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.     

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.     

DNA microarrays in the clinic: how soon,how extensively?

Although DNA microarrays are now widely used in research settings, they have been slow to penetrate clinical practice in spite of their apparent advantages. This is due to the very different requirements for a clinical test in contrast to a research tool, and to a strict necessity for demonstrated clinical utility. There is a clear differentiation between two types of DNA array tests: "genomic" diagnostics, developed to ascertain the presence or absence of mutations, deletions or duplications, and for which clinical evidence is already established, and tests using expression profiling for prognosis or predictive purposes, in which case the clinical correlate must be proven. Most array diagnostics currently used belong, understandably, to the "genomic" variety. It is to be expected that future improvements in tailored technology, as well as a logical trend towards measuring an ever-increasing number of parameters, will ensure an important diagnostic role for DNA arrays in the coming decade.     

Coincidence,coevolution, or causation? DNA content,cellsize, and the C‐value enigma

Variation in DNA content has been largely ignored as a factor in evolution, particularly following the advent of sequence-based approaches to genomic analysis. The significant genome size diversity among organisms (more than 200000-fold among eukaryotes) bears no relationship to organismal complexity and both the origins and reasons for the clearly non-random distribution of this variation remain unclear. Several theories have been proposed to explain this 'C-value enigma' (heretofore known as the 'C-value paradox'), each of which can be described as either a mutation pressure' or 'optimal DNA' theory. Mutation pressure theories consider the large portion of non-coding DNA in eukaryotic genomes as either 'junk' or 'selfish' DNA and are important primarily in considerations of the origin of secondary DNA. Optimal DNA theories differ from mutation pressure theories by emphasizing the strong link between DNA content and cell and nuclear volumes. While mutation pressure theories generally explain this association with cell size as coincidental, the nucleoskeletal theory proposes a coevolutionary interaction between nuclear and cell volume, with DNA content adjusted adaptively following shifts in cell size. Each of these approaches to the C-value enigma is problematic for a variety of reasons and the preponderance of the available evidence instead favours the nucleotypic theory which postulates a causal link between bulk DNA amount and cell volume. Under this view, variation in DNA content is under direct selection via its impacts on cellular and organismal parameters. Until now, no satisfactory mechanism has been presented to explain this nucleotypic effect. However, recent advances in the study of cell cycle regulation suggest a possible 'gene nucleus interaction model' which may account for it. The present article provides a detailed review of the debate surrounding the C-value enigma, the various theories proposed to explain it, and the evidence in favour of a causal connection between DNA content and cell size. In addition, a new model of nucleotypic influence is developed, along with suggestions for further empirical investigation. Finally, some evolutionary implications of genome size diversity are considered, and a broadening of the traditional 'biological hierarchy' is recommended.     

DNA Barcodes for the FIshes of the Narmada,One of India’s Longest Rivers

This study describes the species diversity of fishes of the Narmada River in India. A total of 820 fish specimens were collected from 17 sampling locations across the whole river basin. Fish were taxonomically classified into one of 90 possible species based on morphological characters, and then DNA barcoding was employed using COI gene sequences as a supplemental identification method. A total of 314 different COI sequences were generated, and specimens were confirmed to belong to 85 species representing 63 genera, 34 families and 10 orders. Findings of this study include the identification of five putative cryptic or sibling species and 43 species not previously known from the Narmada River basin. Five species are endemic to India and three are introduced species that had not been previously reported to occur in the Narmada River. Conversely, 43 species previously reported to occur in the Narmada were not found. Genetic diversity and distance values were generated for all of the species within genera, families and orders using Kimura’s 2 parameter distance model followed by the construction of a Neighbor Joining tree. High resolution clusters generated in NJ trees aided the groupings of species corresponding to their genera and families which are in confirmation to the values generated by Automatic Barcode Gap Discovery bioinformatics platform. This aided to decide a threshold value for the discrimination of species boundary from the Narmada River. This study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within complex ecosystems such as the Narmada River.     

