Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells.

Crude extracts of normal human diploid fibroblasts and of human peripheral blood lymphocytes excise thymine dimers from purified ultraviolet-irradiated DNA, or from the DNA presumably present as chromatin in unfractionated cell-free preparations of cells that had been labeled with [3H]thymidine. Extracts of xeroderma pigmentosum cells from complementation groups A, C, and D also excise thymine dimers from purified DNA, but extracts of group A cells do not excise dimers from the DNA of radioactively labeled unfractionated cell-free preparations.     

Nucleotide excision repair by dual incisions in plants

Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26–27 nucleotides are removed by incising ∼20 phosphodiester bonds 5′ and 5 phosphodiester bonds 3′ to the photoproduct.Plants and other organisms that depend on photosynthesis are, by necessity, exposed to more sunlight than other organisms that are chemotrophs or heterotrophs. Hence, plants are expected to receive more exposure to UV wavelengths of light than other organisms. The genotoxic effects of UV are somewhat mitigated by the reflection of UV by the waxy leaf surface and absorbance of UV by the intracellular pigments that are present at high concentration in plant cells, including carotenoids and flavonoids. Nevertheless, plants still receive considerable amounts of DNA-damaging UV radiation and therefore must have the means to cope with the damage to ensure their survival. Indeed, DNA sequencing has revealed that plant genomes contain genes that are homologous to the genes of all major DNA repair pathways, including photoreactivation, nucleotide excision repair, base excision repair, and recombination/double-strand break repair (1–6).However, biochemical studies of these DNA repair mechanisms have been limited. Of significance, Arabidopsis photolyases have been expressed in heterologous systems, purified, and characterized (7–9). Similarly, some of the enzymes of the base excision repair and recombination/double-strand break repair systems have been studied. In contrast, there have been no mechanistic studies on plant nucleotide excision repair, although it is known that plants can remove cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] in a photolyase-independent manner (6, 10, 11), presumably by nucleotide excision repair. Here, we have used an Arabidopsis cell line and the in vivo excision assay recently developed in our laboratory (12–14) to demonstrate that Arabidopsis removes these photoproducts by dual incisions in a manner that is virtually identical to human nucleotide excision repair.     

DNA切除修复酶的表达及与肺癌预后的关系

目的　探讨DNA切除修复鼠缺陷交叉互补基因 2 (ERCC2 )蛋白、尿嘧啶DNA糖基化酶(UDG)、增殖细胞核抗原 (PCNA)在不同肺组织中的表达及与肺癌预后的关系。方法　免疫组化法测ERCC2、UDG、PCNA在良性病变、癌周组织、肿瘤组织中的表达水平 ,并与临床、病理资料进行Ridit分析。结果　肿瘤组织、癌周组织ERCC2、UDG、PCNA表达与良性病变组织相比 ,差异有显著性 (P值均<0 0 5 ) ,其中ERCC2、UDG表达低于后者 ,PCNA表达高于后者 (P <0 0 0 1) ;但癌周组织中UDG的差异未达显著界值 (P >0 0 5 )。肿瘤组织与癌周组织相比 ,ERCC2、UDG、PCNA表达差异无显著性 (P值均 >0 0 5 )。年龄、吸烟、肺癌组织分型、肿瘤转移对ERCC2、UDG、PCNA的表达无显著影响 (P值均 >0 0 5 ) ,但UDG在低分化癌和肿瘤 >3cm的表达水平显著降低 (P <0 0 5 ) ,而PCNA则相反 (P <0 0 5 )。结论　UDG、PCNA的表达水平与肺癌分化、大小有关 ,可作为肺癌预后的指标。     

Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae.

A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae.     

Age-related base excision repair activity in mouse brain and liver nuclear extracts

To assess DNA repair activity relative to age, in vitro base excision repair assays were performed using brain and liver nuclear extracts prepared from mice of various ages. An 85% decline in repair activity was observed in brain nuclear extracts and a 50% decrease in liver nuclear extracts prepared from old mice compared with 6-day-old mice. Brain nuclear extracts prepared from old mice showed a decreased abundance of DNA polymerase-beta, but the addition of purified protein did not restore base excision repair activity. Abundances of other tested base excision repair proteins did not change relative to age. The conclusion is that, during aging, a decline in DNA repair could contribute to increased levels of DNA damage and mutagenesis.     

