobility shift assays show that UV-DDB recognizes these two lesions 4-5 times better than non-damaged DNA. Biochemical studies indicated that UV-DDB stimulated AAG activity on both substrates by 4- to 5-fold. Native gels indicated UV-DDB forms a transient complex with AAG to help facilitate release of AAG from the abasic site product. Single molecule experiments confirmed the interaction and showed that UV-DDB can act to displace AAG from abasic sites. Cells when treated with methyl methanesulfonate resulted in foci containing AAG and UV-DDB that developed over the course of several hours after treatment. While colocalization did not reach 100%, foci containing AAG and UV-DDB reached a maximum at three hours post treatment. Together these data indicate that UV-DDB plays an important role in facilitating the repair of AAG substrates.

Novel CTRP8‐RXFP1‐JAK3‐STAT3 axis promotes Cdc42‐dependent actin remodeling for enhanced filopodia formation and motility in human glioblastoma cells

C1q tumor necrosis factor‐related peptide 8 (CTRP8) is the least studied member of the C1Q‐TNF‐related peptide family. We identified CTRP8 as a ligand of the G protein‐coupled receptor relaxin family peptide receptor 1 (RXFP1) in glioblastoma multiforme (GBM). The CTRP8‐RXFP1 ligand–receptor system protects human GBM cells against the DNA‐alkylating damage‐inducing temozolomide (TMZ), the drug of choice for the treatment of patients with GBM. The DNA protective role of CTRP8 was dependent on a functional RXFP1‐STAT3 signaling cascade and targeted the monofunctional glycosylase N‐methylpurine DNA glycosylase (MPG) for more efficient base excision repair of TMZ‐induced DNA‐damaged sites. CTRP8 also improved the survival of GBM cells by upregulating anti‐apoptotic BCl‐2 and BCL‐XL. Here, we have identified Janus‐activated kinase 3 (JAK3) as a novel member of a novel CTRP8‐RXFP1‐JAK3‐STAT3 signaling cascade that caused an increase in cellular protein content and activity of the small Rho GTPase Cdc42. This is associated with significant F‐actin remodeling and increased GBM motility. Cdc42 was critically important for the upregulation of the actin nucleation complex N‐Wiskott–Aldrich syndrome protein/Arp3/4 and actin elongation factor profilin‐1. The activation of the RXFP1‐JAK3‐STAT3‐Cdc42 axis by both RXFP1 agonists, CTRP8 and relaxin‐2, caused extensive filopodia formation. This coincided with enhanced activity of ezrin, a key factor in tethering F‐actin to the plasma membrane, and inhibition of the actin filament severing activity of cofilin. The F‐actin remodeling and pro‐migratory activities promoted by the novel RXFP1‐JAK3‐STAT3‐Cdc42 axis were blocked by JAK3 inhibitor tofacitinib and STAT3 inhibitor STAT3 inhibitor VI. This provides a new rationale for the design of JAK3 and STAT3 inhibitors with better brain permeability for clinical treatment of the pervasive brain invasiveness of GBM.

Pilot Study to Detect Genes Involved in DNA Damage and Cancer in Humans: Potential Biomarkers of Exposure to E-Cigarette Aerosols.

There is a paucity of data on how gene expression enables identification of individuals who are at risk of exposure to carcinogens from e-cigarette (e-cig) vaping; and how human vaping behaviors modify these exposures. This pilot study aimed to identify genes regulated from acute exposure to e-cig using RT-qPCR. Three subjects (2M and 1F) made three visits to the lab (nTOT = 9 visits); buccal and blood samples were collected before and immediately after scripted vaping 20 puffs (nTOT = 18 samples); vaping topography data were collected in each session. Subjects used their own e-cig containing 50:50 propylene glycol (PG):vegetable glycerine (VG) +3–6 mg/mL nicotine. The tumor suppressor TP53 was significantly upregulated in buccal samples. TP53 expression was puff volume and flow rate dependent in both tissues. In blood, the significant downregulation of N-methylpurine DNA glycosylase (MPG), a base excision repair gene, was consistent across all subjects. In addition to DNA repair pathway, cell cycle and cancer pathways were the most enriched pathways in buccal and blood samples, respectively. This pilot study demonstrates that vaping 20 puffs significantly alters expression of TP53 in human tissues; vaping behavior is an important modifier of this response. A larger study is needed to confirm these relationships.

