Kinetics of circular DNA molecule digestion by restriction endonuclease Computation of kinetic constants from time dependence of fragment concentrations

A model for kinetics of circular substrate cleavage by restriction endonuclease was formulated. The aim of the analysis of the model was to extract kinetic constants for all target sites from time-dependence of fragment concentration in reaction products. That was proved to be possible for molecules with an odd number of fragments only. A symmetry of the molecules with an even number of fragment is the cause. A solution for molecules with an odd number of fragments was found and methods for dealing with the other molecules were suggested.Preliminary results were presented at VIIth CMEA Symposium Biophysics of Nucleic Acids and Proteins, Brno (Czechoslovakia) December 2–6, 1985.     

Discriminating small molecule DNA binding modes by single molecule force spectroscopy

Drugs may interact with double stranded DNA via a variety of binding modes, each mode giving rise to a specific pharmacological function. Here we demonstrate the ability of single molecule force spectroscopy to discriminate between different interaction modes by measuring the mechanical properties of DNA and their modulation upon the binding of small molecules. Due to the unique topology of double stranded DNA and due to its base pair stacking pattern, DNA undergoes several well-characterised structural transitions upon stretching. We show that small molecule binding markedly affects these transitions in ways characteristic to the binding mode and that these effects can be detected at the level of an individual molecule. The minor groove binder berenil, the crosslinker cisplatin and the intercalator ethidium bromide are compared.     

Association between DNA cleavage during apoptosis and regions of chromatin replication

We have addressed the association between the site of DNA cleavage during apoptosis and DNA replication. DNA double strand breaks were introduced into chromatin containing pulse labeled nascent DNA by the induction of apoptosis or autocleavage of isolated nuclei. The location of these breaks in relation to nascent DNA were revealed by Bal 31 exonuclease digestion at the cut sites. Our data show that Bal31 accessible cut sites are directly linked to regions enriched in nascent DNA. We suggest that these regions coincide with the termini of replication domains, possibly linked by strong DNA-matrix interactions with biophysically defined topological structures of 0.5 - 1.3 Mbp in size. The 50 kbp fragments that are commonly observed as products of apoptosis are also enriched in nascent DNA within internal regions but not at their termini. It is proposed that these fragments contain a subset of replicon DNA that is excised during apoptosis through recognition of their weak attachment to the nuclear matrix within the replication domain.J. Cell. Biochem. 70:604-615, 1998. © 1998 Wiley-Liss, Inc. © 1998 Wiley-Liss, Inc.     

An integrative approach for the optical sequencing of single DNA molecules

A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.     

A site-specific endonuclease from Caulobacter crescentus CB-13: restriction endonuclease CcrI

A site-specific restriction endonuclease (CcrI) has been identified from Caulobacter crescentus CB-13. This enzyme has been purified to homogeneity and the cleavage patterns with various DNAs and sequence data show that CcrI recognizes the same sequence as the XhoI restriction endonuclease (5′-C^↓-T-C-G-A-G-3′). Ccr has an absolute requirement for magnesium ions with an optimum concentration of 4 mM. The enzyme is optimally active at pH 8.0 and is stable up to 70°C. CcrI has a molecular weight of 65300 and exists as a monomer in its native state. Most of the physical characteristics observed for CcrI were similar to those observed for XhoI. Kinetic studies on CcrI and XhoI suggest that the enzymes interact with λ DNA in the same manner; however, with ?X-174 R.F. DNA, CcrI has a greater affinity for the supercoiled molecule than XhoI.     

Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and two-site plasmids but has little effect on the three-site plasmid. Taken together the data indicate that EcoRII requires simultaneous binding of three rather than two recognition sites in cis to achieve concerted DNA cleavage at a single site. We show that the orthodox type IIP enzyme PspGI which is an isoschisomer of EcoRII, cleaves different plasmid substrates with equal rates. Data provided here indicate that type IIE restriction enzymes EcoRII and NaeI follow different mechanisms. We propose that other type IIE restriction enzymes may employ the mechanism suggested here for EcoRII.     

Single molecule conformational analysis of DNA G-quadruplexes

Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells.     

A new estimate of sequence divergence of mitochondrial DNA using restriction endonuclease mappings

Summary A new estimate of the sequence divergence of mitochondrial DNA in related species using restriction enzyme maps is constructed. The estimate is derived assuming a simple Posisson-like model for the evolutionary process and is chosen to maximize an expression which is a reasonable approximation to the true likelihood of the restriction map data. Using this estimate, four sets of mitochondrial DNA data are analyzed and discussed.     

Single molecule techniques in DNA repair: A primer

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.     

A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus

Summary. The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus . using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with Mspl digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus , were identical to the common patterns shown by the 14 lines of domestic fowls.     

