Mode of inhibition of the DNA polymerase of Methanococcus vannielii by aphidicolin

The mode of action of aphidicolin on DNA synthesis catalysed by the DNA polymerase of Methanococcus vannielii is competitive for dCTP, noncompetitive for dATP, dGTP and dTTP and uncompetitive for activated DNA. The kinetic data are accounted for by a mechanism in which dCTP and aphidicolin compete for the dCTP-specific binding site on the DNA polymerase. The dissociation constant for the aphidicolin--DNA-polymerase complex is 0.04-0.07 microM. Similar modes of inhibition of DNA synthesis exist for DNA polymerase alpha of higher eucaryotes but not for eubacteria or viruses and suggests a close functional relationship between the DNA polymerase of eucaryotes and of the archaebacterium M. vannielii.     

Immunocytochemical detection of sulphonylated DNA in tissue sections. An alternative to Feulgen staining of DNA

We report here on a new sensitive and highly specific DNA staining technique which we have called sulpho-DNA staining. DNA staining is based on a sulphonylation reaction of 2'-deoxycytidine or cytidine that takes place in the 6th position of cytosine with ensuing immunodetection of the sulphonylated DNA. The specificity of DNA staining is introduced by the use of an antibody recognizing only modified DNA but not modified RNA, by recourse to an additional acid hydrolysis step which destroys RNA but not DNA. We describe here the optimal conditions for the sulphonylation of DNA using O-methylhydroxylamine and metabisulphite as reactants. The new DNA stain labels all nuclei in either normal human tissue or in tumor cells. For nuclear DNA the staining signal is higher for the sulpho-DNA staining than for the Feulgen staining for nuclear DNA. This new DNA staining technique is suitable for use on tissue sections as well as on cytosmears.     

Supercoiling-dependent sequence specificity of mammalian DNA methyltransferase.

Negative supercoiling of substrate DNA dramatically alters the in vitro sequence specificity of mammalian DNA methyltransferase (DNA MeTase). This result suggests that in vivo site selection by DNA MeTase could be regulated by conformational information in the form of alternative secondary structures induced in DNA by local supercoiling or by the binding of specific nuclear proteins. DNA in the left-handed Z-form is shown not to be a substrate for mammalian DNA MeTase. The sensitivity of DNA MeTase to DNA structure may also make it useful as a probe for sequences which undergo supercoiling-dependent structural transitions in vitro.     

DNA recognition properties of the N-terminal DNA binding domain within the large subunit of replication factor C.

Replication Factor C (RFC) is a five-subunit protein complex required for eukaryotic DNA replication and repair. The large subunit within this complex contains a C-terminal DNA binding domain which provides specificity for PCNA loading at a primer-template and a second, N-terminal DNA binding domain of unknown function. We isolated the N-terminal DNA binding domain from Drosophila melanogaster and defined the region within this polypeptide required for DNA binding. The DNA determinants most efficiently recognized by both the Drosophila minimal DNA binding domain and the N-terminal half of the human large subunit consist of a double-stranded DNA containing a recessed 5' phosphate. DNA containing a recessed 5' phosphate was preferred 5-fold over hairpined DNA containing a recessed 3' hydroxyl. Combined with existing data, these DNA binding properties suggest a role for the N-terminal DNA binding domain in the recognition of phosphorylated DNA ends.     

Assessment of the DNA-binding properties of actinomycin and its derivatives by molecular dynamics simulation

A molecular dynamics simulation was used to assess the effect on the elasticity of a DNA fragment and the efficiency of DNA binding for actinomycins (antibiotics that are used in chemotherapy for certain oncology diseases). Hydroxyl and amino groups that were introduced as substituents in the phenoxazine ring of actinomycin were tested for their effect on the dynamic behavior and stability of antibiotic–DNA complexes. The Young modulus was calculated for DNA, DNA–actinomycin, DNA–7-hydroxyactinomycin, and DNA–7-aminoactinomycin. The free energy of complexation with DNA was calculated for actinomycin and its two analogs. The substituents were assumed to structurally stabilize the DNA fragment via additional hydrogen bonding.     

