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.     

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.     

DNA complexes with polycations useful for delivery of DNA into cells

Generation and physicochemical properties of complexes formed by high-molecular thymus DNA and plasmid DNA with synthetic polymers of (dimethyl amino)ethyl methacrylate, (diethyl amino)ethyl methacrylate, and poly(vinyl amine) were studied in solutions of different ionic strength using low-gradient viscometry, electrophoresis, circular dichroism, spectrophotometry, and dynamic light scattering. The complexes were tested for toxicity with T98G cell cultures. Condensation of DNA was shown to occur when the ratio of charged groups in the polycations and DNA exceeded unity. This condensation manifested itself as an increase in the optical density of DNA solutions. Condensation-associated changes in the dimensions of DNA molecules were determined, and phase diagrams of DNA-polycation systems were analyzed in the presence of NaCl. MTT analysis revealed no toxicity of these complexes.     

Processing DNA molecules as text

Polymerase Chain Reaction (PCR) is the DNA-equivalent of Gutenberg’s movable type printing, both allowing large-scale replication of a piece of text. De novo DNA synthesis is the DNA-equivalent of mechanical typesetting, both ease the setting of text for replication. What is the DNA-equivalent of the word processor? Biology labs engage daily in DNA processing—the creation of variations and combinations of existing DNA—using a plethora of manual labor-intensive methods such as site-directed mutagenesis, error-prone PCR, assembly PCR, overlap extension PCR, cleavage and ligation, homologous recombination, and others. So far no universal method for DNA processing has been proposed and, consequently, no engineering discipline that could eliminate this manual labor has emerged. Here we present a novel operation on DNA molecules, called Y, which joins two DNA fragments into one, and show that it provides a foundation for DNA processing as it can implement all basic text processing operations on DNA molecules including insert, delete, replace, cut and paste and copy and paste. In addition, complicated DNA processing tasks such as the creation of libraries of DNA variants, chimeras and extensions can be accomplished with DNA processing plans consisting of multiple Y operations, which can be executed automatically under computer control. The resulting DNA processing system, which incorporates our earlier work on recursive DNA composition and error correction, is the first demonstration of a unified approach to DNA synthesis, editing, and library construction.

Electronic supplementary material

The online version of this article (doi:10.1007/s11693-010-9059-y) contains supplementary material, which is available to authorized users.     

一种快速有效纯化DNA序列分析模板的方法

介绍一种ＤＮＡ序列分析模板的快速、有效的纯化方法。该法对ＤＮＡ模板的回收率可达９５％以上。多次测序结果表明,此法与其他常规纯化方法相比,具有简便、快速、有效、可靠等优点,其测序结果电泳带清晰,无模糊带及“鬼带”出现,重复性及稳定性较好。     

Mitochondrial DNA Repair Pathways

It has long been held that there is no DNA repair in mitochondria. Early observations suggestedthat the reason for the observed accumulation of DNA damage in mitochondrial DNA is thatDNA lesions are not removed. This is in contrast to the very efficient repair that is seen inthe nuclear DNA. Mitochondrial DNA does not code for any DNA repair proteins, but it hasbeen observed that a number of repair factors can be found in mitochondrial extracts. Mostof these participate in the base excision DNA repair pathway which is responsible for theremoval of simple lesions in DNA. Recent work has shown that there is efficient base excisionrepair in mammalian mitochondria and there are also indications of the presence of morecomplex repair processes. Thus, an active field of mitochondrial DNA repair is emerging. Anunderstanding of the DNA repair processes in mammalian mitochondria is an important currentchallenge and it is likely to lead to clarification of the etiology of the common mutations anddeletions that are found in mitochondria, and which are thought to cause various humandisorders and to play a role in the aging phenotype.     

线粒体DNA与DNA甲基化的关系

线粒体DNA（mitochondrial DNA,mtDNA）遗传信息量虽小,却控制着线粒体一些最基本的性质,对细胞及其功能有着重要影响。mtDNA的损伤与衰老、肿瘤等疾病的发生有关。DNA甲基化是调节基因表达的重要方式之一。mtDNA基因的表达受核DNA（nuclear DNA,nDNA）的调控,mtDNA和nDNA协同作用参与机体代谢调节和发病。本文就近年来mtDNA与DNA甲基化的关系作一综述。     

Homologies to chloroplast DNA in the nuclear DNA of a number of Chenopod species

Summary Sequences homologous to chloroplast (ct)DNA have been found in nuclear DNA in five species of the Chenopodiaceae, extending the earlier observations of promiscuous DNA in Spinacia oleracea (Timmis and Scott 1983). Using the 7.7 kbp spinach ctDNA Pst I fragment as a hybridization probe, several separately located homologies to ctDNA were resolved in the nuclear DNA of Beta vulgaris, Chenopodium quinoa, and Enchylaena tomentosa. In Chenopodium album and Atriplex cinerea the major region of homology was to a nuclear Eco RI fragment (6 kbp) indistinguishable from that in ctDNA. These homologies may therefore involve larger tracts of ctDNA because the same restriction sites are apparently retained in the nucleus. This suggests that in these latter two species there is a contrasting, more homogeneous arrangement of ctDNA transpositions in the nucleus.     

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)     

跨损伤合成的DNA聚合酶——一类新的DNA聚合酶

细胞虽然拥有多种修复途径,但有些DNA损伤仍不可避免地会逃避修复而在基因组上保留下来,细胞跨损伤DNA合成的分子机制一直是DNA修复中主要的未解决问题之一.最近通过对一类结构相关性UmuC/DinB蛋白质超家族成员的研究发现它们具有DNA聚合酶功能.这类新发现的DNA聚合酶不同于经典的复制性DNA聚合酶,它们能以易误/突变(error-prone/mutagenic)或无误(error-free)方式进行跨损伤(translesion)DNA合成,并且从细菌到人在进化上功能保守.     