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。     

"What's my DNA worth, anyway?": a response to the commercialization of individuals' DNA information

Extensive enthusiasm surrounds the potential for human DNA information to sustain and enhance the pharmaceutical industry's profitability. Nevertheless, persons whose health makes their DNA of commercial interest are routinely expected simply to give their DNA and the information in it to pharmaceutical or genomics companies or their academic intermediaries, voluntarily and without compensation. This state of affairs is increasingly recognized as paradoxical, but it is favored by conventional bioethical opinion. Given that most DNA information is now collected for commercial purposes and is worth considerably more than is generally imagined, bioethical objections to compensation of individuals for their DNA information are inappropriate. This paper suggests approaches by which individuals and representative governments and patient interest groups can negotiate compensation. Appreciable attitudinal change is required if those individuals personally involved are to be included fairly in the commercialization of human DNA information. Ultimately, however, such change is necessary if commercial genetic research is to respect human dignity and human rights.     

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.     

Flexibility in the order of action and in the enzymology of the nuclease, polymerases, and ligase of vertebrate non-homologous DNA end joining： relevance to cancer, aging, and the immune system

Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.     

Two X family DNA polymerases, λ and μ, in meiotic tissues of the basidiomycete, Coprinus cinereus

The X family DNA polymerases λ (CcPolλ) and μ (CcPolμ) were shown to be expressed during meiotic prophase in the basidiomycete, Coprinus cinereus. These two polymerases are the only members of the X family in the C. cinereus genome. The open reading frame of CcPolλ encoded a predicted product of 800 amino acid residues and that of CcPolμ of 621 amino acid residues. Both CcPolλ and CcPolμ required Mn²⁺ ions for activity, and both were strongly inhibited by dideoxythymidine triphosphate. Unlike their mammalian counterparts, CcPolλ and CcPolμ had no terminal deoxynucleotidyl transferase activity. Immunostaining analysis revealed that CcPolλ was present at meiotic prophase nuclei in zygotene and pachytene cells, which is the period when homologous chromosomes pair and recombine. CcPolμ was present in a slightly wider range of cell stages, zygotene to diplotene. In analyses using D-loop recombination intermediate substrates, we found that both CcPolλ and CcPolμ could promote primer extension of an invading strand in a D-loop structure. Moreover, both polymerases could fully extend the primer in the D-loop substrate, suggesting that D-loop extension is an activity intrinsic to CcPolλ and CcPolμ. Based on these data, we discuss the possible roles of these polymerases in meiosis.     

DNA intercalation without flipping in the specific ThaI–DNA complex

The PD-(D/E)XK type II restriction endonuclease ThaI cuts the target sequence CG/CG with blunt ends. Here, we report the 1.3 Å resolution structure of the enzyme in complex with substrate DNA and a sodium or calcium ion taking the place of a catalytic magnesium ion. The structure identifies Glu54, Asp82 and Lys93 as the active site residues. This agrees with earlier bioinformatic predictions and implies that the PD and (D/E)XK motifs in the sequence are incidental. DNA recognition is very unusual: the two Met47 residues of the ThaI dimer intercalate symmetrically into the CG steps of the target sequence. They approach the DNA from the minor groove side and penetrate the base stack entirely. The DNA accommodates the intercalating residues without nucleotide flipping by a doubling of the CG step rise to twice its usual value, which is accompanied by drastic unwinding. Displacement of the Met47 side chains from the base pair midlines toward the downstream CG steps leads to large and compensating tilts of the first and second CG steps. DNA intercalation by ThaI is unlike intercalation by HincII, HinP1I or proteins that bend or repair DNA.     

Rutin–Nickel Complex: Synthesis,Characterization, Antioxidant,DNA Binding,and DNA Cleavage Activities

The rutin–nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV–visible spectroscopy, IR, mass spectrometry, ¹H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.     

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems.