Preparation of DNA substrates for in vitro mismatch repair

Analyses in vitro of correction of DNA mismatches have been pivotal in biochemical dissection of mismatch repair pathways. However, the complex procedures needed to prepare DNA substrates for mismatch repair have posed substantial barriers to investigators who wish to pursue such analyses. Here we describe a simple, efficient way to prepare a variety of mismatched DNA substrates. We use in our procedure high-copy-number pUC19-derived plasmids, and a newly commercially available endonuclease N.BstNBI that makes site-specific single-strand nicks. The ability to prepare large substrate quantities in a relatively short time and to construct wider ranges of different mismatches in various sequence contexts will facilitate future research. Supported in part by NIH grant ESO94848 to J.B.H. This is Technical Report No. 11680 from the Oregon Agricultural Experiment Station     

DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.     

Mismatch repair in human nuclear extracts. Quantitative analyses of excision of nicked circular mismatched DNA substrates,constructed by a new technique employing synthetic oligonucleotides

Mammalian mismatch repair (MMR) systems respond to broad ranges of DNA mismatches and lesions. Kinetic analyses of MMR processing in vitro have focused on base mismatches in a few sequence contexts, because of a lack of general and quantitative MMR assays and because of the difficulty of constructing a multiplicity of MMR substrates, particularly those with DNA lesions. We describe here simple and efficient construction of 11 different MMR substrates, by ligating synthetic oligomers into gapped plasmids generated using sequence-specific N.BstNBI nicking endonuclease, then using sequence-specific nicking endonuclease N.AlwI to introduce single nicks for initiation of 3' to 5' or 5' to 3' excision. To quantitatively assay MMR excision gaps in base-mispaired substrates, generated in human nuclear extracts lacking exogenous dNTPs, we used position- and strand-specific oligomer probes. Mispair-provoked excision along the shorter path from the pre-existing nick toward the mismatch, either 3' to 5' or 5' to 3', predominated over longer path excision by roughly 10:1 to 20:1. MMR excision was complete within 7 min, was highly specific (90:1) for the nicked strand, and was strongly mispair-dependent (at least 40:1). Nonspecific (mismatch-independent) 5' to 3' excision was considerably greater than nonspecific 3' to 5' excision, especially at pre-existing gaps, but was not processive. These techniques can be used to construct and analyze MMR substrates with DNA mismatches or lesions in any sequence context.     

cosmid克隆筛选的体内同源重组法——一个含有小鼠t复合体连锁DNA顺序的cosmid克隆的分离

一个从cosmid分子克隆库中筛选特别基因顺序的遗传学方法——体内同源重组(invlvo homologous recombination)法。即使探针DNA与分子克隆库中带有与探针同源顺序的克隆发生体内重组,然后以遗传学方法进行筛选。cosmid分子克隆库构建在rec宿主细胞内,经体内包装(in vivo Packaging)成λ噬菌体颗粒,把该噬菌体颗粒转入带有探针DNA的rec~+细胞内,探针是已被克隆在与cosmid载体没有同源顺序的质粒(如PUC8或PUC9)内的。经过一段时间(1—3小时),待重组发生后,把cosmid进行体内包装。此时探针DNA连同质粒已整合入cosmid基因组内,因此它带有原为两个载体所分别带有的双重抗性——Amp~r(氨苄青霉素,PUC8或PUC9)和Kan~r(卡那霉素,cosmid)。这种双重抗性菌落可在含有这2种抗菌素的培养平皿上选出,该重组cosmid借助于λ切除酶的作用将已被整合的探针质粒重新切除,再经体内包装后,该cosmid被还原并纯化,然后可用一含有Xgal的培皿识别和选出。本文用此法以有关DNA探针从cosmid分子克隆库中分离得到含有与小鼠t复合体连锁的基因组顺序的克隆,并对该克隆作了物理图谱分析。     

PEG-mediated one-pot multicomponent reactions for the efficient synthesis of functionalized dihydropyridines and their functional group dependent DNA cleavage activity

Polyethylene glycol (PEG) has been found to be an inexpensive, non-toxic and useful medium for the one pot synthesis of highly functionalized dihydropyridines using multicomponent reactions (MCRs) at room temperature under catalyst free conditions. The notable features of this protocol are: mild reaction condition, applicability to wide range of substrates, reusability of the PEG and good yields. The interaction of the synthesized compounds with pUC19 plasmid DNA was also analyzed. Some of the synthesized compounds showed interesting functional group dependent nuclease activity for plasmid DNA cleavage under physiological conditions.     

