Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2’-O-Methyl RNA and RNA Oligonucleotides

This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes.     

DNA,RNA and protein synthesis in ØLL55-infected Lactobacillus lactis

Lactobacillus lactis cells were infected with the bacteriophage ØLL55. The changes in DNA, RNA and protein synthesis were studied by following a long-term (over 3 h) incorporation of radioactive precursors into acid-insoluble material. Stimulation of DNA synthesis caused by phage occurred 30–35 min after infection and thymidine incorporation continued for about 70 min ceasing 10–20 min before the cells started to lyse. Cumulative (¹⁴C)-uracil incorporation into RNA continued at the level of uninfected cells for 30–40 min before starting to slow up. Protein synthesis in the infected cells followed that of a control culture for 40–50 min before the further incorporation of (¹⁴C)-leucine began to decrease.The additions of antibiotic inhibitors of RNA and protein synthesis (rifampicin and chloramphenicol, respectively) at various times before or during the prereplicative period showed that rifampicin, added up to 15 min after infection and chloramphenicol, added as late as 20–25 min after infection completely prevented the initiation of phage-genome replication. The later addition of these drugs did not prevent the out-burst of thymidine up-take, but promoted, however, a deduction in the initiations of new replication cycles. The results indicate that certain genes of ØLL55 genome must be expressed at the early stages of infection to confirm a proper onset and continuation of phage DNA replication.Abbreviations Rif rifampicin - CAL chloramphenicol - TCA trichloroacetic acid - cpm counts per minute     

Thermodynamic and Kinetic Characterization of Duplex Formation between 2′-O, 4′-C-Methylene-modified Oligoribonucleotides,DNA and RNA

2′-O,4′-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA–DNA mixmers, gabmers as well as full length LNA’s with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg²⁺- ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA’s thus may be a better strategy, than using full length LNA’s to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.     

Structure?Activity Relationship of Polypyridyl Ruthenium(II) Complexes as DNA Intercalators,DNA Photocleavage Reagents,and DNA Topoisomerase and RNA Polymerase Inhibitors

To investigate the relationship between the molecular structure and biological activity of polypyridyl Ru^II complexes, such as DNA binding, photocleavage ability, and DNA topoisomerase and RNA polymerase inhibition, six new [Ru(bpy)₂(dppz)]²⁺ (bpy=2,2′‐bipyridine; dppz=dipyrido[3,2‐a:2,′,3′‐c]phenazine) analogs have been synthesized and characterized by means of ¹H‐NMR spectroscopy, mass spectrometry, and elemental analysis. Interestingly, the biological properties of these complexes have been identified to be quite different via a series of experimental methods, such as spectral titration, DNA thermal denaturation, viscosity, and gel electrophoresis. To explain the experimental regularity and reveal the underlying mechanism of biological activity, the properties of energy levels and population of frontier molecular orbitals and excited‐state transitions of these complexes have been studied by density‐functional theory (DFT) and time‐depended DFT (TDDFT) calculations. The results suggest that DNA intercalative ligands with better planarity, greater hydrophobicity, and less steric hindrance are beneficial to the DNA intercalation and enzymatic inhibition of their complexes.     

Base recognition of gap sites in DNA–DNA and DNA–RNA duplexes by short oligonucleotides

To increase base recognition capability and sensitivity, we propose the separation of a commonly used single-probe system for oligonucleotide analysis into a set of three probes: a fluorophore-labeled probe, a promoter probe, and a short probe. In this study, we found that the probes of only 4 nt in length can selectively bind the corresponding gap site on complexes consisting of the target, fluorophore-labeled probe, and promoter probe, exhibiting a more than 14-fold difference in ligation between the matched and mismatched sequences. Moreover, we demonstrated that the immobilized short probes accurately recognized the sequences of the gap sites.     

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.     

Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5′-phosphate and 3′-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3′-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3′p substrate to generate an RNA 2′,3′-cyclic phosphate or convert DNA3′p to ssDNA^3′pp^5′A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3′p. These modifications of RNA and DNA 3′-phosphates are similar to the activities of RtcA, an RNA 3′-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3′p or DNA 3′p to generate the adenylated intermediate. For RNA ^3′pp^5′A, the third step involves attack of the adjacent 2′ hydroxyl to generate the RNA 2′,3′-cyclic phosphate. These steps are analogous to those in classical 5′ phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3′p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3′-phosphorylated nicks in double-stranded DNA to produce a 3′-adenylated product. These 3′-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5′Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3′-terminal phosphates.     

