Recognition of DNA damage by the Rad4 nucleotide excision repair protein

Mutations in the nucleotide excision repair (NER) pathway can cause the xeroderma pigmentosum skin cancer predisposition syndrome. NER lesions are limited to one DNA strand, but otherwise they are chemically and structurally diverse, being caused by a wide variety of genotoxic chemicals and ultraviolet radiation. The xeroderma pigmentosum C (XPC) protein has a central role in initiating global-genome NER by recognizing the lesion and recruiting downstream factors. Here we present the crystal structure of the yeast XPC orthologue Rad4 bound to DNA containing a cyclobutane pyrimidine dimer (CPD) lesion. The structure shows that Rad4 inserts a beta-hairpin through the DNA duplex, causing the two damaged base pairs to flip out of the double helix. The expelled nucleotides of the undamaged strand are recognized by Rad4, whereas the two CPD-linked nucleotides become disordered. These findings indicate that the lesions recognized by Rad4/XPC thermodynamically destabilize the Watson-Crick double helix in a manner that facilitates the flipping-out of two base pairs.     

(ADP-ribose)n participates in DNA excision repair

Chromatin proteins are covalently modified by at least five different processes; in no case has the precise physiological function been established. One of these post-synthetic, covalent modifications is effected by the enzyme poly(ADP-ribose) polymerase, which uses the coenzyme NAD+ to ADP-ribosylate chromatin proteins. The modification consists largely of mono(ADP-ribose), but long, homopolymer chains of (ADP-ribose) are also present. Various physiological functions have been suggested for (ADP-ribose)n. Here we demonstrate that one function of (ADP-ribose)n is to participate in the cellular recovery from DNA damage. Specific inhibitors of poly(ADP-ribose) polymerase prevent rejoining of DNA strand breaks caused by dimethyl sulphate and cytotoxicity is enhanced thereby. The rejoining of strand breaks is prevented also by nutritionally depleting the cells of NAD.     

Requirement for the replication protein SSB in human DNA excision repair

Replication and repair are essential processes that maintain the continuity of the genetic material. Dissection of simian virus 40 (SV40) DNA replication has resulted in the identification of many eukaryotic replication proteins, but the biochemistry of the multienzyme process of DNA excision repair is less well defined. One protein that is absolutely required for semiconservative replication of SV40 DNA in vitro is human single-stranded DNA-binding protein (SSB, also called RF-A and RP-A). SSB consists of three polypeptides of relative molecular mass 70,000, 34,000 and 13,000, and acts with T antigen and topoisomerases to unwind DNA, allowing the access of other replication proteins. Human SSB can also stimulate the activity of polymerases alpha and delta, suggesting a further role in elongation during DNA replication. We have now found a role for human SSB in DNA excision repair using a cell-free system that can carry out nucleotide excision repair in vitro. Monoclonal antibodies against human SSB caused extensive inhibition of DNA repair in plasmid molecules damaged by ultraviolet light or acetylaminofluorene. Addition of purified SSB reversed this inhibition and further stimulated repair synthesis by increasing the number of repair events. These results show that a mammalian DNA replication protein is also essential for repair.     

Alternative nucleotide incision repair pathway for oxidative DNA damage.

The DNA glycosylase pathway, which requires the sequential action of two enzymes for the incision of DNA, presents a serious problem for the efficient repair of oxidative DNA damage, because it generates genotoxic intermediates such as abasic sites and/or blocking 3'-end groups that must be eliminated by additional steps before DNA repair synthesis can be initiated. Besides the logistical problems, biological evidence hints at the existence of an alternative repair pathway. Mutants of Escherichia coli and mice (ref. 4 and M. Takao et al., personal communication) that are deficient in DNA glycosylases that remove oxidized bases are not sensitive to reactive oxygen species, and the E. coli triple mutant nei, nth, fpg is more radioresistant than the wild-type strain. Here we show that Nfo-like endonucleases nick DNA on the 5' side of various oxidatively damaged bases, generating 3'-hydroxyl and 5'-phosphate termini. Nfo-like endonucleases function next to each of the modified bases that we tested, including 5,6-dihydrothymine, 5,6-dihydrouracil, 5-hydroxyuracil and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine residues. The 3'-hydroxyl terminus provides the proper end for DNA repair synthesis; the dangling damaged nucleotide on the 5' side is then a good substrate for human flap-structure endonuclease and for DNA polymerase I of E. coli.     

Influence of He-Ne laser irradiation on the excision repair of cyclobutyl pyrimidine dimers in the wheat DNA

Using agarose gel electrophoresis and T4-endodeoxyribonuclease-V, which is a kind of restriction endonuclease of cyclobutyl pyrimidine dimer (CPD), the impacts of He-Ne laser (5 mW · mm⁻²) irradiation on DNA excision repair capacity in damaged wheat cells induced by enhanced ultraviolet-B (10.08 kJ ·m⁻² ·d⁻¹) radiation were studied. The results indicated that the content of endonuclease sensitive sites (ESS) was reduced by He-Ne laser irradiation, which formed in cells irradiated by enhanced ultraviolet-B. With the irradiation of He-Ne laser, the excision of CPDs and the reduction of single strand breaks (SSB) contents which were the endonuclease sensitive sites (ESS) digested by T4-endodeoxyribonuclease-V had been stimulated in the wheat cells.     