Involvement of N-methylpurine DNA glycosylase in resistance to temozolomide in patient-derived glioma cells

Chemotherapy for high-grade astrocytic tumors is mainly based on the use of temozolomide (TMZ), whose efficacy is limited by resistance mechanisms. Despite many investigations pointing to O6-methylguanine-DNA-methyltransferase (MGMT) as being responsible for tumor chemo-resistance, its expression does not predict an accurate response in most gliomas, suggesting that MGMT is not the only determinant of response to treatment. In this sense, several reports indicate that N-methylpurine-DNA-glycosylase (MPG) may be involved in that resistance. With that in mind, we evaluated for the first time the degree of resistance to TMZ treatment in 18 patient-derived glioma cells and its association with MGMT and MPG mRNA levels. Viability cell assays showed that TMZ treatment hardly caused growth inhibition in the patient-derived cells, even in high concentrations, indicating that all primary cultures were chemo-resistant. mRNA expression analyses showed that the TMZ-resistant phenotype displayed by cells is associated with an elevated expression of MPG to a greater extent than it is with transcript levels of MGMT. Our findings suggest that not only is MGMT implicated in resistance to TMZ but MPG, the first enzyme in base excision repair processing, is also involved, supporting its potential role as a target in anti-resistance chemotherapy for astrocytoma and glioblastoma.

MPG and NPRL3 Polymorphisms are Associated with Ischemic Stroke Susceptibility and Post-Stroke Mortality.

Ischemic stroke is a complicated disease which is affected by environmental factors and genetic factors. In this field, various studies using whole-exome sequencing (WES) have focused on novel and linkage variants in diverse diseases. Thus, we have investigated the various novel variants, which focused on their linkages to each other, in ischemic stroke. Specifically, we analyzed the N-methylpurine DNA glycosylase (MPG) gene, which plays an initiating role in DNA repair, and the nitrogen permease regulator-like 3 (NPRL3) gene, which is involved in regulating the mammalian target of rapamycin pathway. We took blood samples of 519 ischemic stroke patients and 417 controls. Genetic polymorphisms were detected by polymerase chain reaction (PCR), real-time PCR, and restriction fragment length polymorphism (RFLP) analysis. We found that two NPRL3 polymorphisms (rs2541618 C>T and rs75187722 G>A), as well as the MPG rs2562162 C>T polymorphism, were significantly associated with ischemic stroke. In Cox proportional hazard regression models, the MPG rs2562162 was associated with the survival of small-vessel disease patients in ischemic stroke. Our study showed that NPRL3 and MPG polymorphisms are associated with ischemic stroke prevalence and ischemic stroke survival. Taken together, these findings suggest that NPRL3 and MPG genotypes may be useful clinical biomarkers for ischemic stroke development and prognosis.

The Effect of Allopregnanolone on Enzymatic Activity of the DNA Base Excision Repair Pathway in the Sheep Hippocampus and Amygdala under Natural and Stressful Conditions.

The neurosteroid allopregnanolone (AL) has many beneficial functions in the brain. This study tested the hypothesis that AL administered for three days into the third brain ventricle would affect the enzymatic activity of the DNA base excision repair (BER) pathway in the hippocampal CA1 and CA3 fields and the central amygdala in luteal-phase sheep under both natural and stressful conditions. Acute stressful stimuli, including isolation and partial movement restriction, were used on the last day of infusion. The results showed that stressful stimuli increased N-methylpurine DNA glycosylase (MPG), thymine DNA glycosylase (TDG), 8-oxoguanine glycosylase (OGG1), and AP-endonuclease 1 (APE1) mRNA expression, as well as repair activities for 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC), and 8-oxoguanine (8-oxoG) compared to controls. The stimulated events were lower in stressed and AL-treated sheep compared to sheep that were only stressed (except MPG mRNA expression in the CA1 and amygdala, as well as TDG mRNA expression in the CA1). AL alone reduced mRNA expression of all DNA repair enzymes (except TDG in the amygdala) relative to controls and other groups. DNA repair activities varied depending on the tissue—AL alone stimulated the excision of εA in the amygdala, εC in the CA3 and amygdala, and 8-oxoG in all tissues studied compared to controls. However, the excision efficiency of lesioned bases in the AL group was lower than in the stressed and stressed and AL-treated groups, with the exception of εA in the amygdala. In conclusion, the presented modulating effect of AL on the synthesis of BER pathway enzymes and their repair capacity, both under natural and stressful conditions, indicates another functional role of this neurosteroid in brain structures.

