植物细胞核DNA,叶绿体DNA和线粒体DNA的比较

植物一般有细胞核,叶绿体和线粒体三套遗传体系,本文结合近年来植物分子生物学研究的最新进展,系统比较了细胞核ＤＮＡ,叶绿体ＤＮＡ和线粒体ＤＮＡ在组织结构,遗传方式,基因表达（转录,翻译,ＲＮＡ加工）等方面的差异。     

Physical Interactions between Mcm10, DNA, and DNA Polymerase ��

Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase α (pol α), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol α. However, the mechanism by which Mcm10 interacts with pol α on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID·ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286–310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol α within the replication fork.To maintain their genomic integrity, cells must ensure complete and accurate DNA replication once per cell cycle. Consequently, DNA replication is a highly regulated and orchestrated series of molecular events. Multiprotein complexes assembled at origins of replication lead to assembly of additional proteins that unwind chromosomal DNA and synthesize nascent strands. The first event is the formation of a pre-replicative complex, which is composed of the origin recognition complex, Cdc6, Cdt1, and Mcm2–7 (for review, see Ref. 1). Initiation of replication at the onset of S-phase involves the activity of cyclin- and Dbf4-dependent kinases concurrent with recruitment of key factors to the origin. Among these, Mcm10 (2, 3) is recruited in early S-phase and is required for loading of Cdc45 (4). Mcm2–7, Cdc45, and the GINS complex form the replicative helicase (5–8). Origin unwinding is followed by loading of RPA,³ And-1/Ctf4, and pol α onto ssDNA (9–12). In addition, recruitment of Sld2, Sld3, and Dpb11/TopBP1 are essential for replication initiation (13, 14), and association of topoisomerase I, proliferating cellular nuclear antigen (PCNA), replication factor C, and the replicative DNA polymerases δ and ϵ completes the replisome (for review, see Ref. 15).Mcm10 is exclusive to eukaryotes and is essential to both initiation and elongation phases of chromosomal DNA replication (6, 8, 16). Mutations in Mcm10 in yeast result in stalled replication, cell cycle arrest, and cell death (2, 3, 17–19). These defects can be explained by the number of genetic and physical interactions between Mcm10 and many essential replication proteins, including origin recognition complex, Mcm2–7, and PCNA (3, 12, 20–24). In addition, Mcm10 has been shown to stimulate the phosphorylation of Mcm2–7 by Dbf4-dependent kinase in vitro (25). Thus, Mcm10 is an integral component of the replication machinery.Importantly, Mcm10 physically interacts with and stabilizes pol α and helps to maintain its association with chromatin (16, 26, 27). This is a critical interaction during replication because pol α is the only enzyme in eukaryotic cells that is capable of initiating DNA synthesis de novo. Indeed, Mcm10 stimulates the polymerase activity of pol α in vitro (28), and interestingly, the fission yeast Mcm10, but not Xenopus Mcm10, has been shown to exhibit primase activity (29, 30). Mcm10 is composed of three domains, the N-terminal (NTD), internal (ID), and C-terminal (CTD) domains (29). The NTD is presumably an oligomerization domain, whereas the ID and CTD both interact with DNA and pol α (29). The CTD is not found in yeast, whereas the ID is highly conserved among all eukaryotes. The crystal structure of Mcm10-ID showed that this domain is composed of an oligonucleotide/oligosaccharide binding (OB)-fold and a zinc finger motif, which form a unified DNA binding platform (31). An Hsp10-like motif important for the interaction with pol α has been identified in the sequence of Saccharomyces cerevisiae Mcm10-ID (16, 26).DNA pol α-primase is composed of four subunits: p180, p68, p58, and p48. The p180 subunit possesses the catalytic DNA polymerase activity, and disruption of this gene is lethal (32, 33). p58 and p48 form the DNA-dependent RNA polymerase (primase) activity (34, 35), whereas the p68 subunit has no known catalytic activity but serves a regulatory role (36, 37). Pol α plays an essential role in lagging strand synthesis by first creating short (7–12 nucleotide) RNA primers followed by DNA extension. At the critical length of ∼30 nucleotides, replication factor C binds to the nascent strand to displace pol α and loads PCNA with pols δ and ϵ (for review, see Ref. 38).The interaction between Mcm10 and pol α has led to the suggestion that Mcm10 may help recruit the polymerase to the emerging replisome. However, the molecular details of this interaction and the mechanism by which Mcm10 may recruit and stabilize the pol α complex on DNA has not been investigated. Presented here is the high resolution structure of the conserved Mcm10-ID bound to ssDNA together with NMR chemical shift perturbation competition data for pol α binding in the presence of ssDNA. Collectively, these data demonstrate a shared binding site for DNA and pol α in the OB-fold cleft of Mcm10-ID, with a preference for ssDNA over pol α. In addition, we have mapped the Mcm10-ID binding site on pol α to a 24-residue segment of the N-terminal domain of p180. Based on these results, we propose Mcm10 helps to recruit pol α to origins of replication by a molecular hand-off mechanism.     

