DNA knots and DNA supercoiling

Comment on: Witz G, et al. Proc Natl Acad Sci USA 2011; 108:3608-11.     

DNA topoisomerases and DNA repair

DNA topoisomerases are enzymes that can modify, and may regulate, the topological state of DNA through concerted breaking and rejoining of the DNA strands. They have been believed to be directly involved in DNA excision repair, and perhaps to be required for the control of repair as well. The vicissitudes of this hypothesis provide a noteworthy example of the dangers of interpreting cellular phenomena without genetic information and vice versa.     

真核生物的DNA甲基转移酶与DNA甲基化

真核生物的ＤＮＡ甲基化就是在ＤＮＡ的ＣｐＧ二核苷酸胞嘧啶的第 5位碳原子上加上甲基 ,催化这一过程的是ＤＮＡ甲基转移酶 (Ｄｎｍｔ)。ＤＮＡ的甲基化修饰参与基因表达调控、胚胎发育、细胞分化、基因组印迹、Ｘ染色体灭活和细胞记忆等诸多重要生物学过程[1,2 ] 。在不同组织或同一类型细胞的不同发育阶段 ,基因组ＤＮＡ上各ＣｐＧ位点甲基化状态的差异即构成基因组的ＤＮＡ甲基化谱。根据催化反应类型。可以将ＤＮＡ甲基转移酶分为三类 :第一类将腺嘌呤转化成Ｎ6 甲基腺嘌呤 ;第二类将胞嘧啶转化成Ｎ4 甲基胞嘧啶 ;第三类将胞嘧啶转化成…     

DNA杂交与DNA指纹技术

Southern印迹杂交和DNA指纹技术在分子生物学研究以及疾病的诊断、亲缘关系鉴定、犯罪分子确认等过程中发挥了重要作用。回顾了2种技术的发明、发展历程和在生命科学研究中的作用,并探讨了可能的发展方向,从中可以从一个侧面了解分子生物学的发展历程和体会科学家的智慧在科学技术发展中所起的重要作用。     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that function. Received: 20 April 1998 / Accepted: 8 May 1998     

Gapped DNA and cyclization of short DNA fragments

We use the cyclization of small DNA molecules, approximately 200 bp in length, to study conformational properties of DNA fragments with single-stranded gaps. The approach is extremely sensitive to DNA conformational properties and, being complemented by computations, allows a very accurate determination of the fragment's conformational parameters. Sequence-specific nicking endonucleases are used to create the 4-nt-long gap. We determined the bending rigidity of the single-stranded region in the gapped DNA. We found that the gap of 4 nt in length makes all torsional orientations of DNA ends equally probable. Our results also show that the gap has isotropic bending rigidity. This makes it very attractive to use gapped DNA in the cyclization experiments to determine DNA conformational properties, since the gap eliminates oscillations of the cyclization efficiency with the DNA length. As a result, the number of measurements is greatly reduced in the approach, and the analysis of the data is greatly simplified. We have verified our approach on DNA fragments containing well-characterized intrinsic bends caused by A-tracts. The obtained experimental results and theoretical analysis demonstrate that gapped-DNA cyclization is an exceedingly sensitive and accurate approach for the determination of DNA bending.     

Active DNA demethylation and DNA repair

DNA methylation on cytosine is an epigenetic modification and is essential for gene regulation and genome stability in vertebrates. Traditionally DNA methylation was considered as the most stable of all heritable epigenetic marks. However, it has become clear that DNA methylation is reversible by enzymatic “active” DNA demethylation, with examples in plant cells, animal development and immune cells. It emerges that “pruning” of methylated cytosines by active DNA demethylation is an important determinant for the DNA methylation signature of a cell. Work in plants and animals shows that demethylation occurs by base excision and nucleotide excision repair. Far from merely protecting genomic integrity from environmental insult, DNA repair is therefore at the heart of an epigenetic activation process.     

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the presence or absence of high mobility group proteins), the methylation of m5C-deficient DNA and that of hemimethylated DNA were compared. Although hemimethylated , m5C-rich DNAs were much better substrates than were m5C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extract, a DNA-binding protein of unknown function was isolated which binds to m5C-rich DNA in preference to the analogous m5C-poor DNA.     

The origin of DNA genomes and DNA replication proteins

In recent years, it has became clear that most proteins involved in cellular DNA precursor synthesis or DNA replication have been 'invented' more than once, indicating that the transition from RNA to DNA genomes was more complex than previously thought. Several authors have suggested that DNA viruses, which often encode their own version of these proteins, played an important role in this process. The nature of the genome of the last universal cellular ancestor (LUCA) -- that is, RNA or DNA, prokaryotic-like or eukaryotic-like -- remains in dispute. A hyperthermophilic LUCA would have suggested a circular, double-stranded DNA genome; however, recent data favor a mesophilic or moderately thermophilic LUCA.     