ACTR-43. GENOMIC ANALYSIS OF RESPONDERS OF PHASE II TRIAL OF TEMOZOLOMIDE AND TRC-102 (BASE EXCISION REPAIR INHIBITOR) IN BEVACIZUMAB-NAÏVE GLIOBLASTOMA AT FIRST RECURRENCE

Abstract BACKGROUND Temozolomide forms O6-methylguanine (O6mG), 7-methylguanine (N7mG), and 3- methyladenine (N3mA) DNA adducts. The O6mG DNA adduct is repaired by MGMT. N7mG and N3mA DNA adducts are removed by the base excision repair (BER) pathway, initiated by N- methylpurine DNA glycosolase (MPG). TRC-102 is a BER inhibitor that binds to the apurinic site created through the action of MPG. METHODS A phase II study of adult glioblastoma in first recurrence was performed in the Adult Brain Tumor Consortium with temozolomide, 150 mg/ m2 and TRC-102, 150 mg (1–5/ 28 days). Primary objective included radiographic response rate. Secondary objectives included safety and PFS-6. Exploratory objectives included tumor expression of N-methylpurine DNA glycosylase (MPG). The study tested hypothesis that combination therapy will achieve 30% RR. To understand the context of vulnerability to TRC102 we performed RNA sequencing on treatment naïve tissue from 7 patients. RESULTS Nineteen patients were enrolled in first stage. Median age was 60 years (range: 48–76), 53% females, median KPS was 80 (range: 70–90). Median cycles of treatment was 2 (range: 1–12). No responses were observed. Median OS was 11.0 months (95% CI: 8–18 months), median PFS was 2.0 months (95% CI: 1.8–3.6 months). PFS-6 rate was 10.5 % (2/19). The combination was safe. MPG staining was negative in six, 1+ in five and 2+ in three patients. PFS of 11 + months in two patients (exceptional responders) was associated with MPG expression. Preliminary analysis on RNA sequencing revealed significant enrichment for DNA Damage Response pathways (MsigDB), chromosomal instability gene signature (CIN70 and CIN25), and proliferative gene signature (PCNA25) in these 2 patients. CONCLUSIONS TRC 102 with temozolomide has acceptable safety but did not meet the primary endpoint of response. Gene signature of MsigDB, CIN70, CIN25 and PCNA25 was seen in exceptional responders and biomarker driven study is planned.

Congenic mapping and candidate gene analysis for streptozotocin-induced diabetes susceptibility locus on mouse chromosome 11.

Streptozotocin (STZ) has been widely used to induce diabetes in rodents. Strain-dependent variation in susceptibility to STZ has been reported; however, the gene(s) responsible for STZ susceptibility has not been identified. Here, we utilized the A/J-11SM consomic strain and a set of chromosome 11 (Chr. 11) congenic strains developed from A/J-11SM to identify a candidate STZ-induced diabetes susceptibility gene. The A/J strain exhibited significantly higher susceptibility to STZ-induced diabetes than the A/J-11SM strain, confirming the existence of a susceptibility locus on Chr. 11. We named this locus Stzds1 (STZ-induced diabetes susceptibility 1). Congenic mapping using the Chr. 11 congenic strains indicated that the Stzds1 locus was located between D11Mit163 (27.72Mb) and D11Mit51 (36.39Mb). The Mpg gene, which encodes N-methylpurine DNA glycosylase (MPG), a ubiquitous DNA repair enzyme responsible for the removal of alkylated base lesions in DNA, is located within the Stzds1 region. There is a close relationship between DNA alkylation at an early stage of STZ action and the function of MPG. A Sanger sequence analysis of the Mpg gene revealed five polymorphic sites in the A/J genome. One variant, p.Ala132Ser, was located in a highly conserved region among rodent species and in the minimal region for retained enzyme activity of MPG. It is likely that structural alteration of MPG caused by the p.Ala132Ser mutation elicits increased recognition and excision of alkylated base lesions in DNA by STZ.