Distinct kinetics of human DNA ligases I,IIIα, IIIβ, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIIIβ and the hLigIIIα/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.     

Cloning and Characterization of the Human Mitochondrial DNA Polymerase, DNA Polymerase γ

The nuclear-encoded DNA polymerase γ (DNA POLγ) is the sole DNA polymerase required for the replication of the mitochondrial DNA. We have cloned the cDNA for human DNA POLγ and have mapped the gene to the chromosomal location 15q24. Additionally, the DNA POLγ gene fromDrosophila melanogasterand a partial cDNA for DNA POLγ fromGallus gallushave been cloned. The predicted human DNA POLγ polypeptide is 1239 amino acids, with a calculated molecular mass of 139.5 kDa. The human amino acid sequence is 41.6, 43.0, 48.7, and 77.6% identical to those ofSchizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster,and the C-terminal half ofG. gallus,respectively. Polyclonal antibodies raised against the polymerase portion of the protein reacted specifically with a 140-kDa protein in mitochondrial extracts and immunoprecipitated a protein with DNA POLγ like activity from mitochondrial extracts. The human DNA POLγ is unique in that the first exon of the gene contains a CAG₁₀trinucleotide repeat.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 2. Formation of Topotecan–DNA Complex Containing Several DNA Molecules

This study is a continuation of a series of papers dealing with topotecan interaction with double-stranded polydeoxyribonucleotides. We showed earlier that topotecan molecules form dimers in solution at concentration above 10^–5(per base pair). Topotecan interaction with calf thymus DNA in solutions of low ionic strength was studied by fluorescence, circular dichroism, and linear flow dichroism. The data obtained indicate that topotecan forms two types of complex with DNA, DNA molecules combining with each other during formation of one of these complexes. The association constant of two topotecan-filled DNA molecules with each other was estimated at 10⁴M^–1(per base pair) in 1 mM sodium cacodylate buffer, pH 6.8, at 20°C. A possibility of modulation of DNA topoisomerase I activity by topotecan due to complexation with several sites of a supercoiled DNA molecule is discussed.     

DNA指纹法可靠,但需标准化

在鉴定个人案件中,DNA测试(常常被称为DNA指纹鉴定法)的可靠性受到了法庭挑战,部分地是因为遗传鉴定被作为发生于弗罗里达、得克萨斯及乔治亚等州的死亡争吵中为同居者定罪的主要依据.尽管美国于1986年才开始引用此法,但在其49个州和哥     

端粒DNA与细胞的衰老,死亡

端粒ＤＮＡ与细胞的衰老、死亡白丽荣（河北衡水师专生物系,０５３０００）端粒ＤＮＡ是真核生物染色体的天然末端,它的生物学功能是保持染色体稳定。近年来研究发现,端粒ＤＮＡ决定了细胞寿命,并与细胞的自然凋亡及癌化有关。端粒（Ｔｅｌｏｍｅｒｅ）是指真核细胞线...     

DNA polymerase mu,a candidate hypermutase?

A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining.     

Binding of indium,used in the perturbed γ-γ angular correlation studies,on DNA and DNA moieties

The two successive gamma rays emitted from bound indium (¹¹¹In) are utilized in the Perturbed γ-γ Angular Correlation (PAC) method for the study of the molecular dynamics of cellular macromolecules such as DNA. In this paper, evidence is presented indicating that indium binds on both phosphate and the base moieties of DNA and of the nucleotides and that, the binding of indium, under the conditions used in the PAC method, does not alter the hydrodynamic properties of the DNA macromolecule or its UV absorbance spectrum.     