Comparative effect of microwaves and boiling on the denaturation of DNA

The effect of heat and microwave denaturation of small volumes of double-stranded plasmid DNA has been compared. Samples of intact plasmid DNA had plasmid DNA linearized by digestion with EcoRI were conventionally denatured in a boiling water bath or denatured by 2450 MHz of microwave energy for 0-300 s. Heat denaturation for periods longer than 120 s caused breakdown of linearized plasmid DNA; however, microwave denaturation for 10-300 s caused no apparent degradation of linearized DNA. Breakdown of DNA forms II and III was noted in plasmid DNA subjected to 300 s of either heat or microwave denaturation but breakdown of forms II and III occurred more quickly with heat than with microwave treatment. Microwave treatment was also found to be better than heat to denature 32P-labeled DNA probes subsequently used to detect homologous DNA samples immobilized on nitrocellulose filters. A microwave-treated 32P-labeled DNA probe was able to hybridize to DNA samples 20 times more dilute than a heat-treated 32P-labeled DNA probe. Depending on the form of DNA to be analyzed, these results indicate that small volumes of DNA solutions and radiolabeled DNA probes can be effectively denatured in a conventional microwave oven.     

Inhibition of DNA polymerase activity by methyl methanesulfonate

Methyl methanesulfonate (MMS) inhibits both thymidine incorporation into DNA in mitogen-activated human lymphocytes and deoxythymidine triphosphate incorporation into template DNA by DNA polymerase-alpha in a cell-free system. When MMS-modified DNA was used as the template for DNA synthesis utilizing unmodified DNA polymerase-alpha, nucleotide incorporation into template DNA was not inhibited. When unmodified DNA was used as the template for DNA synthesis utilizing MMS-modified DNA polymerase-alpha, nucleotide incorporation was differentially inhibited dependent on the MMS concentration. An analysis of the kinetics of DNA polymerase-alpha inhibition showed that incorporation of all 4 deoxynucleoside triphosphates into DNA template was noncompetitively inhibited by MMS, which is consistent with nonspecific MMS modification of the enzyme. These data indicate that MMS modification of DNA polymerase-alpha alone is sufficient to inhibit the incorporation of deoxynucleoside triphosphates into template DNA in vitro. The data further indicate that alkylation of both DNA polymerase-alpha and DNA template synergistically increases inhibition of DNA synthesis.     

Effect of actinomycin D on nucleic acid hybridization: the cause of erroneous DNA elongation during DNA synthesis of RNA tumor viruses in vitro

Actinomycin D, known for its suppression of cellular RNA synthesis and for the reduction of the rate of synthesis of double-stranded DNA by the RNA tumor virus RNA-dependent DNA polymerase, was found to interact with single-stranded DNA in such a way as to inhibit DNA . DNA and DNA . RNA hybridizations. This finding is discussed in the light of the observation that DNA elongation during DNA synthesis of RNA tumor viruses is blocked in vitro in the presence of actinomycin D. It thus supports the model that hybridization is a necessary step during RNA tumor virus DNA synthesis.     

Calorimetric analysis of binding of two consecutive DNA strands to RecA protein illuminates mechanism for recognition of homology

RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change (DeltaH) with temperature (heat capacity change, DeltaC(p)). In the presence of the ATP analogue ATPgammaS, the DeltaH for the binding of the first DNA strand depends upon temperature (large DeltaC(p)) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced DeltaC(p), indicating the absence of further reorganisation of the RecA-DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the DeltaH of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the DeltaH is larger than that for non-complementary DNA strand, but less than the DeltaH of the annealing of the complementary DNA without RecA. This small DeltaH could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The DeltaH of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands.     

