A robust luminescent assay for screening alkyladenine DNA glycosylase inhibitors to overcome DNA repair and temozolomide drug resistance

Temozolomide(TMZ)is an anticancer agent used to treat glioblastoma,typically following radiation therapy and/or surgical resection.However,despite its effectiveness,at least 50%of patients do not respond to TMZ,which is associated with repair and/or tolerance of TMZ-induced DNA lesions.Studies have demonstrated that alkyladenine DNA glycosylase(AAG),an enzyme that triggers the base excision repair(BER)pathway by excising TMZ-induced N3-methyladenine(3meA)and N7-methylguanine lesions,is overexpressed in glioblastoma tissues compared to normal tissues.Therefore,it is essential to develop a rapid and efficient screening method for AAG inhibitors to overcome TMZ resistance in glioblastomas.Herein,we report a robust time-resolved photoluminescence platform for identifying AAG inhibitors with improved sensitivity compared to conventional steady-state spectroscopic methods.As a proof-of-concept,this assay was used to screen 1440 food and drug administration-approved drugs against AAG,resulting in the repurposing of sunitinib as a potential AAG inhibitor.Sunitinib restored glioblastoma(GBM)cancer cell sensitivity to TMZ,inhibited GBM cell proliferation and stem cell characteristics,and induced GBM cell cycle arrest.Overall,this strategy offers a new method for the rapid identification of small-molecule inhibitors of BER enzyme activities that can prevent false negatives due to a fluorescent background.

Biochemical Characterization of Uracil-DNA Glycosylase from Pyrococcus furiosus

We report the characterization of a uracil-DNA glycosylase（UDG） from the hyperthermophilic archaea Pyrococcus furiosus（P, furiosus）. P. furiosus UDG（PfUDG） has high sequence similarity to the families IV and V UDGs（thermostable UDG family and PaUDG-b family）. PfUDG excises uracil from various DNA substrates with the following order： U/T=U/C〉U/G=U/AP=U/-〉U/U=U/I=U/A. The optimal temperature and pH value for uracil exci- sion by PfUDG are 70 ℃ and 9.0, respectively. The removal of U is inhibited by the divalent ions of Fe, Ca, Zn, Cu, Co, Ni and Mn, as well as a high concentration of NaC1. The phosphorothioates near uracil strongly inhibit the exci- sion of uracil by PfUDG. Interestingly, pfuDNA（Pyrococcusfuriosus DNA） polymerase, which tightly binds the ura- cil-carrying oligonucleotide, does not inhibit the excision by Pfl.IDG, suggesting PfUDG in vivo functions as the re- pair enzyme to excise uracil damage in genome.

Fusion of a rice endogenous N-methylpurine DNA glycosylase to a plant adenine base transition editor ABE8e enables A-toK 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.

Ginsenoside Rd Attenuates DNA Damage by Increasing Expression of DNA Glycosylase Endonuclease VIII-like Proteins after Focal Cerebral Ischemia

Background： Ginsenoside Rd （GSRd）, one of the main active ingredients in traditional Chinese herbal Panax ginseng, has been found to have therapeutic effects on ischemic stroke. However, the molecular mechanisms of GSRd＇s neuroprotective function remain unclear. Ischemic stroke-induced oxidative stress results in DNA damage, which triggers cell death and contributes to poor prognosis. Oxidative DNA damage is primarily processed by the base excision repair （BER） pathway. Three of the five major DNA glycosylases that initiate the BER pathway in the event of DNA damage from oxidation are the endonuclease VIII-like （NELL） proteins. This study aimed to investigate the effect of GSRd on the expression ofDNA glycosylases NEILs in a rat model of focal cerebral ischemia. Methods： NEIL expression patterns were evaluated by quantitative real-time polymerase chain reaction in both normal and middle cerebral artery occlusion （MCAO） rat models. Survival rate and Zea-Longa neurological scores were used to assess the effect of GSRd administration on MCAO rats. Mitochondrial DNA （mtDNA） and nuclear DNA （nDNA） damages were evaluated by the way of real-time analysis of mutation frequency. NEIL expressions were measured in both messenger RNA （mRNA） and protein levels by quantitative polymerase chain reaction and Western blotting analysis. Apoptosis level was quantitated by the expression of cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay. Results： We found that GSRd administration reduced mtDNA and nDNA damages, which contributed to an improvement in survival rate and neurological function; significantly up-regulated NEIL1 and NEIL3 expressions in both mRNA and protein levels of MCAO rats; and reduced cell apoptosis and the expression of cleaved caspase-3 in rats at 7 days after MCAO. Conclusions： Our results indicated that the neuroprotective function of GSRd for acute ischemic stroke might be partially explained by the up-regulation of NEILI and NEIL3 expressions.