Endoplasmic reticulum stress-inducing drugs sensitize glioma cells to temozolomide through downregulation of MGMT, MPG, and Rad51

Endoplasmic reticulum (ER) stress results from protein misfolding imbalance and has been postulated as a therapeutic strategy. ER stress activates the unfolded protein response which leads to a complex cellular response, including the upregulation of aberrant protein degradation in the ER, with the goal of resolving that stress. O(6)-methylguanine DNA methyltransferase (MGMT), N-methylpurine DNA glycosylase (MPG), and Rad51 are DNA damage repair proteins that mediate resistance to temozolomide in glioblastoma. In this work we sought to evaluate whether ER stress-inducing drugs were able to downmodulate DNA damage repair proteins and become candidates to combine with temozolomide. MTT assays were performed to evaluate the cytotoxicity of the treatments. The expression of proteins was evaluated using western blot and immunofluorescence. In vivo studies were performed using 2 orthotopic glioblastoma models in nude mice to evaluate the efficacy of the treatments. All statistical tests were 2-sided. Treatment of glioblastoma cells with ER stress-inducing drugs leads to downregulation of MGMT, MPG, and Rad51. Inhibition of ER stress through pharmacological treatment resulted in rescue of MGMT, MPG, and Rad51 protein levels. Moreover, treatment of glioblastoma cells with salinomycin, an ER stress-inducing drug, and temozolomide resulted in enhanced DNA damage and a synergistic antitumor effect in vitro. Of importance, treatment with salinomycin/temozolomide resulted in a significant antiglioma effect in 2 aggressive orthotopic intracranial brain tumor models. These findings provide a strong rationale for combining temozolomide with ER stress-inducing drugs as an alternative therapeutic strategy for glioblastoma.

Germ Line Variants of Human N-Methylpurine DNA Glycosylase Show Impaired DNA Repair Activity and Facilitate 1,N6-Ethenoadenine-induced Mutations

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N(6)-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (KD) of hMPG toward 1,N(6)-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.

Naturally occurring polyphenol, morin hydrate, inhibits enzymatic activity of N-methylpurine DNA glycosylase, a DNA repair enzyme with various roles in human disease

Interest in the mechanisms of DNA repair pathways, including the base excision repair (BER) pathway specifically, has heightened since these pathways have been shown to modulate important aspects of human disease. Modulation of the expression or activity of a particular BER enzyme, N-methylpurine DNA glycosylase (MPG), has been demonstrated to play a role in carcinogenesis and resistance to chemotherapy as well as neurodegenerative diseases, which has intensified the focus on studying MPG-related mechanisms of repair. A specific small molecule inhibitor for MPG activity would be a valuable biochemical tool for understanding these repair mechanisms. By screening several small molecule chemical libraries, we identified a natural polyphenolic compound, morin hydrate, which inhibits MPG activity specifically (IC50=2.6μM). Detailed mechanism analysis showed that morin hydrate inhibited substrate DNA binding of MPG, and eventually the enzymatic activity of MPG. Computational docking studies with an x-ray derived MPG structure as well as comparison studies with other structurally-related flavonoids offer a rationale for the inhibitory activity of morin hydrate observed. The results of this study suggest that the morin hydrate could be an effective tool for studying MPG function and it is possible that morin hydrate and its derivatives could be utilized in future studies focused on the role of MPG in human disease.

DNA breaks and chromosomal aberrations arise when replication meets base excision repair.

Exposures that methylate DNA potently induce DNA double-strand breaks (DSBs) and chromosomal aberrations, which are thought to arise when damaged bases block DNA replication. Here, we demonstrate that DNA methylation damage causes DSB formation when replication interferes with base excision repair (BER), the predominant pathway for repairing methylated bases. We show that cells defective in the N-methylpurine DNA glycosylase, which fail to remove N-methylpurines from DNA and do not initiate BER, display strongly reduced levels of methylation-induced DSBs and chromosomal aberrations compared with wild-type cells. Also, cells unable to generate single-strand breaks (SSBs) at apurinic/apyrimidinic sites do not form DSBs immediately after methylation damage. In contrast, cells deficient in x-ray cross-complementing protein 1, DNA polymerase β, or poly (ADP-ribose) polymerase 1 activity, all of which fail to seal SSBs induced at apurinic/apyrimidinic sites, exhibit strongly elevated levels of methylation-induced DSBs and chromosomal aberrations. We propose that DSBs and chromosomal aberrations after treatment with N-alkylators arise when replication forks collide with SSBs generated during BER.