Condensation of DNA—A putative obstruction for repair process in abasic clustered DNA damage

Clustered DNA damages are defined as two or more closely located DNA damage lesions that may be present within a few helical turns of the DNA double strand. These damages are potential signatures of ionizing radiation and are often found to be repair resistant. Types of damaged lesions frequently found inside clustered DNA damage sites include oxidized bases, abasic sites, nucleotide dimers, strand breaks or their complex combinations. In this study, we used a bistranded two-lesion abasic cluster DNA damage model to access the repair process of DNA in condensate form.Oligomer DNA duplexes (47 bp) were designed to have two deoxyuridine in the middle of the sequences, three bases apart in opposite strands. The deoxyuridine residues were converted into abasic sites by treatment with UDG enzyme creating an abasic clustered damage site in a precise position in each of the single strand of the DNA duplex. This oligomer duplex having compatible cohesive ends was ligated to pUC19 plasmid, linearized with HindIII restriction endonuclease. The plasmid–oligomer conjugate was transformed into condensates by treating them with spermidine. The efficiency of strand cleavage action of ApeI enzyme on the abasic sites was determined by denaturing PAGE after timed incubation of the oligomer duplex and the oligomer–plasmid conjugate in presence and absence of spermidine. The efficiency of double strand breaks was determined similarly by native PAGE. Quantitative gel analysis revealed that rate of abasic site cleavage is reduced in the DNA condensates as compared to the oligomer DNA duplex or the linear ligated oligomer–plasmid conjugates. Generation of double strand break is significantly reduced also, suggesting that their creation is not proportionate to the number of abasic sites cleaved in the condensate model. All these suggest that the ApeI enzyme have difficulty to access the abasic sites located deep into the condensates leading to repair refractivity of the damages. In addition, we found that presence of a polyamine such as spermidine has no notable effect in the incision activity of ApeI enzyme in linear oligomer DNA duplexes in our experimental concentration.     

Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker

The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5alpha fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the SmaI site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins.     

Generation of DNA double-strand breaks and inhibition of somatic embryogenesis by tungsten microparticles in wheat

Particles of metallic tungsten, known also as tungsten microprojectiles, are routinely used to deliver foreign DNA into target cells and tissues. Some side effects of biolistic transformation have been observed but never studied in detail. Here we present evidence that intact tungsten particles can promote a breakage of phosphodiester bonds in native DNA, at a limited number of sites. A single, double-strand break appeared within almost each of the circular pUC119 molecules after a short incubation of plasmid DNA with a suspension of tungsten particles. No further DNA cutting could be induced even if the reaction rate was accelerated by increasing the concentration of tungsten in the incubation mixture. Indirect evidence indicates that similar lesions may be generated in cellular DNA of bombarded tissues. These lesions are rapidly repaired, as evidenced by increasing incorporation of labelled DNA precursors in bombarded wheat embryos. The rate of repair is, however, not high enough to restore all the genome functions. Neither germination of mature embryos nor initiation of callus tissues from immature embryos was inhibited by biolistic bombardment. Nevertheless, the frequency of formation of somatic embryos in calli derived from bombarded embryos was markedly lower than in calli derived from control embryos. Both immediate (generation of a limited number of double-strand breaks) and remote (selective inhibition of somatic embryogenesis) side effects of the biolistic process strongly suggest that biological activity of tungsten deserves special attention. This revised version was published online in June 2006 with corrections to the Cover Date.     

Variety of molecular conformation of plasmid pUC18 DNA and solenoidally supercoiled DNA

The plasmid pUC18 DNA isolated from Escherichia coli HB101 were analyzed by two-dimensional agarose gel electrophoresis and hybridization. The results show that the DNA sample can be separated into six groups of different structural components. The plectonemically and solenoidally supercoiled pUC18 DNA coexist in it. These two different conformations of supercoiled DNA are interchangeable with the circumstances (ionic strength and type, etc.). The amount of solenoidally supercoiled pUC18 DNA in the samples can be changed by treatment of DNA topoisome rases. Under an electron microscope, the solenoidal supercoiling DNA has a round shape with an average diameter of 45 nm. The facts suggest that solenoidaUy supercoiled DNA be a structural entity independent of histones. The polymorphism of DNA structure may be important to packing of DNA in vivo.     