Mutations in DNA repair genes are associated with the Haarlem lineage of Mycobacterium tuberculosis independently of their antibiotic resistance

The analysis of the DNA repair genes ogt and ung was carried out in 117 Mycobacterium tuberculosis clinical isolates from Argentina and Colombia in order to explore correlation between mutations in these genes and multi-drug resistance. With the exception of two Beijing family isolates, the rest of the strains harbored either two wild-type or two mutant alleles with identical single nucleotide polymorphisms (SNPs) in each gene (ogt44 and ung501). These ogt44 and ung501 mutations were not associated with multi-drug resistance and occurred simultaneously in circulating Haarlem genotype M. tuberculosis strains. We therefore propose the use of these markers as tools in phylogenetic and epidemiologic studies.     

切除修复交叉互补基因1、生存素与顺铂耐药的研究进展

顺铂耐药有多种复杂机制参与,大量研究表明DNA修复途径的关键基因切除修复交叉互补基因1和参与细胞凋亡和有丝分裂调控的生存素基因与顺铂耐药密切相关.     

核苷酸切除修复基因表达水平与肺癌易感性关系的研究

目的 研究核苷酸切除修复基因表达水平与肺癌易感性关系,在分子水平探讨肺癌发病的相关危险因素.方法 采用病例-对照研究方法,应用实时荧光定量PCR技术检测50例原发性肺癌患者和32名健康者外周血淋巴细胞核苷酸切除修复基因(XPA、XPB、XPC、XPG、ERCC1)表达水平,分析这些基因表达水平与肺癌易感性的关系.结果 肺癌组XPB mRNA表达水平低于健康对照组(P＜0.05),XPB低表达的人群患肺癌的相对危险度是高表达人群的2.57倍.结论 XPB低表达的人群肺癌发病风险增高.     

Folate depletion impairs DNA excision repair in the colon of the rat

Background/Aims—Diminishedfolate status appears to promote colonic carcinogenesis by, as of yet,undefined mechanisms. Impaired DNA repair plays a significant role inthe evolution of many colon cancers. Since folate is essential for thede novo synthesis of nucleotides and sincefolate depletion has previously been associated with excessive DNAstrand breaks, it was hypothesised that folate depletion may impair DNArepair. Studies were therefore performed to examine whether folatedepletion affects the two major categories of DNA repair.
Methods—Study 1: eightweanling male Sprague-Dawley rats were fed on diets containing either 0 or 8 mg folate/kg diet with 1% succinylsulphathiazole for four weeks.After viable colonocytes had been harvested, DNA excision repair wasevaluated by a single cell gel electrophoresis assay. Study 2: eighteenanimals were fed on similar diets for five weeks. Also in study 2, 18 additional rats were fed on the same defined diet withoutsuccinylsulphathiazole for 15 weeks. Weekly injections with theprocarcinogen, 1,2-dimethylhydrazine (20 mg base/kg), were administeredto the latter group of animals. Five microsatellite loci from differentchromosomes were investigated for instability in hepatic and colonic DNA.
Results—In study 1, asignificantly retarded rate of DNA excision repair was observed in thefolate deficient colonocytes compared with controls (p<0.05). In study2, there was no evidence of instability at the five microsatellite lociassociated with either short or long term folate depletion.
Conclusions—Folatedeficiency impairs DNA excision repair in rat colonic mucosa; a similardegree of deficiency, even when administered in conjunction with acolonic carcinogen, did not produce evidence of a widespread defect inmismatch repair.