Tobacco nuclear DNA contains long tracts of homology to chloroplast DNA

Summary Long tracts of DNA with high sequence homology to chloroplast DNA were isolated from nuclear genomic libraries of Nicotiana tabacum. One lambda EMBL4 clone was characterised in detail and assigned to nuclear DNA. The majority of the 15.5-kb sequence is greater than 99% homologous with its chloroplast DNA counterpart, but a single base deletion causes premature termination of the reading frame of the psaA gene. One region of the clone contains a concentration of deleted regions, and these were used to identify and quantify the sequence in native nuclear DNA by polymerase chain reaction (PCR) methods. An estimated 15 copies of this specific region are present in a 1c tobacco nucleus.     

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIα-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions −1 and −2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.     

DNA 的电荷转移

在双链DNA分子中,电荷常出现远距离的迁移现象,这与很多生物学功能有密切的关系,本文说明了DNA电荷转移的形成机制,分析了误配、绞联、三股螺旋及DNA结合蛋白对DNA电荷转移效率的影响,同时论述了DNA电荷转移的生物学意义。     

Biodegradable microcapsules with entrapped DNA for development of new DNA vaccines

In this study an universal method for preparation of biodegradable microcapsules for antigen entrapment was proposed and optimized. The multilayer microcapsules were prepared by layer-by-layer adsorption of various polyelectrolytes (such as alginate, poly-L-lysine, κ-carrageenan, chitosan and dextran derivatives). High entrapment efficiency of protein and plasmid DNA (non less than 90%) was shown. To carry out in vivo tests, a set of microcapsules with entrapped pTKShi plasmid encoding the E2 polypeptide of classical swine fever was prepared. It was shown that an injection of these microcapsules into mice induced an immune response. The highest antibody titers of mouse blood sera were got after immunization by microcapsules based on modified dextran/carrageenan and modified chitosan/carrageenan. The proposed method for antigen entrapment in biodegradable microcapsules could be used for development of encapsulated vaccines of a new generation (DNA-vaccines).     

线粒体DNA与DNA甲基化

线粒体是除细胞核之外唯一携带遗传物质的细胞器,其线粒体DNA（mitochondrial DNA,mtDNA）控制着线粒体一些最基本的性质,对细胞功能有着重要影响.DNA甲基化是调节基因表达的重要方式之一.研究表明mtDNA存在CpG位点的低甲基化,并且mtDNA基因的表达受核DNA（nuclear DNA,nDNA）及线粒体自身DNA甲基化的调控,mtDNA和nDNA协同作用参与机体代谢调节和疾病发生发展过程.就近年来mtDNA与DNA甲基化的关系作一综述.     

Distributions of Topological States in Circular DNA

A distinctive feature of closed circular DNA molecules is their particular topological state, which cannot be altered by any conformational rearrangement short of breaking at least one strand. This topological constraint opens unique possibilities for experimental studies of the distributions of topological states created in different ways. Primarily, the equilibrium distributions of topological properties are considered in the review. It is described how such distributions can be obtained and measured experimentally, and how they can be computed. Comparison of the calculated and measured equilibrium distributions over the linking number of complementary strands, equilibrium fractions of knots and links formed by circular molecules has provided much valuable information about the properties of the double helix. Study of the steady-state fraction of knots and links created by type II DNA topoisomerases has revealed a surprising property of the enzymes: their ability to reduce these fractions considerably below the equilibrium level.     

Involvement of DNA polymerase beta in DNA replication and mutagenic consequences

Overexpression in mammalian cells of the error-prone DNA polymerase beta (Pol beta) has been found to increase the spontaneous mutagenesis. Here, we investigated a possible mechanism used by Pol beta to be a genetic instability enhancer: its interference in replicative DNA synthesis, which is normally catalysed by the DNA polymerases alpha, delta and epsilon. By taking advantage of the ability to incorporate efficiently into DNA the chain terminator ddCTP as well as the oxidised nucleotide 8-oxo-dGTP, we show here that purified Pol beta can compete with the replicative DNA polymerases during replication in vitro of duplex DNA when added to human cell extracts. We found that involvement of Pol beta lowers replication fidelity and results in a modified error-specificity. Furthermore, we demonstrated that involvement of Pol beta occurred during synthesis of the lagging strand. These in vitro data provide one possible explanation of how overexpression of the enzyme could perturb the genetic instability in mammalian cells. We discuss these findings within the scope of the up-regulation of Pol beta in many cancer cells.     

DNA生物传感器及其研究进展

就ＤＮＡ生物传感器的工作原理,分类、ＤＮＡ探针的固化方法,以及电化学ＤＮＡ生物传感器、光学ＤＮＡ生物传感器及压电ＤＮＡ生物传感器的研究进展、优缺点和发展趋势加以介绍。     

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.     

DNA甲基化微阵列

DNA甲基化微阵列是近年发展起来的高通量分析基因组水平DNA甲基化状态和模式的新型技术,已成为肿瘤表观遗传学组研究的重要工具之一。利用DNA甲基化微阵列研究某种疾病状态下异常甲基化的基因有利于进一步明确该疾病的表观遗传学异常机制,发现与之相关的表观遗传学标志物。现有的DNA甲基化微阵列主要包括CpG岛微阵列和甲基化寡聚核苷探针微阵列,根据已有的文献资料,较为详细地阐述了上述技术的原理、特点和适用范围,对于研究者根据自己的研究目的选择适当的DNA甲基化微阵列技术具有一定的指导价值。