1-Methyl-4-phenyl-pyridinium time-dependently alters expressions of oxoguanine glycosylase 1 and xeroderma pigmentosum group F protein in PC12 cells

Objective To determine if DNA excision repair enzymes oxoguanine glycosylase 1 （OGG1） and xeroderma pigmentosum group F protein （XPF） are involved in the pathogenesis of Parkinson＇s disease （PD） in a cell model. Methods PC12 cells were treated with 1-Methyl-4-phenylpyridine ion （MPP＋） for various periods of time to induce oxidative DNA damage. MTT assay was used to determine cell viability. Immunocytochemistry with antibody against 8-hydroxy-2＇- deoxyguanosine （8-oxodG） was used to evaluate oxidative DNA damage. Immunoblotting was used to detect the protein levels of OGG1 and XPF. Results MPP＋ treatment （1 mmol/L） for 18 h and 24 h reduced cell viability to 78.6% and 70.3% of the control, respectively, in a time-dependent way. MPP＋ increased the immunoreactivity of 8-oxodG in the cytoplasm at 3 h and in the nucleus at 24 h of treatment. With the treatment of MPP＋, the expression of OGG1 was significantly increased at 1 h, reaching a peak at 3 h, and then it was decreased at 24 h, as compared to that with vehicle treatment. The same effect was exerted on XPF level, except that the XPF level reached a peak at 18 h of MPP＋ treatment. Moreover, the maximally-increased protein level of OGG1 by MPP＋ was approximately 2-fold higher than that of XPF. Conclusion MPP＋ treatment could time- dependently induce increases in OGG1 and XPF expressions in PC12 cells. Also, this study indicates that the base and nucleotide excision repair pathways may be compensatorily activated in the early stage of pathogenesis in the cells after MPP＋ treatment.

Recent advances in DNA glycosylase assays

Genomic deoxyribonucleic acid(DNA)is selected as the ideal carrier for preserving and transmitting the genetic information over the course of evolution.However,the genomic DNA is constantly exposed to various endogenous and environmental threats,causing a diversity of damaged bases,lesions,mismatches and base-pair modifications in the genome,eventually leading to genomic instability and cancers.Base excision repair(BER)is the most important repair mechanism,repairing a variety of DNA damages arising from oxidation,alkylation,methylation,deamination,and hydrolysis reactions.DNA glycosylases are responsible for initiating the first step of the BER pathway through cleaving the N-glycosidic bond between the damaged base and the DNA backbone.However,abnormal DNA glycosylases are associated with a variety of diseases such as cancer,cardiovascular disease,neurological disease and inflammation,suggesting the important role of DNA glycosylases in cancer diagnosis and treatment.Therefore,it is highly desirable to monitor the activity of DNA glycosylases,gaining a deep understanding of the restoration process of damaged DNA and clinical diagnosis.Recently,a series of novel DNA glycosylases detection methods with excellent performance have been developed.In this minireview,we summarize the recent advances in DNA glycosylase assays including amplification-free assay and amplification-assisted assay.Firstly,a brief introduction of amplification-free assay for DNA glycosylase is given.Then,amplification-assisted assays for DNA glycosylases are discussed in detail.Ultimately,the conclusion and prospects of the directions of DNA glycosylase assays are provided.

