Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC₅₀ (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml⁻¹) and by HeLa, HT29 and JM cells with IC₅₀ in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml⁻¹, all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.     

火球菌8-氧鸟嘌呤DNA糖苷酶基因克隆、表达纯化和酶学性质研究

【目的】在大肠杆菌中表达火球菌8-氧鸟嘌呤DNA糖苷酶,纯化得到重组火球菌8-氧鸟嘌呤DNA糖苷酶,在此基础上系统研究火球菌8-氧鸟嘌呤DNA糖苷酶的酶学特征。【方法】构建8-氧鸟嘌呤DNA糖苷酶重组表达质粒,将重组质粒转化Escherichia coli Rosetta(DE3),利用IPTG诱导表达重组蛋白,通过Ni2+亲和层析柱纯化重组蛋白;最后利用含8-氧鸟嘌呤损伤的寡核苷酸作为底物,测定8-氧鸟嘌呤DNA糖苷酶的酶学性质。【结果】在大肠杆菌中成功诱导表达了重组火球菌8-氧鸟嘌呤DNA糖苷酶,经Ni2+亲和纯化后蛋白纯度大于95%。在体外鉴定了重组火球菌8-氧鸟嘌呤DNA糖苷酶的酶学性质。结果表明重组火球菌8-氧鸟嘌呤DNA糖苷酶可以切除DNA中的8-氧鸟嘌呤(8-Oxo-G,GO)损伤碱基,并且具有AP裂解酶活性。重组火球菌8-氧鸟嘌呤DNA糖苷酶催化反应的最适pH值和温度分别是pH 8.5和55°C。除Zn2+对火球菌8-氧鸟嘌呤DNA糖苷酶的酶促反应有明显的抑制作用外,实验中测定的其它二价离子(Mn2+,Mg2+,Ca2+,Ni2+,Co2+,Cu2+)对其没有明显的影响。离子强度在50-100 mmol/L范围内对其酶促反应影响不大,超过100 mmol/L时有明显的抑制作用。与8-氧鸟嘌呤互补的碱基差异对火球菌8-氧鸟嘌呤DNA糖苷酶切除8-氧鸟嘌呤损伤的效率影响不大;但与单链DNA相比,双链DNA是优选底物,切割效率如下:GO/C≈GO/G≈GO/T≈GO/AGO/-。【结论】在大肠杆菌中成功表达,并Ni2+亲和纯化了火球菌8-氧鸟嘌呤DNA糖苷酶,生化研究表明制备的重组蛋白具有8-氧鸟嘌呤DNA糖苷酶活性,可能负责切除火球菌基因组DNA中的8-氧鸟嘌呤损伤。     

TET-TDG Active DNA Demethylation at CpG and Non-CpG Sites

In mammalian genomes, cytosine methylation occurs predominantly at CG (or CpG) dinucleotide contexts. As part of dynamic epigenetic regulation, 5-methylcytosine (mC) can be erased by active DNA demethylation, whereby ten-eleven translocation (TET) enzymes catalyze the stepwise oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), thymine DNA glycosylase (TDG) excises fC or caC, and base excision repair yields unmodified cytosine. In certain cell types, mC is also enriched at some non-CG (or CH) dinucleotides, however hmC is not. To provide biochemical context for the distribution of modified cytosines observed in biological systems, we systematically analyzed the activity of human TET2 and TDG for substrates in CG and CH contexts. We find that while TET2 oxidizes mC more efficiently in CG versus CH sites, this context preference can be diminished for hmC oxidation. Remarkably, TDG excision of fC and caC is only modestly dependent on CG context, contrasting its strong context dependence for thymine excision. We show that collaborative TET-TDG oxidation-excision activity is only marginally reduced for CA versus CG contexts. Our findings demonstrate that the TET-TDG-mediated demethylation pathway is not limited to CG sites and suggest a rationale for the depletion of hmCH in genomes rich in mCH.     

Isolation of a formamidopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12

Summary The fpg ⁺ gene of Escherichia coli coding for formamidopyrimidine-DNA glycosylase was previously cloned on a multicopy plasmid. The plasmid copy of the fpg ⁺ gene was inactivated by cloning a kanamycin resistance gene into the open reading frame, yielding the fpg-1:: Kn^r mutation. This mutation was transferred to the chromosome in the following steps: (i) linearization of the plasmid bearing the fpg-1::Kn^r mutation and transformation of competent bacteria (recB recC sbcB); (ii) selection for chromosomal integration of the fpg-1::Kn^r mutation; (iii) phage P1 mediated transduction of the fpg-1::Kn^r mutation in the AB1157 background. The resulting fpg ^- mutant exhibited no detectable Fapy-DNA glycosylase activity in crude lysates. The insertion mutation was localized by means of genetic crosses between mtl and pyrE, at 81.7 min on the E. coli linkage map. Sequence analysis confirmed this mapping and further showed that fpg is adjacent to rpmBG in the order fpg, rpmGB, pyrE. The formamidopyrimidine-DNA glycosylase defective strain does not show unusual sensitivity to the following DNA damaging treatments: (i) methylmethanesulfonate, (ii) N-methyl-N-nitro-N-nitrosoguanidine, (iii) ultraviolet light, (iv) -radiation. The fpg gene is neither part of the SOS regulon nor the adaptive response to alkylating agents.     