Keywords:folate; colon cancer; DNA repair; single cell gelelectrophoresis; microsatellite instability; rat

     

Aag-initiated base excision repair promotes ischemia reperfusion injury in liver,brain, and kidney

Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag^−/− mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag^−/− mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag^−/− liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.Ischemia reperfusion (I/R)-induced tissue injury is one of the most common examples of acute, sterile inflammation-induced tissue damage. Health events that incur I/R include ischemic stroke, acute liver or kidney failure, myocardial infarction, various forms of circulatory shock, sickle cell disease, and organ transplantations. These conditions are frequently accompanied by profound morbidity and mortality worldwide (1). The need for effective approaches to manage patients with I/R-induced organ damage is highlighted by the fact that current treatment is primarily supportive care (1).During I/R, a burst of reactive oxygen and nitrogen species (RONS) occurs within the first moments of reperfusion, and this burst is thought to be primarily responsible for collateral tissue damage (2). Furthermore, RONS are also generated during the preceding ischemia, despite the low oxygen tension (3). I/R alters cellular metabolism and redox state, and induces so-called “sterile inflammation” by activating the innate immune system. The activation of macrophages and recruitment of neutrophils to sites of I/R by intravascular danger signals results in the release of an arsenal of RONS capable of inducing DNA damage and lipid peroxidation, thus causing major collateral tissue damage. Base excision repair (BER), initiated by various DNA glycosylases, is critical for the repair of RONS-associated DNA damage, including oxidized, deaminated, and etheno (ε)-adducted DNA bases (4, 5). BER involves the orchestration of the following enzymatic steps: damaged bases are excised by DNA glycosylase, followed by cleavage of the DNA backbone at the resulting abasic site; DNA ends are trimmed to generate a 3′OH and 5′P; the gap is then filled by DNA polymerase and the remaining nick sealed by DNA ligase to complete BER (6, 7) (Fig. 1A). The murine alkyladenine DNA glycosylase (Aag; also known as Mpg) acts efficiently on 1,N⁶-ethenodeoxyadenosine (ε-A) lesions and deaminated adenosine (hypoxanthine, Hx) (8, 9), and with lower efficiency on 8-oxoguanine (8-oxoG) (10), all of which are induced directly or indirectly by RONS (11). Aag therefore seemed likely to provide resistance to I/R-induced toxicity. In support of this hypothesis, we previously found that Aag^−/− mice suffer more inflammation-associated intestinal tissue damage and colon carcinogenesis than WT mice (5, 12); with this in mind, we set out to examine whether the Aag DNA glycosylase modulates I/R-induced tissue injury in three distinct tissue environments—namely liver, brain, and kidney. To our surprise, we find that Aag-initiated BER actually exacerbates, rather than attenuates, I/R tissue injury in all three organs.Open in a separate windowFig. 1.Aag deficiency protects liver against I/R-induced tissue damage. (A) The BER pathway initiated by various DNA glycosylases consists of four key enzymatic steps: (i) glycosylase-mediated excision of a damaged base generating an abasic site; (ii) incision of the sugar-phosphate backbone at the abasic site by APE, yielding a 3′-OH adjacent to a 5′-dRP moiety; (iii) 5′-dRP removal and DNA synthesis by DNA polymerase β (Pol β); and (iv) sealing of the DNA nick by DNA ligase (LIG). (B) Serum ALT concentrations after 90 min of ischemia and 24 h of reperfusion (n = 3 in sham groups and n = 7–8 in I/R groups). (C) Representative images of necrotic areas 24 h after I/R in Aag^−/− (n = 7) or WT livers (n = 8); liver sections (4 μm) were stained with H&E. (D) Mean necrotic area in ischemic liver samples and (E) serum LDH concentrations 24 h after reperfusion (n = 3 in sham groups and n = 7–8 in I/R groups); necrosis analyzed by ImageJ (necrotic areas indicated by green color). (F) Serum ALT in control mice (no surgery) or after 60 min of ischemia plus either 6- or 24-h reperfusion in WT and Aag^−/− mice. Error bars represent the mean ± SEM of at least three independent experiments.     