GENETIC VARIATION AND DNA REPLICATION TIMING,OR WHY IS THERE LATE REPLICATING DNA?

Mutation rates vary significantly within the genome and across species. Recent studies revealed a long suspected replication-timing effect on mutation rate, but the mechanisms that regulate the increase in mutation rate as the genome is replicated remain unclear. Evidence is emerging, however, that DNA repair systems, in general, are less efficient in late replicating heterochromatic regions compared to early replicating euchromatic regions of the genome. At the same time, mutation rates in both vertebrates and invertebrates have been shown to vary with generation time (GT). GT is correlated with genome size, which suggests a possible nucleotypic effect on species-specific mutation rates. These and other observations all converge on a role for DNA replication checkpoints in modulating generation times and mutation rates during the DNA synthetic phase (S phase) of the cell cycle. The following will examine the potential role of the intra-S checkpoint in regulating cell cycle times (GT) and mutation rates in eukaryotes. This article was published online on August 5, 2011. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected October 4, 2011.     

800?000 year old mammoth DNA,modern elephant DNA or PCR artefact?

Poulakakis and colleagues (Poulakakis et al. 2006: Biol. Lett. 2, 451-454), report the recovery of 'authentic' mammoth DNA from an 800,000-year-old fragment of bone excavated on the island of Crete. In light of results from other ancient DNA studies that indicate how DNA survival is unlikely in samples, which are recovered from warm environments and are relatively old (e.g. more than 100,000 years), these findings come as a great surprise. Here, we show that problems exist with the methodological approaches used in the study. First, the nested PCR technique as reported is nonsensical--one of the second round 'nested' primers falls outside the amplicon of the first round PCR. More worryingly, the binding region of one of the first round primers (Elcytb320R) falls within the short 43 base pair reported mammoth sequence, specifically covering two of the three reportedly diagnostic Elephas polymorphisms. Finally, we demonstrate using a simple BLAST search in GenBank that the claimed 'uniquely derived character state' for mammoths is in fact also found within modern elephants.     

DNA Polymerase β Ribonucleotide Discrimination: INSERTION,MISINSERTION, EXTENSION,AND CODING*

DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase β to utilize nucleotides with modified sugars. DNA polymerase β readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3′-endo sugar pucker facilitates nucleotide insertion, whereas a 2′-endo conformation inhibits insertion.     

DNA芯片的基本原理,方法及应用前景

ＤＮＡ芯片是近年分子生物学技术的重要进展,该技术是由把巨大数量的寡核苷酸探针或ｃＤＮＡ探针固定在一块面积很小的基板上而组成一个高密,二维的阵列所得。本文综述了它的基本原理,制作 方法,存在问题和新近发展及其应用。     

Bst DNA聚合酶大片段的克隆,表达,性质及其应用

用耐高温的ＤＮＡ聚合酶进行酶法ＤＮＡ序列测定在分子生物学中具有十分重要的实际应用价值,从耐高温的嗜热脂肪芽孢杆菌（Ｂａｃｉｌｌｕｓｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ）特异菌株中克隆分离了编码去除了５′－３′外切酶活性的ＢｓｔＤＮＡ聚合酶大片段的结构基因。经重组于表达质粒载体ｐＹＺ２３,在大肠杆菌ＪＦ１１２５（ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＪＦ１１２５）中得到了稳定的高效表达,经分离纯化,此基因工     

贵州汉族,苗族,布依族和水族人群线粒体DNA多态性研究

本文运用１６种限制性内切酶对来自贵州的汉族、苗族、布依族和水族的１５０个样本进行了ｍｔＤＮＡ的限制性片段长度多态性（ＲＦＬＰ）分析。共检测到３１种限制性格局（Ｒｅｓｔｒｉｃｔｉｏｎｐａｔｔｅｒｎ）,其中ＨａｅＩＩ－１３型、ＥｃｏＲＶ－３型和ＰｓｔＩ－４型３种限制性格局为新报道综合这些限制性格局,共得出２８种ｍｔＤＮＡ类型（ｍｔＤＮＡｔｙｐｅ）。运用ＵＰＧ法和简约法分析了各ｍｔＤＮＡ类型之间、各人群之间的聚类关系,结果表明：水族人群的ｍｔＤＮＡ变异度较大;汉族和苗族的亲缘关系最近,布依族和水族有着较远的亲缘关系。