Oxidative stress and male reproductive health

One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also limits the fertilizing potential of these cells as a result of collateral damage to proteins and lipids in the sperm plasma membrane. The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high leve｜s of superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to respond to such an attack because they only possess the first enzyme in the base excision repair （BER） pathway, 8-oxoguanine glycosylase 1 （OGG1）. The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further because they lack the downstream proteins （APE1, XRCC1） needed to complete the repair process. It is the responsibility of the oocyte to continue the BER pathway prior to initiation of S-phase of the first mitotic division. If a mistake is made by the oocyte at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the spermatozoa of male infertility patients may have implications for the health of children conceived in vitro and serves as a driver for current research into the origins of free radical generation in the germ line.

hOGG1 Ser326Cys polymorphism modifies the significance of the environmental risk factor for colon cancer

AIM:To determine the association of hOGG1 (8-oxoguanine glycosylase I,OGG1) polymorphism of Ser326Cys substitution with colon cancer risk and possible interaction with known environmental risk factors. METHODS:A case-control study with 125 colon cancer cases and 247 controls was conducted, RESULTS:There was no major difference in Ser326Cys genotype distribution between cases and controls.The meat intake tended to increase the odds ratio for colon cancer with an OR of 1.72 (95 % confidence interval;CI=1.12-2.76). Such tendency was more prominent in Cys/Cys carriers (OR=4.31,95 % CI=1.64-11.48),but meat intake was not a significant risk factor for colon cancer in Ser/Ser or Ser/ Cys carriers.The OR for colon cancer was elevated with marginal significance in smokers who were Cys/Cys carriers (OR=2.75,95 % CI=1.07-7.53) but not in Ser/Ser or Ser/ Cys carriers. CONCLUSION:These results suggest that the hOGG1 Ser326Cys polymorphism is probably not a major contributor to individual colon cancer susceptibility overall,but the Cys/ Cys genotype may alter the impact of some environmental factors on colon cancer development.

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Base excision repair （BER） is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged （oxidized or aikylated） or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species （ROS）. BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease （APE） to generate 3＇ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA iigase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEI, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3＇ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and iigases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organeile targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

Structure and mechanism for DNA lesion recognition

A fundamental question in DNA repair is how a lesion is detected when embedded in millions to billions of normal base pairs. Extensive structural and functional studies reveal atomic details of DNA repair protein and nucleic acid interactions. This review summarizes seemingly diverse structural motifs used in lesion recognition and suggests a general mechanism to recognize DNA lesion by the poor base stacking. After initial recognition of this shared structural feature of lesions, different DNA repair pathways use unique verification mechanisms to ensure correct lesion identification and removal.

HOGG1 polymorphism in atrophic gastritis and gastric cancer after Helicobacter pylori eradication

AIM:To investigate the association between Ser326Cys human oxoguanine glycosylase 1(hOGG1) polymorphism and atrophic gastritis and gastric cancer after Helicobacter pylori(H.pylori) eradication.METHODS:A total of 488 subjects(73 patients with gastric cancer,160 with atrophic gastritis after H.pylori eradication and 255 controls) were prospectively collected.Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to distinguish hOGG1 Ser326Cys polymorphism.Statistical analysis was conducted by two-sample t test for continuous variables and χ2 test for categorical variables.Logistic regression models were used to f ind the risk factors for gastric cancer and atrophic gastritis.RESULTS:Neither the hOGG1 Ser/Cys nor the Cys/Cys genotype was associated with gastric cancer.Compared with the Ser/Ser genotype,odds ratio(OR) for Ser/Cys was 0.96,(95% CI:0.51-1.84) and OR for Cys/Cys was 1.1(95% CI:0.48-2.1).No association was detected between hOGG1 polymorphism and Lauren type of gastric cancer(P=0.61) either.However,Ser/Cys and Cys/Cys were signif icantly associated with atrophic gastritis with OR:1.76 for Ser/Cys(95% CI:1.03-3.0) and 2.38 for Cys/Cys(95% CI:1.34-4.23).After controlling for age,gender,smoking and alcohol,there were still signif icant associations with OR:2.05 for Ser/Cys(95% CI:1.14-3.68) and 2.76 for Cys/Cys(95% CI:1.47-5.18).CONCLUSION:HOGG1 polymorphisms(Cys/Cys and Ser/Cys) are associated with atrophic gastritis.No significant association is detected between hOGG1 polymorphisms(Cys/Cys or Ser/Cys) and gastric cancer.