Decreased RNA, and Increased RNA Oxidation, in Ribosomes from Early Alzheimer’s Disease

All cells rely on efficient protein synthesis in order to maintain cellular homeostasis. Recent studies from our laboratory indicate that declines in protein synthesis and ribosome function occur in the earliest stage of Alzheimer’s disease (AD). Additional studies indicate a potential role for ribosomal RNA oxidation as a potential mediator of decreased protein synthesis in AD. The ribosome is a complex of proteins and nucleic acids that mediates all protein synthesis. At present it is unclear if significant alterations in ribosomal RNA occurs within the ribosome complex during the progression of AD. In this study we examined the amount of ribosomal RNA in the different ribosomal fractions generated from control subjects, individuals with mild cognitive impairment (MCI), and individuals with AD. Studies were conducted in the inferior parietal lobule of each subject. Together, these data demonstrate that during the progression of AD there is a gross decline in the amount of ribosomal RNA within the ribosome complex. Additionally, these studies provide evidence for gross elevations in RNA oxidation within the ribosome complex of MCI and AD. Together, these data strongly suggest a role for RNA alterations within the ribosome as a mediator of decreased protein synthesis in both MCI and AD.     

Nucleases of the Metallo-β-lactamase Family and Their Role in DNA and RNA Metabolism

ABSTRACT

Proteins of the metallo-β-lactamase family with either demonstrated or predicted nuclease activity have been identified in a number of organisms ranging from bacteria to humans and has been shown to be important constituents of cellular metabolism. Nucleases of this family are believed to utilize a zinc-dependent mechanism in catalysis and function as 5′ to 3′ exonucleases and or endonucleases in such processes as 3′ end processing of RNA precursors, DNA repair, V(D)J recombination, and telomere maintenance. Examples of metallo-β-lactamase nucleases include CPSF-73, a known component of the cleavage/polyadenylation machinery, which functions as the endonuclease in 3′ end formation of both polyadenylated and histone mRNAs, and Artemis that opens DNA hairpins during V(D)J recombination. Mutations in two metallo-β-lactamase nucleases have been implicated in human diseases: tRNase Z required for 3′ processing of tRNA precursors has been linked to the familial form of prostate cancer, whereas inactivation of Artemis causes severe combined immunodeficiency (SCID). There is also a group of as yet uncharacterized proteins of this family in bacteria and archaea that based on sequence similarity to CPSF-73 are predicted to function as nucleases in RNA metabolism. This article reviews the cellular roles of nucleases of the metallo-β-lactamase family and the recent advances in studying these proteins.     

《反义RNA和DNA》

《反义RNA和DNA》(Antisense RNA and DNA)由James A.H.Murray编著,1992年 Wiley-Liss出版社出版,401页。反义RNA和DNA是对基因活动进行高度选择性操作的技术。“反义”顺序互补编码链,能特殊地阻断基因表达。反义顺序通过碱基配对,同编码RNA结合,干扰蛋白质的翻译,从而降低蛋白质生产总量。该技术同选择作用相结合,有着广泛应用,可以降低或完全阻断任何基因表达。它对在生物体内,操纵基因功能,进行科研、医药或农业活动,都具有巨大潜力。     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.     

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.     

Ribonucleotide reductases: the link between an RNA and a DNA world?

The structures of a class III ribonucleotide reductase (RNR) and pyruvate formate lyase exhibit striking homology within their active site domains with respect to each other and to the previously published structure of a class I RNR. The common structures and the common complex-radical-based chemistry of these systems, as well as of the class II RNRs, suggest that RNRs evolved by divergent evolution and provide an essential link between the RNA and DNA world.     

When, where, and in what environment could the RNA world appear and evolve?

The environment necessary for the existence, amplification, and evolution of the RNA world, the difficulties of the abiogenous synthesis of RNA, and paradoxical situations with the stability of RNA, its functions, and the place of RNA in the geological history of the Earth are discussed. The chemical instability of the covalent structure of RNA in the aqueous medium is incompatible with the necessity of water for formation of its functionally active conformations (“water paradox”). The stable double-helical structure of RNA required for replication is incompatible with the stable compact conformations of single-stranded RNA molecules that are necessary for catalytic functions (conformational paradox). There was a very short time gap (or no gap at all) between the end of the massive meteorite bombardment of the Earth (3.9 Ga ago) and the appearance of the first evidence of cellular life (bacteria) in the Earth’s rocks (3.8–3.85 Ga ago or even earlier) (geological paradox). It is concluded that the RNA world could not appear, exist, or evolve into cellular forms of life on the Earth. This paper briefly discusses the possibility of an extraterrestrial origin of the RNA world and its extraterrestrial evolution with a subsequent distribution in space (mainly by comets) of the cellular form of life as more resistant to the environment as compared with free RNA.     

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.