关于植物DNA条形码研究技术规范

DNA条形码是利用标准的基因片段对物种进行快速鉴定的技术,已经成功用于生物物种分类和鉴定、生态学调查和生物多样性评估等研究领域。尽管生命条形码数据（BOLD）系统提供了主要针对动物类群DNA条形码研究的技术规范,但由于植物本身的生物学特性与所使用的条形码不同,因此已有技术规范并不完全适用于植物DNA条形码的研究。本文根据植物DNA条形码研究的特点与我国的实际情况,编写了植物DNA条形码研究技术标准和规范指南,具体包括十个方面的内容,即植物DNA条形码研究的样品采集策略;植物标本和野外数据的采集规范;植物标本图像信息的采集规范;植物DNA材料的采集规范;植物DNA材料的干燥与保存规范;植物总DNA的质量标准及保存规范;植物标准DNA条形码的选择与通用引物;DNA条形码的扩增与测序;DNA条形码数据的命名、编辑和提交规范;以及DNA条形码数据分析。我们期望通过这些标准规范的实施和在实践中的不断修订和完善,能为我国学者开展植物DNA条形码和iFlora研究提供参考和借鉴。
关键词：植物DNA条形码;技术规范;物种鉴定;标准;新一代植物志     

Assembly of DNA polymerase delta and epsilon holoenzymes depends on the geometry of the DNA template.

To study in details the assembly of DNA polymerases delta and epsilon holoenzymes a circular double-stranded DNA template containing a gap of 45 nucleotides was constructed. Both replication factor C and proliferating cell nuclear antigen were absolutely required and sufficient for assembly of DNA polymerase delta holoenzyme complex on DNA. On such a circular DNA substrate replication protein A (or E. coli single-strand DNA binding protein) was neither required for assembly of DNA polymerase delta holoenzyme complex nor for the gap-filling reaction. A circular structure of the DNA substrate was found to be absolutely critical for the ability of auxiliary proteins to interact with DNA polymerases. The linearization of the circular DNA template resulted in three dramatic effects: (i) DNA synthesis by DNA polymerase delta holoenzyme was abolished, (ii) the inhibition effect of replication factor C and proliferating cell nuclear antigen on DNA polymerase alpha was relieved and (iii) DNA polymerase epsilon could not form any longer a holoenzyme with replication factor C and proliferating cell nuclear antigen. The comparison of the effect of replication factor C and proliferating cell nuclear antigen on DNA polymerases alpha, delta and epsilon indicated that the auxiliary proteins appear to form a mobile clamp, which can easily slide along double-stranded DNA.     

Structure and function of 2:1 DNA polymerase.DNA complexes

DNA polymerases are required for DNA replication and DNA repair in all of the living organisms. Different DNA polymerases are responsible different stages of DNA metabolism, and many of them are multifunctional enzymes. It was generally assumed that the different reactions are catalyzed by the same enzyme molecule. In addition to 1:1 DNA polymerase.DNA complex reported by crystallization studies, 2:1 and higher order DNA polymerase.DNA complexes have been identified in solution studies by various biochemical and biophysical approaches. Further, abundant evidences for the DNA polymerase-DNA interactions in several DNA polymerases suggested that the 2:1 complex represents the more active form. This review describes the current status of this emerging subject and explores their potential in vitro and in vivo functional significance, particularly for the 2:1 complexes of mammalian DNA polymerase beta (Pol beta), the Klenow fragment of E. coli DNA polymerase I (KF), and T4 DNA polymerase.     

DNA unwinding by replication protein A is a property of the 70 kDa subunit and is facilitated by phosphorylation of the 32 kDa subunit.

Replication protein A (RP-A) is a heterotrimeric single-stranded DNA binding protein with important functions in DNA replication, DNA repair and DNA recombination. We have found that RP-A from calf thymus can unwind DNA in the absence of ATP and MgCl2, two essential cofactors for bona fide DNA helicases (Georgaki, A., Strack, B., Podust, V. and Hübscher, U. FEBS Lett. 308, 240-244, 1992). DNA unwinding by RP-A was found to be sensitive to MgCl2, ATP, heating and freezing/thawing. Escherichia coli single stranded DNA binding protein at concentrations that coat the single stranded regions had no influence on DNA unwinding by RP-A suggesting that RP-A binds fast and tightly to single-stranded DNA. DNA unwinding by RP-A did not show directionality. Experiments with monoclonal antibodies strongly suggested that the 70kDa subunit is responsible for DNA unwinding. Phosphorylation of the 32kDa subunit of RP-A by chicken cdc2 kinase facilitated DNA unwinding indicating that this posttranslational modification might be important for modulating this activity of RP-A. Finally, DNA unwinding of a primer recognition complex for DNA polymerase delta which is composed of proliferating cell nuclear antigen, replication factor C and ATP bound to a singly-primed M13DNA slightly inhibited DNA unwinding. An important role for DNA unwinding by RP-A in processes such as initiation of DNA replication, fork propagation, DNA repair and DNA recombination is discussed.     

Forms of Deoxyribonucleic Acid Produced by Virions of the Ribonucleic Acid Tumor Viruses

The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two fractions were analyzed for their content of single-stranded DNA, double-stranded DNA, and DNA-ribonucleic acid (RNA) hybrid by (i) digestion with enzymes of known specificity and (ii) equilibrium centrifugation in Cs(2)SO(4) gradients. The major fraction early in the reaction contained equal amounts of single-stranded DNA and DNA-RNA hybrid and little double-stranded DNA. The major fraction after extensive synthesis contained equal amounts of single-and double-stranded DNA and little hybrid. In the presence of actinomycin D, the predominant product was single-stranded DNA. To account for these various forms of DNA, we postulate the following model: the first DNA synthesis occurs in a replicative complex containing growing DNA molecules attached to an RNA molecule. Each DNA molecule is displaced as single-stranded DNA by the synthesis of the following DNA strand, and the single-stranded DNA is copied to form double-stranded DNA either before or after release of the single strand from the RNA. Actinomycin blocks this conversion of single-to double-stranded DNA.     

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.     

Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA.

A modification of the asymmetric PCR method is described, which reliably facilitates sequencing of PCR-amplified DNA. This procedure produces single-stranded DNA fragments as long as two kilobases that are suitable for dideoxy DNA sequencing. First, a PCR for double-stranded DNA is preformed under optimal conditions (double-stranded PCR). Then, a 5-10-microliters fraction of the double-stranded PCR and a single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer is approximately 0.4 microM. Usually 15 to 25 cycles of single-stranded PCR are optimal to produce single-stranded DNA for four to eight sequencing reactions. The single-stranded DNA is purified by centrifugal ultrafiltration and used directly in dideoxy sequencing. This method was employed to produce high-quality single-stranded DNA templates from a variety of organisms for efficient DNA sequencing of PCR-amplified DNA.     

Identification of conserved residues contributing to the activities of adenovirus DNA polymerase

Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase alpha family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3'-5'-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087-1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.     

DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.     

Effects of substitutions of arginine residues on the basic surface of herpes simplex virus UL42 support a role for DNA binding in processive DNA synthesis

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14- to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.     

Effects of DNA Topology on Transformation Efficiency of Bacillus subtilis ISW1214 by Electroporation

We report an investigation of electrotransformation by three different topological isomers, circular supercoiled (sc DNA), circular relaxed (cr DNA), and linearized (In DNA) forms of the plasmids pUB110 (4.5 kbp) and pBDR331T (12.6 kbp), of a Gram-positive bacterium, Bacillus subtilis ISW1214. Treatment of the sc DNA with calf thymus topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of sc DNA with restriction endonuclease linearized the DNA. The transformation with the sc DNA of pUB110 resulted in the maximum efficiency of (2.6±0.6) × 10⁵ transformants per μg DNA higher than that ((2.0±0.3) × 10⁴ transformants per μg DNA) for the cr DNA, using the DNA concentration of 20 μg/ml at an electric field strength of 7kV/cm and a capacitance of 10 μF with a single decayed pulse. The transformation efficiency (TE) for the In DNA was zero. The variations of TE for different topological forms of DNA reflected their relative stability in the host cells. The molecular efficiency (ME, transformants per molecule) for sc DNA was nearly one order of magnitude greater for the lower molecular size of pUB110 DNA than that for the higher molecular size of pBDR331T DNA.