Transforming growth factor-β and toll-like receptor-4 polymorphisms are not associated with fibrosis in haemochromatosis

AIM:To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis.METHODS:A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied,with all subjects having liver biopsy data and DNA available for testing.This study assessed the association of eight single nucleotide polymorphisms(SNPs)in a total of six genes including toll-like receptor 4(TLR4),transforming growth factor-beta(TGF-β),oxoguanine DNA glycosylase,monocyte chemoattractant protein 1,chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity.Genotyping was performed using high resolution melt analysis and sequencing.The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration.RESULTS:There were significant associations between the cofactors of male gender(P=0.0001),increasing age(P=0.006),alcohol consumption(P=0.0001),steatosis(P=0.03),hepatic iron concentration(P<0.0001)and the presence of hepatic fibrosis.Of the candidate gene polymorphisms studied,none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors.We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied.Importantly,in this large,well characterised cohort of patients there was no association between SNPs for TGF-βor TLR4and the presence of fibrosis,cirrhosis or increasing fibrosis stage in multivariate analysis.CONCLUSION:In our large,well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis.

Genetic and environmental determinants of risk for cholangiocarcinoma in Thailand

Cholangiocarcinoma(CCA) is a difficult cancer to diagnose in the early stage and to treat by curative resec-tion. The incidence of CCA in the northeast of Thailand is the highest in the world. To make progress in detecting a high risk group and in the prevention and detection of CCA, we have been analyzing the risk factors for CCA. Although liver fluke infection is known to be a risk factor, there are patients who are not infected with the liver fluke and not all people infected with the liver fluke will suffer from the disease. Therefore, it is of the utmost importance to analyze the risk factors and the mechanism to prevent the disease and also to detect the disease in its early stage to save patients' lives. Through collaboration among Thai and Japanese researchers, we analyzed the genetic and environmental determinants of risks for CCA. Also, we have been trying to develop methods to detect the disease in a non-invasive way. Without repeating findings reported in various reviews on CCA, we will first discuss the environmental and genetic determinants of the risks for CCA. Second, we will discuss the properties of CCA, including the etiological agents and the mechanism of cholangiocarcinogenesis, and finally, we will discuss future approaches to prevent and cure CCA from the standpoint of evidence-based medicine. We will discuss these points by including the data from our laboratories. We would like to emphasize the importance of the genetic data, especially whole genome approaches, to understand the properties of CCA, to find a high risk population for CCA and to develop effective preventative methods to stop the carcinogenic steps toward CCA in the near future. In addition, it is of the upmost importance to develop a non-invasive, specific and sensitive method to detect CCA in its early stage for the application of modern medical approaches to help patients with CCA.

A novel strategy to protect prokaryotic cells from virus infection

The recent discovery of the CRISPR-Cas-mediated acquired immunity system highlights the fact that our knowl-edge of phage/virus defense mechanisms encoded in bacterial and archaeal genomes is far from complete.Indeed,new prokaryotic immune systems are now continually being discovered.A recent report described a novel glyco-sylase that recognizesα-glycosyl-hydroxymethyl cytosin(α-Glu-hmC),a modified base observed in the T4 phage genome,where it produces an abasic site,thereby inhibiting the phage propagation.

OGG1 inhibition suppresses African swine fever virus replication

African swine fever virus(ASFV)is an important pathogen that causes a highly contagious and lethal disease in swine,for which neither a vaccine nor treatment is available.The DNA repair enzyme 8-oxoguanine DNA glycosylase 1(OGG1),which excises the oxidative base lesion 8-oxo-7,8-dihydroguanine(8-oxoG),has been linked to the pathogenesis of different diseases associated with viral infections.However,the role of OGG1-base excision repair(BER)in ASFV infection has been poorly investigated.Our study aimed to characterize the alteration of host reactive oxygen species(ROS)and OGG1 and to analyse the role of OGG1 in ASFV infection.We found that ASFV infection induced high levels and dynamic changes in ROS and 8-oxoG and consistently increased the expression of OGG1.Viral yield,transcription level,and protein synthesis were reduced in ASFV-infected primary alveolar macrophages(PAMs)treated by TH5487 or SU0268 inhibiting OGG1.The expression of BER pathway associated proteins of ASFV was also suppressed in OGG1-inhibited PAMs.Furthermore,OGG1 was found to negatively regulate interferonβ(IFN-β)production during ASFV infection and IFN-βcould be activated by OGG1 inhibition with TH5487 and SU0268,which blocked OGG1 binding to 8-oxoG.Additionally,the interaction of OGG1 with viral MGF360-14-L protein could disturb IFN-βproduction to further affect ASFV replication.These results suggest that OGG1 plays the crucial role in successful viral infection and OGG1 inhibitors SU0268 or TH5487 could be used as antiviral agents for ASFV infection.