甲基异丁酮萃取—火焰原子吸收光度法测定砷

研究了酸度、钼酸铵用量、乙酸丁酯和甲基异丁酮萃取时间、乙酸丁酯萃取次数以及酸洗用酸量与洗涤时间等对分离、萃取和测定砷的影响,提出在硝酸介质和钼酸铵存在下,用乙酸丁酯萃取硅和磷后,再用甲基异丁酮萃取砷,萃取砷的有机相用火焰原子吸收光度法测定。在1.5 mol/LHNO3介质中,钼酸铵(50 g/L)、乙酸丁酯和甲基异丁酮的用量分别为10 mL,乙酸丁酯和甲基异丁酮的萃取时间分别为1 min,乙酸丁酯萃取为2次,硅、磷和砷能分别被乙酸丁酯和甲基异丁酮完全萃取。硝酸(1+10)洗涤甲基异丁酮萃砷液,用量为10 mL、洗涤时间为30 s对砷的测定无明显影响。     

三氧化二砷抑制肝癌细胞端粒酶活性的实验研究

目的：观察三氧化二砷对肝癌细胞株BEL-7402和SMMC-7721端粒酶活性的影响。方法：应用端粒酶多聚酶联反应一酶联免疫测定(PCR-ELISA)方法,检测三氧化二砷作用后,肝癌细胞系BEL-7402和SMMC-7721细胞端粒酶活性的变化并比较两种细胞端粒酶对三氧化二砷敏感性的差异。结果：0．25～2．00μmol／L三氧化二砷(24～96h)可抑制肝癌细胞系BFL-7402的端粒酶活性,抑制作用与时间、剂量呈依赖性关系;0．25～0．50μmol／L三氧化二砷对SMMC-7721细胞端粒酶活性没有影响(96h以内),1．00～2．00μmol/L三氧化二砷可抑制SMMC-7721细胞端粒酶活性(4H8～96h),有时间、剂量依赖关系。结论：三氧化二砷可以抑制肝癌细胞系BEL-7402和SMMC-7721细胞端粒酶的活性;BEL-7402细胞端粒酶对三氧化二砷的敏感性较SMMC-7721细胞端粒酶高。     

采用硫化砷渣制备三氧化二砷工艺

硫化砷渣经氢氧化钠溶液浸出、空气氧化脱硫和SO2还原制备得到As2O3。研究结果表明：当NaOH与AS2S3物质的量比为7．2：1,固体质量与液体体积之比为1：6,反应温度为90℃,反应时间为2h,转速为300r／min时,用氢氧化钠溶液浸取硫化砷渣,其砷的浸取率达到95．90％;过滤后在碱浸液中通空气脱除碱浸液中Na3AsS3中的硫;当反应时间为10h,反应温度为30℃,空气流量为120L／h,对苯二酚和高锰酸钾质量浓度分别为1．5g／L和0．5g/L,木质素磺酸钠质量浓度为0．13g／L时,脱硫率可达到96．00％;当pH值为0,反应时间为1h,反应温度为30℃,砷质量浓度为60．00g/L时通入SO2还原溶液中AsO4^3-,产物中As2O3含量和砷回收率分别达到92．14％和95．21％;稀硫酸洗涤后,As2O3纯度达95．14％。     

Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain

Xeroderma pigmentosum (XP) is an autosomal recessive disease, characterized by a high incidence of sunlight-induced skin cancer. Cells from people with this condition are hypersensitive to ultraviolet because of a defect in DNA repair. There are nine genetic complementation groups of XP, groups A-H and a variant. We have cloned the mouse DNA repair gene that complements the defect of group A, the XPAC gene. Here we report molecular cloning of human and mouse XPAC complementary DNAs. Expression of XPAC cDNA confers ultraviolet-resistance on several group A cell lines, but not on lines of other XP groups. Almost all group A lines tested showed abnormality or absence of XPAC messenger RNAs. These results indicate that a defective XPAC gene causes group A XP. The human and mouse XPAC genes are located on chromosome 9q34.1 and chromosome 4C2, respectively. Human XPAC cDNA encodes a protein of 273 amino acids with a zinc-finger motif.     

香草醛对酪氨酸酶活性的抑制

在25℃，pH=6．8的Na2HPO4-NaH2PO4缓冲体系中，采用酶动力学方法研究了香草醛对酪氨酸酶单酚酶和二酚酶活性的抑制效应．实验结果表明：香草醛对酪氨酸酶单酚酶和二酚酶活性均有抑制作用，其半数抑制浓度（IC50）约分别为2．7和4．1mmol／L；香草醛能明显延长单酚酶的迟滞时间，4mmol／L香草醛能使迟滞时间由1．1min延长至3min；Lineweaver-Burk图显示香草醛对二酚酶的抑制作用表现为混合性抑制，对游离酶的抑制常数和对酶-底物络合物的抑制常数分别为3．48和15．1mmol／L．