DNA damage and repair in mammalian cells exposed to p-hydroxyphenylpyruvic acid

Tyrosinemia type 1 (HT1) is an autosomal recessive disorder of the tyrosine metabolism in which the fumarylacetoacetate hydrolase enzyme is defective. This disease is clinically heterogeneous and a chronic and acute form is discerned. Characteristic of the chronic form is the development of cellular hepatocarcinoma. Although p-hydroxyphenylpyruvic acid (pHPPA) is used as one of the diagnostic markers of this disease, it was suggested that it is unlikely to be involved in the pathophysiology of HT1 as it is present in other disorders that does not have hepatorenal symptoms. It was the aim of this study to investigate the possible effect of pHPPA on DNA damage and repair in mammalian cells. The comet assay was used to establish the genotoxicity of pHPPA in human peripheral blood lymphocytes and isolated rat hepatocytes after their exposure to pHPPA. At first glance the damage to DNA caused by pHPPA seemed reparable in both cell types, however, after challenging the DNA repair capacity of metabolite-treated cells with treatment with H(2)O(2), a marked impairment in the DNA repair capability of these cells was observed. We suggest that the main effect of pHPPA is the long-term impairment of the DNA repair machinery rather than the direct damage to DNA and that this effect of pHPPA, together with the other characteristic metabolites, e.g., FAA and MAA, causes cellular hepatocarcinoma to develop in the chronic form of HT1.     

New Noncleavable Analogs of 8-Oxoguanine-DNA Glycosylase Substrates

8-Oxoguanine-DNA glycosylases play a key role in repairing oxidatively damaged DNA. Excision repair enzymes Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein) and human 8-oxoguanine-DNA glycosylase (hOGG1) catalyze excision of 7,8-dihydro-8-oxoguanine (oxoG) from DNA and subsequent cleavage of the sugar–phosphate backbone. Contacts between DNA phosphate groups and amino acid residues of the active centers of the enzymes are of importance for specific binding and catalysis. To construct noncleavable analogs of Fpg protein and hOGG1 substrates, modifications of phosphate groups bound to a damaged nucleotide were tested for their effect on the substrate properties of modified DNA duplexes. New oxoG-containing analogs of Fpg protein and hOGG1 substrates were synthetic DNA duplexes that contained a pyrophosphate or a substituted pyrophosphate group bound with the 5- or 3-OH of 8-oxoguanosine. The duplexes proved to be recognized and specifically bound by Fpg protein and hOGG1. Analysis of the mechanism of their interaction with Fpg protein and hOGG1 showed that modification of the internucleotide phosphate bound with 3-OH of 8-oxoguanosine prevents oxoG excision from DNA. Yet both enzymes efficiently cleaved the DNA duplexes when the modified phosphate was bound with the 5-OH of 8-oxoguanosine. DNA duplexes with a pyrophosphate or substituted pyrophosphate group at 3-OH of 8-oxoguanosine are noncleavable analogs of 8-oxoguanine-DNA glycosylase substrates and can be used to study the structures of catalytically active forms of Fpg protein and hOGG1 and their prokaryotic or eukaryotic homologs in complex with oxoG-containing DNA.     

Human 3-methyladenine-DNA glycosylase: effect of sequence context on excision, association with PCNA, and stimulation by AP endonuclease

Human 3-methyladenine-DNA glycosylase (MPG protein) is involved in the base excision repair (BER) pathway responsible mainly for the repair of small DNA base modifications. It initiates BER by recognizing DNA adducts and cleaving the glycosylic bond leaving an abasic site. Here, we explore several of the factors that could influence excision of adducts recognized by MPG, including sequence context, effect of APE1, and interaction with other proteins. To investigate sequence context, we used 13 different 25 bp oligodeoxyribonucleotides containing a unique hypoxanthine residue (Hx) and show that the steady-state specificity of Hx excision by MPG varied by 17-fold. If APE1 protein is used in the reaction for Hx removal by MPG, the steady-state kinetic parameters increase by between fivefold and 27-fold, depending on the oligodeoxyribonucleotide. Since MPG has a role in removing adducts such as 3-methyladenine that block DNA synthesis and there is a potential sequence for proliferating cell nuclear antigen (PCNA) interaction, we hypothesized that MPG protein could interact with PCNA, a protein involved in repair and replication. We demonstrate that PCNA associates with MPG using immunoprecipitation with either purified proteins or whole cell extracts. Moreover, PCNA binds to both APE1 and MPG at different sites, and loading PCNA onto a nicked, closed circular substrate with a unique Hx residue enhances MPG catalyzed excision. These data are consistent with an interaction that facilitates repair by MPG or APE1 by association with PCNA. Thus, PCNA could have a role in short-patch BER as well as in long-patch BER. Overall, the data reported here show how multiple factors contribute to the activity of MPG in cells.