DNA cytosine methylation in plant development

Cytosine bases of the nuclear genome in higher plants are often extensively methylated.Cytosine methylation has been implicated in the silencing of both transposable elements （TEs） and endogenous genes,and loss of methylation may have severe functional consequences.The recent methylation profiling of the entire Arabidopsis genome has provided novel insights into the extent and pattern of cytosine methylation and its relationships with gene activity.In addition,the fresh studies also revealed the more dynamic nature of this epigenetic modification across plant development than previously believed.Cytosine methylation of gene promoter regions usually inhibits transcription,but methylation in coding regions （gene-body methylation） does not generally affect gene expression.Active demethylation （though probably act synergistically with passive loss of methylation） of promoters by the 5-methyl cytosine DNA glycosylase or DEMETER （DME） is required for the uni-parental expression of imprinting genes in endosperm,which is essential for seed viability.The opinion that cytosine methylation is indispensible for normal plant development has been reinforced by using single or combinations of diverse loss-of-function mutants for DNA methyltransferases,DNA glycosylases,components involved in siRNA biogenesis and chromatin remodeling factors.Patterns of cytosine methylation in plants are usually faithfully maintained across organismal generations by the concerted action of epigenetic inheritance and progressive correction of strayed patterns.However,some variant methylation patterns may escape from being corrected and hence produce novel epialleles in the affected somatic cells.This,coupled with the unique property of plants to produce germline cells late during development,may enable the newly acquired epialleles to be inherited to future generations,which if visible to selection may contribute to adaptation and evolution.

hOGG1 Ser326Cys and XRCC1 Arg399Gln polymorphisms associated with chronic obstructive pulmonary disease

Background Cigarette-smoke induced DNA damage can cause airway cell apoptosis and death, which may be associated with the development of chronic obstructive pulmonary disease （COPD）. However, only 20%-30% of smokers develop COPD, suggesting that different degrees of DNA repair produce different outcomes in smokers, i.e., part of them develop COPD. We investigated the association between polymorphisms in DNA repair genes hOGG1 （Ser326Cys） and XRCC1 （Arg399GIn）, alone or in combination, and susceptibility of COPD. Methods Altogether 201 COPD patients and 309 controls were recruited and frequency-matched on age and sex. hOGG1 and XRCC1 genotypes were determined by PCR-restriction fragment length polymorphism analysis. Results The risk of COPD was not significantly different among individuals with Ser/Cys and Cys/Cys genotypes compared with those with hOGG1 Ser/Ser genotype. The risk of COPD was not significantly different among individuals with Gin/Gin genotype compared with those with XRCC1 Arg/Arg genotype, but it was significantly elevated among individuals with Arg/GIn genotype （adjusted odds ratios （OR）=1.55, 95% confidence intervals （CI） 1.05-2.29, P=0.029）. Assessment of smoking status in current smokers compared with those with hOGG1 Ser/Ser genotype revealed that the risk of COPD was significantly elevated among individuals with Cys/Cys genotype （adjusted OR=5.07, 95% CI 1.84-13.95, P=0.002）. Compared with those with XRCC1 Arg/Arg genotype, the risk of COPD was significantly elevated among individuals with Arg/GIn genotype （adjusted OR=2.77, 95% CI 1.52-5.07, P=-0.001）. Assessment of smoking exposure in light smokers compared with those with hOGG1 Ser/Ser genotype showed that the risk of COPD was significantly elevated among individuals with Cys/Cys genotype （adjusted OR=4.02, 95% CI 1.05-16.80, P=0.042）. Compared with those with XRCC1 Arg/Arg genotype, the risk of COPD was significantly elevated among individuals with Gin/Gin genotype （adjusted OR=4.48, 95% CI 1.35-14.90, P=0.014）. In heavy smokers compared with those with XRCC1 Arg/Arg genotype, the risk of COPD was significantly elevated among individuals with Arg/GIn genotype （adjusted OR= 2.55, 95% CI 1.42-4.58, P=0.002）. When hOGG1 Ser326Cys and XRCC1 Arg399GIn polymorphisms were evaluated together, compared with those with 0-1 of hOGG1 326Cys and XRCC1 399Gin alleles, the risk of COPD was significantly elevated among individuals with 3-4 of hOGG1 326Cys and XRCC1 399Gin alleles （adjusted OR=3.18, 95% CI 1.86-5.43, P=0.000）. Assessment of smoking status and smoking exposure in current/light/heavy smokers showed that the risk of COPD was significantly elevated among individuals with 3-4 of hOGG1 326Cys and XRCC1 399Gin alleles （adjusted OR=8.32, 95% CI 3.59-19.27, P=0.000; OR=5.46, 95% CI 2.06-14.42, P=0.001; OR=2.93, 95% CI 1.43-6.02, P=0.003; respectively）. Conclusions hOGG1 Ser326Cys and XRCC1 Arg399GIn polymorphisms are associated with the susceptibility to COPD. The risk of COPD is significantly elevated among current/light smokers with hOGG1 326Cys and XRCC1 399Gin.