Discrimination and versatility in mismatch repair

Evolutionarily-conserved mismatch-repair (MMR) systems correct all or almost all base-mismatch errors from DNA replication via excision-resynthesis pathways, and respond to many different DNA lesions. Consideration of DNA polymerase error rates and possible consequences of excess gratuitous excision of perfectly paired (homoduplex) DNA in vivo suggests that MMR needs to discriminate against homoduplex DNA by three to six orders of magnitude. However, numerous binding studies using MMR base-mispair-recognition proteins, bacterial MutS or eukaryotic MSH2.MSH6 (MutSalpha), have typically shown discrimination factors between mismatched and homoduplex DNA to be 5-30, depending on the binding conditions, the particular mismatches, and the DNA-sequence contexts. Thus, downstream post-binding steps must increase MMR discrimination without interfering with the versatility needed to recognize a large variety of base-mismatches and lesions. We use a complex but highly MMR-active model system, human nuclear extracts mixed with plasmid substrates containing specific mismatches and defined nicks 0.15 kbp away, to measure the earliest quantifiable committed step in mismatch correction, initiation of mismatch-provoked 3'-5' excision at the nicks. We compared these results to binding of purified MutSalpha to synthetic oligoduplexes containing the same mismatches in the same sequence contexts, under conditions very similar to those prevailing in the nuclear extracts. Discrimination against homoduplex DNA, only two-to five-fold in the binding studies, increased to 60- to 230-fold or more for excision initiation, depending on the particular mismatches. Remarkably, the mismatch-preference order for excision initiation was substantially altered from the order for hMutSalpha binding. This suggests that post-binding steps not only strongly discriminate against homoduplex DNA, but do so by mechanisms not tightly constrained by initial binding preferences. Pairs of homoduplexes (40, 50, and 70 bp) prepared from synthetic oligomers or cut out of plasmids showed virtually identical hMutSalpha binding affinities, suggesting that high hMutSalpha binding to homoduplex DNA is not the result of misincorporations or lesions introduced during chemical synthesis. Intrinsic affinities of MutS homologs for perfectly paired DNA may help these proteins efficiently position themselves to carry out subsequent mismatch-specific steps in MMR pathways.     

Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC → AT transitions and GC → TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to asses the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.     

Novel DNA glycosylases from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Oxidized bases are removed from DNA of Escherichia coli by enzymes formamidopyrimidine DNA glycosylase (Eco-Fpg) and endonuclease VIII (Eco-Nei) of the same structural family Fpg/Nei. New homologs of these enzymes not characterized earlier have been found in genomes of Actinobacteria. We have cloned and expressed two paralogs (Mtu-Nei2 and Mtu-Fpg2) from 36KAZ and KHA94 isolates of Mycobacterium tuberculosis and studied their ability to participate in DNA repair. Under heterologous expression in E. coli, Mtu-Nei2 decreased the rate of spontaneous mutagenesis in the rpoB gene, whereas Mtu-Fpg2 moderately increased it, possibly due to absence of residues crucially important for catalysis in this protein. Mtu-Nei2 was highly active toward double-stranded DNA substrates containing dihydrouracil residues and apurine-apyrimidine sites and was less efficient in cleavage of substrates containing 8-oxoguanine and uracil residues. These lesions, as well as 8-oxoadenine residues, were also recognized and removed by the enzyme from single-stranded DNA. Fpg and Nei homologs from M. tuberculosis can play an important role in protection of bacteria against genotoxic stress caused by oxidative burst in macrophages.     

Endonuclease activity from tobacco nuclei specific for ultraviolet radiation-damaged DNA

Endonuclease activity specific for UV damaged DNA was isolated from tobacco leaf nuclei and detected by relaxation of supercoiled pUC 19 plasmid DNA. The activity did not require divalent cations or ATP. It acted on photoproducts induced by as little as 24 J m⁻² of UV-C (primarily 254 nm) radiation. but not on photoproducts produced by UV-B (290–320 nm) radiation in the presence of acetophenone and a N₂ atmosphere or by UV-A (320–400 nm) radiation in the presence of 4'-methoxy-methyltrioxsalen in a N₂ atmosphere and not on the products of OsO₄ oxidation of the DNA. Using end-labeled DNA of defined sequence, it was possible to identify sites in UV-C-irradiated DNA that were cut by the endonuclease preparation: most sites were assocrated with pyrimidine pairs. Cleavage by the tobacco endonuclease was not eliminated by treatment with Escherichia coli photolyase and light, suggesting that the endonuclease did not recognize cyclobutadipyrimidines.     

Origin of adeno-associated virus DNA replication is a target of carcinogen-inducible DNA amplification.

DNA amplification of the helper-dependent parvovirus AAV (adeno-associated virus) can be induced by a variety of genotoxic agents in the absence of coinfecting helper virus. Here we investigated whether the origin of AAV type 2 DNA replication cloned into a plasmid is sufficient to promote replication activity in cells treated by the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A pUC19-based plasmid, designated pA2Y1, which contains the left terminal repeat sequences (TRs) representing the AAV origin of replication and the p5 and p19 promoter but lacks any functional parvoviral genes is shown to confer replication activity and to allow selective DNA amplification in carcinogen-treated cells. Following transfection of plasmid pA2Y1 or plasmid pUC19 as a control, density labeling by a bromodeoxyuridine and DpnI resistance assay suggested a semi-conservative mode of replication of the AAV origin-containing plasmid. Furthermore, the amount of DpnI-resistant full-length pA2Y1 DNA molecules was increased by MNNG treatment of cells in a dose-dependent manner. In addition, DNA synthesis of plasmid pA2Y1 was studied in vitro. Extracts derived from MNNG-treated CHO-9 and L1210 cells displayed greater synthesis of DpnI-resistant full-length pA2Y1 molecules than did nontreated controls. Experiments with specific enzyme inhibitors suggested that the reaction is largely dependent on DNA polymerase alpha, DNA primase, and DNA topoisomerase I. Furthermore, restriction endonuclease mapping analysis of the in vitro reaction products revealed the occurrence of specific initiation at the AAV origin of DNA replication. Though elongation was not very extensive, extracts from carcinogen-treated cells markedly amplified the AAV origin region. Our results, including electron microscopic examination, suggest that the AAV origin/terminal repeat structure is recognized by the cellular DNA replicative machinery induced or modulated by carcinogen treatment in the absence of parvoviral gene products.     

Efficient long DNA gap-filling in a mammalian cell-free system: A potential new in vitro DNA replication assay

In vitro assay of mammalian DNA replication has been variously approached. Using gapped circular duplex substrates containing a 500-base single-stranded DNA region, we have constructed a mammalian cell-free system in which physiological DNA replication may be reproduced. Reaction of the gapped plasmid substrate with crude extracts of human HeLaS3 cells induces efficient DNA synthesis in vitro. The induced synthesis was strongly inhibited by aphidicolin and completely depended on dNTP added to the system. In cell extracts in which PCNA was depleted step-wise by immunoprecipitation, DNA synthesis was accordingly reduced. These data suggest that replicative DNA polymerases, particularly pol delta, may chiefly function in this system. Furthermore, DNA synthesis is made quantifiable in this system, which enables us to evaluate the efficiency of DNA replication induced. Our system sensitively and quantitatively detected the reduction of the DNA replication efficiency in the DNA substrates damaged by oxidation or UV cross-linking and in the presence of a potent chain terminator, ara-CTP. The quantitative assessment of mammalian DNA replication may provide various advantages not only in basic research but also in drug development.     

A new vector for insertion of any DNA fragment into the chromosome of transformable Neisseriae

A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence which is required for natural transformation; (4) a promoter capable of functioning in Neisseria; and (5) several unique restriction sites useful for cloning. pNLE1 integrates into the leuS region of the neisserial chromosome at high frequencies by transformation-mediated recombination. The usefulness of this vector has been demonstrated by cloning the tetracycline-resistance gene (tet) and subsequently inserting the tet gene into the meningococcal chromosome.     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

Functional analysis of ori1 and repA of the R-plasmid pSJ5.6 from Neisseria gonorrhoeae

The functional ori1 of the 5.6kb gonococcal R-plasmid pSJ5.6 contains an A-T rich region followed by four 22bp direct repeats and one 19bp inverted repeat. The replication region of the plasmid also contains a gene encoding for a 39kD RepA protein. We have further assessed the functionality of the replication region in pSJ5.6, an-iteron type plasmid, using in vivo complementation assays in Escherichia coli. A 2.1kb PstI-RsaI fragment containing the ori1 and repA gene of pSJ5.6 was cloned into vector pZErO -2 to obtain pZA-MRR. The pUC origin in pZA-MRR was deleted to render the plasmid dependable on the cis-acting ori1 for replication. The resulting plasmid, pMRR, was capable of replication and maintenance in E. coli. We also cloned the ori1 and repA gene separately to obtain pA-Ori and pZG-Rep, respectively. Using in vivo complementation assays, we demonstrated that the ori1(+) plasmid (pA-Ori) was maintained only when the RepA protein was supplied in trans by the high copy number plasmid pZG-Rep.     

新城疫C3d-P29分子佐剂F基因疫苗的构建

C3d是补体C3的裂解产物之一,与抗原相连时可以明显提高抗原分子的免疫原性.CR2/CD21在其功能发挥过程中起到重要作用,P29为编码C3d与CR2结合区域的基因.从健康AA肉鸡肝脏中RT-PCR克隆C3d cDNA,设计引物克隆P29至pUC19载体,利用同裂酶BamH Ⅰ和Bgl Ⅱ构建pUC-P29.n.酶切获得P29.n,将其克隆至真核表达载体pCDNA3.1( ).最后RT-PCR扩增新城疫F基因,定向克隆至真核表达载体pCDNA-P29.n中P29.n的上游,构建完成新城疫F基因疫苗.3周龄SPF鸡进行基因免疫,结果pCDNA-F-P29.4、pCDNA-F-P29.6较pCDNA-F都能够提高HI抗体水平及保护力,虽然HI抗体水平不及灭活苗,但是能够抵抗致死量病毒的攻击,并且pCDNA-F-P29.6效果更好.目前发表的关于C3d的佐剂作用的文章多是关于鼠C3d,相应的抗原不能够自然感染鼠类,关于鸡C3d的报道较少.研究结果为进一步开发和利用鸡C3d奠定了基础.