中国人线粒体DNA的八种限制酶图及其电镜结构

尽管Anderson等人(1981)已测定了人mtDNA的全部序列,但还不能全面地反映整个人类mtDNA核苷酸序列的情况。因此,在具有代表特征的中国人mtDNA序列被测定之前,为了开展对中国人mtDNA的遗传学研究,笔者构建了中国人mtDNA的8种限制酶图。并通过电镜技术对mtDNA进行了研究,发现了一种周长为2μm的小环状DNA,推测它可能在核DNA和mtDNA之间的信息传递或衰老发生中起到某些作用。     

黑麦属种间叶绿体DNA的变异性和种系发生的关系

为了获得有关黑麦属种间关系、黑麦属与小麦属和山羊草属种系发牛关系的新资料,应用Ban HI等8种限制性核酸内切酶酶解黑麦属5个种的叶绿体DNA,进行琼脂糖凝胶电泳,分析其酶解图谱。结果表明,黑麦属种叶绿体基因组的大小与小麦属和山羊草属的叶绿体基因组非常相似。根据黑麦属5个种遗传距离的估算,并与小麦属和山羊草属已知叶绿体基因组间所观察到的遗传距离相比,黑麦属叶绿体基因组的分化很少;这些资料进一步证实黑麦属的近代起源;并表明黑麦属、小麦属和山羊草属间的亲缘关系是非常密切的。     

Purification and properties of a site-specific restriction endonuclease AaaI from Acetobacter aceti subsp. aceti No. 1023

Abstract A type II restriction endonuclease, named Aaa I, was purified from Acetobacter aceti subsp. aceti No. 1023. The optimum pH and temperature were determined to be 8.5 and 37°C, respectively. The enzyme activity was stimulated by the addition of either NaCl or KCl and their optimum concentrations were 100 mM for both cations. Aaa I recognized the hexanucleotide sequence and cleaved it at the positions indicated by the arrows. Aaa I was an isoschizomer of Xma III from Xanthomonas malvacaerum and Eco 52I from Escherichia coli .     

A nonradioactive DNA methyltransferase assay adaptable to high-throughput screening

We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.     

Surface trapping and AFM detection of DNA topological intermediates generated from an oxidative chemical nuclease

Direct observation of DNA topological intermediates generated from a 'chemical nuclease' treatment has been made by atomic force microscopy (AFM). The intermediates were trapped at the mica-water interface and imaging was carried out in the dynamic force mode. Complete conversion from supercoiled circular state to relaxed circular/linear state has been observed over a time scale of 8 min. Implication of such studies in complementing gel electrophoresis data has been predicted.     

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.     

A hingeless Fc fusion system for site-specific cleavage by IdeS

Fusion of proteins to the Fc region of IgG is widely used to express cellular receptors and other extracellular proteins, but cleavage of the fusion partner is sometimes required for downstream applications. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a protease with exquisite specificity for human IgG, and it can also cleave Fc-fusion proteins at a single site in the N-terminal region of the CH2 domain. However, the site of IdeS cleavage results in the disulfide-linked hinge region partitioning with the released protein, complicating downstream usage of the cleaved product. To tailor the Fc fragment for release of partner proteins by IdeS treatment, we investigated the effect of deleting regions of IgG-derived sequence that are upstream of the cleavage site. Elimination of the IgG-derived hinge sequence along with several residues of the CH2 domain had negligible effects on expression and purity of the fusion protein, while retaining efficient processing by IdeS. An optimal Fc fragment comprising residues 235–447 of the human IgG1 heavy chain sufficed for efficient production of fusion proteins and minimized the amount of residual Ig-derived sequence on the cleavage product following IdeS treatment. Pairing of this truncated Fc fragment with IdeS cleavage enables highly specific cleavage of Fc-fusion proteins, thus eliminating the need to engineer extraneous cleavage sequences. This system should be helpful for producing Fc-fusion proteins requiring downstream cleavage, particularly those that are sensitive to internal miscleavage if treated with alternative proteases.     

鳜及大眼鳜线粒体DNA比较研究

采用10种限制性内切酶HindⅢ、EcoRⅠ、BglⅠ、BglⅡ、BamHⅠ、PvuⅡ、PstⅠ、SalⅠ、XbaⅠ、XhoⅠ对鳜及大眼鳜肝脏线粒体DNA（mtDNA）进行了分析。鳜鱼mtDNA分子量为9.703×106u,大小为16．19kb;大眼鳜mtDNA分子量为9．603×106u,大小为16．02kb。根据单、双酶切结果构建了鳜及大眼鳜mtDNA10种酶限制性酶切图谱,并对两种鱼的mtDNA酶切图谱进行了比较。     

A new S4-ligated zinc-peptide 1:2 complex for the hydrolytic cleavage of DNA

A new sulfur-ligated Zn-peptide 1:2 complex, [Zn(II)(Boc-NH-Cys-Gly-Cys-OMe)2]2- (2), was prepared, characterized, and tested for its DNA-binding and -cleavage properties. Complex 2 was found to cleave DNA hydrolytically. The negative charge in 2 reduces the affinity of the complex for DNA, and enhances its binding specificity.