Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II.

A rapid and simple method has been developed which allows detection and isolation of covalent DNA/protein adducts. The method is based upon the use of an ionic detergent, SDS, to neutralize cationic sites of weakly bound proteins thereby resulting in their dissociation off the helix. Proteins tightly or covalently bound to DNA that are not dissociable by SDS, result in the precipitation of the DNA fragment by the addition of KCl; however, free nucleic acid does not precipitate. The method is particularly useful as an analytical tool to titrate the binding of prototypic covalent binding proteins, topoisomerase I and II; thus, quantitation of topoisomerase activity is possible under defined conditions. As an analytical tool the method can be used as a general assay in the purification of as yet unidentified topoisomerases or other activities that bind DNA covalently. Moreover, the technology can be adapted for use in a preparative mode to separate covalent complexes from free DNA in a single step.     

Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure

The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.     

DNA sequencing by partial ribosubstitution

A new rapid method for DNA sequence analysis has been devised. In this method, base-specific cleavage is achieved at partially substituted ribonucleotides which are introduced by DNA polymerase extension in the presence of Mn2⁺. Access to a target sequence and label incorporation are achieved by extending a restriction fragment primer with DNA polymerase I. After a short initial incorporation with [α-³²P]deoxynucleotide triphosphates to label the 5′ region of the target sequence, the triphosphates are removed and the reaction mixture is divided four ways for a second primed extension. The second extension is a cold chase in the presence of Mn²⁺, all four deoxynucleotides and one of the four ribonucleotides under conditions that result in about 2% ribonucleotides substitution at each position. After cleavage at the restriction site and alkali cleavage at the positions of partial ribosubstitution, each reaction mixture is analysed by electrophoresis on a high-resolution denaturing acrylamide gel. As in the other rapid DNA sequencing methods the extent of DNA sequence that can be determined from a single experiment is limited only by the resolution of the analysing gels. At present some 100 nucleotides of sequence can be determined from a single priming reaction.     

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents.     

Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions.

We have developed an in vitro DNA polymerase forward mutation assay using damaged DNA templates that contain the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. The quantitative method uses complementary strand hybridization to gapped duplex DNA molecules and chloramphenicol selection. This design ensures exclusive analysis of mutations derived from the DNA strand produced during in vitro synthesis. We have examined the accuracy of DNA synthesis catalyzed by calf thymus polymerase alpha-primase, polymerase beta and exonuclease-deficient Klenow polymerase. Using unmodified DNA templates, polymerase beta displays a unique specificity for the loss of two bases in a dinucleotide repeat sequence within the HSV-tk locus. Treatment of the DNA template with N-ethyl-N-nitrosourea resulted in a dose-dependent inhibition of DNA synthesis concomitant with an increased mutation frequency. Similar dose-response curves were measured for the three polymerases examined; thus the identity of the DNA polymerase does not appear to affect the mutagenic potency of ethyl lesions. The HSV-tk system is unique in that damage-induced mutagenesis can be analyzed both quantitatively and qualitatively in human cells, in bacterial cells and in in vitro DNA synthesis reactions at a single target sequence.     

Bacterial cell killing mediated by topoisomerase I DNA cleavage activity

DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerase can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such a stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS-inducing and cell-killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents.     

Cleavage of DNA by mammalian DNA topoisomerase II

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.     

Amino acid changes coded by bacteriophage T4 DNA polymerase mutator mutants. Relating structure to function

Previous studies on the selection of bacteriophage T4 mutator mutants have been extended and a method to regulate the mutator activity of DNA polymerase mutator strains has been developed. The nucleotide changes of 17 bacteriophage T4 DNA polymerase mutations that confer a mutator phenotype and the nucleotide substitutions of several other T4 DNA polymerase mutations have been determined. The most striking observation is that the distribution of DNA polymerase mutator mutations is not random; almost all mutator mutations are located in the N-terminal half of the DNA polymerase. It has been shown that the T4 DNA polymerase shares several regions of homology at the protein sequence level with DNA polymerases of herpes, adeno and pox viruses. From studies of bacteriophage T4 and herpes DNA polymerase mutants, and from analyses of similar protein sequences from several organisms, we conclude that DNA polymerase synthetic activities are located in the C-terminal half of the DNA polymerase and that exonucleolytic activity is located nearer the N terminus.     

Identification by a random sequencing strategy of the fowlpoxvirus DNA polymerase gene, its nucleotide sequence and comparison with other viral DNA polymerases.

The nucleotide sequence of the DNA polymerase gene of the avipoxvirus fowlpox is presented and the predicted amino acid sequence compared with that of the orthopoxvirus vaccinia. The results have brought to light an error in the vaccinia sequence which has resulted in the ommission of 44 amino acids from the carboxy-terminus of the vaccinia DNA polymerase. There has been extensive conservation of amino acids throughout the enzymes, and regions identified as being present in DNA polymerases from a wide range of viruses are again present here. The method used to identify the fowlpoxvirus gene could have applications towards defining genomic organisations in other viral systems.     