Nucleotide excision repair pathway gene polymorphisms are associated with risk and prognosis of colorectal cancer

BACKGROUND Single nucleotide polymorphisms(SNPs)are universally present in nucleotide excision repair(NER)pathway genes,which could make impacts on colorectal carcinogenesis and prognosis.AIM To explore the association of all tagSNPs in NER pathway genes with colorectal cancer(CRC)risk and prognosis in a northern Chinese population by a two-stage case-control design composed of a discovery and validation stage.METHODS Genotyping for NER SNPs was performed using kompetitive allele specific PCR.In the discovery stage,39 tagSNPs in eight genes were genotyped in 368 subjects,including 184 CRC cases and 184 individual-matched controls.In the validation stage,13 SNPs in six genes were analyzed in a total of 1712 subjects,including 854 CRC cases and 858 CRC-free controls.RESULTS Two SNPs(XPA rs10817938 and XPC rs2607775)were associated with an increased CRC risk in overall and stratification analyses.Significant cumulative and interaction effects were also demonstrated in the studied SNPs on CRC risk.Another two SNPs(ERCC2 rs1052555 and ERCC5 rs2228959)were newly found to be associated with a poor overall survival of CRC patients.CONCLUSION Our findings suggest novel SNPs in NER pathway genes that can be predictive for CRC risk and prognosis in a large-scale Chinese population.The present study has referential values for the identification of all-round NER-based genetic biomarkers in predicting the susceptibility and clinical outcome of CRC.     

胸腔积液游离核酸检测在结核性胸膜炎早期诊断中的价值

目的: 评价胸腔积液结核分枝杆菌游离核酸检测技术(cell-free Mycobacterium tuberculosis DNA test,CF.TB)在结核性胸膜炎中的诊断价值。方法: 选取2016年7月至2018年12月收住于首都医科大学附属北京胸科医院初诊不明原因胸腔积液患者216例。所有患者均在B超引导下进行胸腔穿刺术或置管术留取胸腔积液,并同时进行胸腔积液CF.TB、SAT-PCR、GeneXpert MTB/RIF及腺苷脱氨酶(adenosine deaminase,ADA)检测和血结核感染T细胞斑点试验(T-SPOT.TB)检测。以临床复合标准(composite reference standard,CRS)为参考标准,评价胸腔积液CF.TB、SAT-PCR、GeneXpert MTB/RIF、ADA及血T-SPOT.TB检测诊断结核性胸膜炎的敏感度、特异度、阳性预测值、阴性预测值。评价胸腔积液CF.TB方法单独或与ADA联合检测对结核性胸膜炎早期诊断的价值。结果: 216例患者中,165例患者临床确诊为结核性胸膜炎,51例患者确诊为其他病因胸腔积液。以CRS为参考标准,CF.TB、SAT-PCR、GeneXpert MTB/RIF、ADA及T-SPOT.TB方法对结核性胸膜炎诊断的敏感度分别为70.30%(116/165)、7.88%(13/165)、12.12%(20/165)、67.27%(111/165)和87.27%(144/165),特异度分别为100.00%(51/51)、100.00%(51/51)、100.00%(51/51)、92.16%(47/51)和62.75%(32/51),阳性预测值分别为100.00%(116/116)、100.00%(13/13)、100.00%(20/20)、96.52%(111/115)和88.34%(144/163),阴性预测值分别为51.00%(51/100)、25.12%(51/203)、26.02%(51/196)、46.53%(47/101)和60.38%(32/53)。CF.TB检测胸腔积液MTB的敏感度(70.30%)明显高于SAT-PCR(7.88%)、 GeneXpert MTB/RIF(12.12%)和ADA(67.27%)(χ²=1.350、1.153、1.025,P值均=0.000),差异均有统计学意义;而且CF.TB与ADA联合检测(88.48%, 146/165)可明显提高CF.TB(70.30%, 116/165)诊断结核性胸膜炎患者的敏感度,差异有统计学意义(χ²=16.670,P=0.000)。结论: 胸腔积液CF.TB检测技术对结核性胸膜炎诊断的敏感度优于SAT-PCR及GeneXpert MTB/RIF技术,特异度优于ADA及T-SPOT.TB方法,而且CF.TB与ADA联合检测可明显提高其在结核性胸膜炎中的诊断价值,建议CF.TB技术可作为结核性胸膜炎的辅助诊断。