DNA ligases in the repair and replication of DNA

DNA ligases are critical enzymes of DNA metabolism. The reaction they catalyse (the joining of nicked DNA) is required in DNA replication and in DNA repair pathways that require the re-synthesis of DNA.Most organisms express DNA ligases powered by ATP, but eubacteria appear to be unique in having ligases driven by NAD(+). Interestingly, despite protein sequence and biochemical differences between the two classes of ligase, the structure of the adenylation domain is remarkably similar. Higher organisms express a variety of different ligases, which appear to be targetted to specific functions. DNA ligase I is required for Okazaki fragment joining and some repair pathways; DNA ligase II appears to be a degradation product of ligase III; DNA ligase III has several isoforms, which are involved in repair and recombination and DNA ligase IV is necessary for V(D)J recombination and non-homologous end-joining. Sequence and structural analysis of DNA ligases has shown that these enzymes are built around a common catalytic core, which is likely to be similar in three-dimensional structure to that of T7-bacteriophage ligase. The differences between the various ligases are likely to be mediated by regions outside of this common core, the structures of which are not known. Therefore, the determination of these structures, along with the structures of ligases bound to substrate DNAs and partner proteins ought to be seen as a priority.     

DNA Methyltransferases and Structural-Functional Specificity of Eukaryotic DNA Modification

Properties of the main families of mammalian, plant, and fungal DNA methyltransferases are considered. Structural-functional specificity of eukaryotic genome sequences methylated by DNA methyltransferases is characterized. The total methylation of cytosine in DNA sequences is described, as well as its relation with RNA interference. Mechanisms of regulation of expression and modulation of DNA methyltransferase activity in the eukaryotic cell are discussed.__________Translated from Biokhimiya, Vol. 70, No. 7, 2005, pp. 885–899.Original Russian Text Copyright © 2005 by Buryanov, Shevchuk.This article was not published in the journal special issue devoted to the 70th anniversary of B. F. Vanyushin (Biochemistry (Moscow) (2005) 70, No. 5) because of limiting volume of the journal.     

Selective interaction of tirapazamine with DNA bases and DNA

An electrochemical model has been used to study the reductive activation of the hypoxic cell cytotoxin tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine-1,4-dioxide). Cyclic voltammetry and controlled potential electrolysis have been used to generate and study the 1-electron reduction product, the assumed biologically active species. Cyclic voltammetry of tirapazamine in dimethylformamide shows a quasi-reversible 1-electron reduction with the product showing a tendency to participate in a following chemical reaction. Controlled potential electrolysis to generate the 1-electron reduction product was unsuccessful due to the formation of a new redox-active species at less negative reduction potentials. However, the cyclic voltammetry of tirapazamine in the presence of E. coli DNA shows a decrease in the lifetime of the radical anion, signifying direct interaction with the DNA. The radical lifetime also decreased in the presence of adenine, thymine and guanine, but increased upon addition of cytosine and ribose. The study shows that cyclic voltammetry is an extremely useful tool for investigating the interaction between bio-reductive drugs and biological target molecules.     

DNA binding and antigenic specifications of DNA gyrase.

Complexes of DNA gyrase and minichromosomal DNA containing the origin of replication of Escherichia coli (oriC) can be formed without metabolic energy and visualised by electron microscopy. The A subunit, part of the A2B2-DNA gyrase complex is the binding protein. Various binding sites are scattered around the minichromosomal DNA including oriC. The minimal origin contains the only prominent and reproducible binding site. Binding to this site is suppressed by oxolinic acid and the ATP analogue beta-y-imido ATP. If gyrase isolated from the gram-positive bacterium Bacillus subtilis is used no binding to oriC is seen. This observation is consistent with antigenic differences between the A subunits of the two microorganisms. The binding to oriC might reflect a requirement for DNA gyrase during the initiation of DNA replication.     

Perspectives of DNA microarray and next-generation DNA sequencing technologies

DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation DNA sequencing method was first introduced by the 454 Company in 2003, immediately followed by the establishment of the Solexa and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology, with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles of these two technologies as well as their application and perspectives in genome research. Supported by the National High-Tech Research Program of China (Grant No.2006AA020704) and Shanghai Science and Technology Commission (Grant No. 05DZ22201)