DNA断裂检测方法──单细胞凝胶电泳法

单细胞凝胶电泳(single cell gel electrophoresis assay,SCGE)也叫彗星试验(comet assay),是一种快速、敏感、简便、廉价的检测单个哺乳动物细胞DNA断裂的技术,目前已用于检测氧化、紫外线和电离辐射引起的损伤,以及三氯乙烷、丙烯酰胺等化学物及老化、吸烟所致损害的研究.文章介绍SCGE的发展、检测分析方法、原理及其在DNA损伤与修复、生物监测、遗传毒理研究、肿瘤治疗方案优化和疗效研究方面的应用前景．     

Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence-independent amplification of DNA

A method that permits the in vitro amplification and cloning of DNA dissected from specific regions of a chromosome and does not require prior knowledge of the DNA sequence is described. DNA from several different chromosomal loci in the Drosophila melanogaster genome has been isolated by this method. Although the procedure was developed to permit the isolation of DNA sequences from serial sections of a single microdissected polytene chromosome, it should be useful for obtaining DNA clones from specific regions of the nonpolytene chromosomes of other organisms as well.     

DNA polymerase lambda from calf thymus preferentially replicates damaged DNA

A new gene (POLL), has been identified encoding the novel DNA polymerase lambda and mapped to mouse chromosome 19 and at human chromosome 10. DNA polymerase lambda contains all the critical residues involved in DNA binding, nucleotide binding, nucleotide selection, and catalysis of DNA polymerization and has been assigned to family X based on sequence homology with polymerase beta, lambda, mu, and terminal deoxynucleotidyltransferase. Here we describe a purification of DNA polymerase lambda from calf thymus that preferentially can replicate damaged DNA. By testing polymerase activity on non-damaged and damaged DNA, DNA polymerase lambda was purified trough five chromatographic steps to near homogeneity and identified as a 67-kDa polypeptide that cross-reacted with monoclonal antibodies against DNA polymerase beta and polyclonal antibodies against DNA polymerase lambda. DNA polymerase lambda had no detectable nuclease activities and, in contrast to DNA polymerase beta, was aphidicolin-sensitive. DNA polymerase lambda was a 6-fold more accurate enzyme in an M13mp2 forward mutation assay and 5-fold more accurate in an M13mp2T90 reversion system than human recombinant DNA polymerase beta. The biochemical properties of the calf thymus DNA polymerase lambda, described here for the first time, are discussed in relationship to the proposed role for this DNA polymerase in vivo.     

Enzymatic syntheses of DNA-silicas using DNA polymerase.

Oligothymidylic acids couple to an activated ester silica (N-hydroxysuccinimidyl-silica) only when they contain an added aminoalkyl group. Heteropolymeric oligomers containing other nucleotide bases were shown to also couple by way of the nucleotide base (adenine, cytosine, or guanine); however, when a heteropolymeric oligonucleotide also contains a 5'-aminoalkyl moiety, coupling by way of the latter is the favored reaction. When duplex hybrids of oligonucleotides are formed, the nucleotide bases are protected from chemical coupling. Coupling by way of nucleotide bases would be detrimental to some chromatography experiments. On the basis of these observations, two different procedures were developed to produce DNA-silicas in which a single strand of the DNA is coupled by only its 5'-terminus. In the first of these, the polymerase chain reaction was used with a 5'-aminoalkyl primer to make a duplex DNA with one strand containing the 5'-aminoalkyl group and the duplex DNA is then coupled to the activated ester silica. This yielded a silica containing about 0.17 nmol of a 242-mer per gram silica which bound only probes specific for the coupled strand. In the other procedure, a template DNA strand was poly(A) tailed and hybridized to (dT)18-silica. DNA polymerase I (Klenow large fragments) was then used to copy the template-specified sequence directly onto the 3'-terminus of the (dT)18. This procedure yielded about 1.2 to 2.7 nmol DNA copied/g of silica of a specific 21-mer sequence. The DNA-silica produced selectively hybridized only with complementary sequences and not with DNA lacking that sequence. Either of these procedures thus produces DNA-silicas from heteropolymeric DNA sequences with a predetermined, specific 5'-terminal site of attachment.     

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.     

Chick-embryo DNA polymerase gamma. Identity of gamma-polymerases purified from nuclei and mitochondria

The level of DNA polymerase gamma as compared to DNA polymerases alpha and beta has been determined in chick embryo by means of specific tests: the amount of gamma-polymerase in the 12-day-old chick embryo reaches about 15% of the total polymerase activity. This enzyme is mainly localized in nuclei and mitochondria, where it represents the prevailing if not the unique DNA polymerase activity. The mitochondrial DNA polymerase gamma is likely to be associated with the internal membrane or the matrix of this organelle since it is not removed by digitonin treatment. The gamma-polymerases have been purified from chick embryo nuclei and mitochondria 500-700 times by means of DEAE-cellulose, phosphocellulose and hydroxyapatite chromatographies. The purified mitochondrial DNA polymerase gamma is closely related to the homologous enzyme purified from the nuclei of the same cells. So far, they cannot be distinguished on the basis of their sedimentation, catalytical properties and response to inhibitors or denaturating agents. The purified gamma enzymes are distinct from the chick embryo DNA polymerases alpha and beta and are not inhibited by antibodies prepared against the latter enzymes. The nuclear and mitochondrial gamma-polymerases do not respond to the oncogenic RNA virus DNA polymerase assay with natural mRNAs.     