My Journey to DNA Repair

I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have dis- covered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mam- malian exonucleases DNase III （TREX1） and IV （FEN1）. Interestingly, expression of TREXI was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O6-methylguanine （O6mG） is repaired without removing the guanine from DNA, identifying a sur- prising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation.

DNA demethylases remodel DNA methylation in rice gametes and zygote and are required for reproduction

Fertilization constitutes a critical step in the plant life cycle during which the gamete genomes undergo chromatin dynamics in preparation for embryogenesis. In mammals, parental chromatin is extensively reprogrammed through the global erasure of DNA methylation. However, in flowering plants it remains unclear whether and how DNA methylation is remodeled in gametes and after fertilization in the zygote. In this study, we characterize DNA methylation patterns and investigate the function of DNA glycosylases in rice eggs, sperm, and unicellular zygotes and during embryogenesis. We found that DNA methylation is locally reconfigured after fertilization and is intensified during embryogenesis. Genetic, epigenomic, and transcriptomic analysis revealed that three rice DNA glycosylases, DNG702, DNG701, and DNG704, demethylate DNA at distinct genomic regions in the gametes and the zygote, and are required for zygotic gene expression and development. Collectively, these results indicate that active DNA demethylation takes place in the gametes and the zygote to locally remodel DNA methylation, which is critical for egg and zygote gene expression and reproduction in rice.

A TDG/CBP/RARα Ternary Complex Mediates the Retinoic Acid-dependent Expression of DNA Methylation-sensitive Genes

The thymine DNA glycosylase （TDG） is a multifunctional enzyme,which is essential for embryonic development.It mediates the base excision repair （BER） of G：T and G：U DNA mismatches arising from the deamination of 5-methyl cytosine （5-MeC） and cytosine,respectively.Recent studies have pointed at a role of TDG during the active demethylation of 5-MeC within CpG islands.TDG interacts with the histone acetylase CREB-binding protein （CBP） to activate CBP-dependent transcription.In addition,TDG also interacts with the retinoic acid receptor α （RARα）,resulting in the activation of RARα target genes.Here we provide evidence for the existence of a functional ternary complex containing TDG,CBP and activated RARα.Using global transcriptome profiling,we uncover a coupling of de novo methylation-sensitive and RA-dependent transcription,which coincides with a significant subset of CBP target genes.The introduction of a point mutation in TDG,which neither affects overall protein structure nor BER activity,leads to a significant loss in ternary complex stability,resulting in the deregulation of RA targets involved in cellular networks associated with DNA replication,recombination and repair.We thus demonstrate for the first time a direct coupling of TDG＇s epigenomic and transcription regulatory function through ternary complexes with CBP and RARα.