Preferential access to genetic information from endogenous hominin ancient DNA and accurate quantitative SNP-typing via SPEX

The analysis of targeted genetic loci from ancient, forensic and clinical samples is usually built upon polymerase chain reaction (PCR)-generated sequence data. However, many studies have shown that PCR amplification from poor-quality DNA templates can create sequence artefacts at significant levels. With hominin (human and other hominid) samples, the pervasive presence of highly PCR-amplifiable human DNA contaminants in the vast majority of samples can lead to the creation of recombinant hybrids and other non-authentic artefacts. The resulting PCR-generated sequences can then be difficult, if not impossible, to authenticate. In contrast, single primer extension (SPEX)-based approaches can genotype single nucleotide polymorphisms from ancient fragments of DNA as accurately as modern DNA. A single SPEX-type assay can amplify just one of the duplex DNA strands at target loci and generate a multi-fold depth-of-coverage, with non-authentic recombinant hybrids reduced to undetectable levels. Crucially, SPEX-type approaches can preferentially access genetic information from damaged and degraded endogenous ancient DNA templates over modern human DNA contaminants. The development of SPEX-type assays offers the potential for highly accurate, quantitative genotyping from ancient hominin samples.     

Primary structure of the catalytic subunit of calf thymus DNA polymerase delta: sequence similarities with other DNA polymerases.

The 125- and 48-kDa subunits of bovine DNA polymerase delta have been isolated by SDS-polyacrylamide gel electrophoresis and demonstrated to be unrelated by partial peptide mapping with N-chlorosuccinimide. A 116-kDa polypeptide, usually present in DNA polymerase delta preparations, was shown to be a degraded form of the 125-kDa catalytic subunit. Amino acid sequence data from Staphylococcus aureus V8 protease, cyanogen bromide, and trypsin digestion of the 125- and 116-kDa polypeptides were used to design primers for the polymerase chain reaction to determine the nucleotide sequence of a full-length cDNA encoding the catalytic subunit of bovine DNA polymerase delta. The predicted polypeptide is 1106 amino acids in length with a calculated molecular weight of 123,707. This is in agreement with the molecular weight of 125,000 estimated from SDS-polyacrylamide gel electrophoresis. Comparison of the deduced amino acid sequence of the catalytic subunit of bovine DNA polymerase delta with that of its counterpart from Saccharomyces cerevisiae showed that the proteins are 44% identical. The catalytic subunit of bovine DNA polymerase delta contains the seven conserved regions found in a number of bacterial, viral, and eukaryotic DNA polymerases. It also contains five additional regions that are highly conserved between bovine and yeast DNA polymerase delta, but these regions share little or no homology with the alpha polymerases. Four of these additional regions are also highly homologous to the herpes virus family of DNA polymerases, whereas one region is not homologous to any other DNA polymerase that has been sequenced thus far.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA-directed DNA polymerase of Rous sarcoma virus: initiation of synthesis with 70 S viral RNA as template

DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus with 70 S viral RNA as template initiates by the covalent attachment of dAMP to the 3′ terminal adenosine of an RNA molecule. Initiation continues throughout the course of a 90-minute enzymatic reaction, and chain propagation occurs on most if not all of the dAMP residues attached to primer RNA. The nature of the primer molecules was established in two ways. First, the RNA was tagged by attachment of radioactive mono- and oligodeoxynucleotides. Second, primers were isolated directly from their covalent complexes with nascent DNA. The results of both procedures indicate that DNA synthesis initiates on the 3′ termini of 4 S RNA molecules hydrogen-bonded to 70 S RNA. Purified primer RNA has a nucleotide composition (G + C = 64%) different from that (G + C = 60%) of other 4 S RNAs found hydrogen-bonded to the 70 S RNA of Rous sarcoma virus.     

An endogenous protein inhibitor of DNA polymerase alpha in normal and neoplastic rat mammary tissues

Extracts of whole tissue or isolated nuclei from lactating rat mammary gland that has diminished cell replication capacity were more active than the corresponding extracts of pregnant rat mammary gland that contains actively replicating cells in causing a dose-dependent inhibition of DNA polymerase alpha in vitro. Purification of the inhibitor from both tissue and nuclear extracts using a sequence of Sephacryl S200, DEAE-cellulose and CM52 columns confirmed the above assay results. Using the same assay and purification procedures, both tissue and nuclear extracts from the rapidly growing transplanted R3220AC mammary tumors exhibited very little or no inhibitor activity. The partially purified mammary inhibitor (mol. wt of 155kD, high A280 nm/A260 nm ratio, heat labile) was equally inhibitory to the purified DNA polymerase alpha from either R3230AC tumor or calf thymus, and to the nuclear matrix bound DNA polymerase alpha of R3230AC tumor.