Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation by Thermophilic DNA Polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: Cellular Roles of DNA Polymerase Beta

Since its discovery and purification in 1971, DNA polymerase ß (Pol ß) is one of the most well-studied DNA polymerases. Pol ß is a key enzyme in the base excision repair (BER) pathway that functions in gap filling DNA synthesis subsequent to the excision of damaged DNA bases. A major focus of our studies is on the cellular roles of Pol ß. We have shown that germline and tumor-associated variants of Pol ß catalyze aberrant BER that leads to genomic instability and cellular transformation. Our studies suggest that Pol ß is critical for the maintenance of genomic stability and that it is a tumor suppressor. We have also shown that Pol ß functions during Prophase I of meiosis. Pol ß localizes to the synaptonemal complex and is critical for removal of the Spo11 complex from the 5’ ends of double-strand breaks. Studies with Pol ß mutant mice are currently being undertaken to more clearly understand the function of Pol ß during meiosis. In this review, we will highlight our contributions from our studies of Pol ß germline and cancer-associated variants.     

The Rate-limiting Step of DNA Synthesis by DNA Polymerase Occurs in the Fingers-closed Conformation

DNA polymerases maintain genomic integrity by copying DNA with high fidelity, part of which relies on the polymerase fingers opening-closing transition, a series of conformational changes during the DNA synthesis reaction cycle. Fingers opening and closing has been challenging to study, mainly due to the need to synchronise molecular ensembles. We previously studied fingers opening-closing on single polymerase-DNA complexes using single-molecule FRET; however, our work was limited to pre-chemistry reaction steps. Here, we advance our analysis to extensible substrates, and observe DNA polymerase (Pol) conformational changes across the entire DNA polymerisation reaction in real-time, gaining direct access to an elusive post-chemistry step rate-limiting for DNA synthesis. Our results showed that Pol adopts the fingers-closed conformation during polymerisation, and that the post-chemistry rate-limiting step occurs in the fingers-closed conformation. We found that fingers-opening in the Pol-DNA binary complex in the absence of polymerisation is slow (～5.3 s^?1), and comparable to the rate of fingers-opening after polymerisation (3.4 s^?1); this indicates that the fingers-opening step itself could be largely responsible for the slow post-chemistry step, with the residual rate potentially accounted for by pyrophosphase release. We also observed that DNA chain-termination of the 3′ end of the primer increases substantially the rate of fingers-opening in the Pol-DNA binary complex (5.3 → 29 s^?1), demonstrating that the 3′-OH residue is important for the kinetics of fingers conformational changes. Our observations offer mechanistic insight and tools to offer mechanistic insight for all nucleic acid polymerases.     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

利用Taq DNA聚合酶反转录cDNA第一链的研究

应用Taq DNA聚合酶(Thermus aquaticus DNA polymerase)直接对RNA进行反转录成cDNA第一链,然后用特异引物进行PCR扩增,结果表明,反转录达到AMV逆转录酶的效果,且可能得到比AMV逆转录酶更完整的cDNA第一链。     

核酶对人巨细胞病毒mRNA片段的体外切割

引导序列GSs(GuideSequences)是能与mRNA互补,引导核酶RNaseP催化核心M1RNA对互补区域特异切割的小片段游离RNA。针对人巨细胞病毒HCMV(humancytomegalovirus)DNA聚合酶mRNA序列设计GS,共价结合到大肠杆菌来源M1RNA中,构建成M1GST7核酶。通过对巨细胞病毒DNA聚合酶亚克隆片段转录产物体外切割实验,表明该核酶具备对DNA聚合酶mRNA片段的特异切割能力 。     

利用套叠PCR和高保真DNA聚合酶进行基因多位点突变的研究

利用套叠PCR和高保真DNA聚合酶对人工合成的牛口蹄疫病毒VPI基因(FMDV-VPI,Foot-Mouth Disease Vims-VPI)的5个突变位点进行修复,修复的成功率为100％。     

NGS-based analysis of base-substitution signatures created by yeast DNA polymerase eta and zeta on undamaged and abasic DNA templates in vitro

Translesion synthesis (TLS) is the mechanism in which DNA polymerases (TLS polymerases) bypass unrepaired template damage with high error rates. DNA polymerase η and ζ (Polη and Polζ) are major TLS polymerases that are conserved from yeast to humans. In this study, we quantified frequencies of base-substitutions by yeast Polη and Polζ on undamaged and abasic templates in vitro. For accurate quantification, we used a next generation sequencing (NGS)-based method where DNA products were directly analyzed by parallel sequencing. On undamaged templates, Polη and Polζ showed distinct base-substitution profiles, and the substitution frequencies were differently influenced by the template sequence. The base-substitution frequencies were influenced mainly by the adjacent bases both upstream and downstream of the substitution sites. Thus we present the base-substitution signatures of these polymerases in a three-base format. On templates containing abasic sites, Polη created deletions at the lesion in more than 50% of the TLS products, but the formation of the deletions was suppressed by the presence of Polζ. Polζ and Polη cooperatively facilitated the TLS reaction over an abasic site in vitro, suggesting that these two polymerases can cooperate in efficient and high fidelity TLS.     

Mechanisms of mutagenesis induced by DNA lesions: multiple factors affect mutations in translesion DNA synthesis

Abstract

Environmental mutagens lead to mutagenesis. However, the mechanisms are very complicated and not fully understood. Environmental mutagens produce various DNA lesions, including base-damaged or sugar-modified DNA lesions, as well as epigenetically modified DNA. DNA polymerases produce mutation spectra in translesion DNA synthesis (TLS) through misincorporation of incorrect nucleotides, frameshift deletions, blockage of DNA replication, imbalance of leading- and lagging-strand DNA synthesis, and genome instability. Motif or subunit in DNA polymerases further affects the mutations in TLS. Moreover, protein interactions and accessory proteins in DNA replisome also alter mutations in TLS, demonstrated by several representative DNA replisomes. Finally, in cells, multiple DNA polymerases or cellular proteins collaborate in TLS and reduce in vivo mutagenesis. Summaries and perspectives were listed. This review shows mechanisms of mutagenesis induced by DNA lesions and the effects of multiple factors on mutations in TLS in vitro and in vivo.     

The Loss of Genetic Diversity in Sichuan Taimen as Revealed by DNA Fingerprinting

Species endangerment often derives from the “endangerment” of genetic diversity, thus loss of genetic diversity is an important cause of species extinction. Since historical specimens were unavailable, previous studies mainly described the genetic diversity status in the current population rather than the loss of genetic variation over time. In this study, we collected samples during1998–1999 and obtained historical specimens from 1957 to 1958. Based on the two sets of fish, we determined the changes in genetic diversity of Sichuan taimen using DNA fingerprinting. The differences in genetic parameters between the present samples and historical taimens revealed their loss of genetic variation. As a result, the existing populations have lower viability, and proper management has to be implemented to preserve genetic diversity.     

PCR 扩增人型结核杆菌 DNA 及结核杆菌属 DNA 探针的制备

用多聚酶链反应(PCR)方法扩增人型、牛型结核杆菌基因组 DNA,获得特异的158 bpDNA 片段,而从另外十三种分枝杆菌未见到特异的扩增产物.回收158 bpDNA 片段作探针,它除与人型、牛型结核杆菌有特异的杂交信号外,与金黄色葡萄球菌、绿脓杆菌及一些分枝杆菌皆没有杂交反应.结果表明,PCR 可用于检测结核杆菌基因组 DNA,扩增产物158 bp DNA 片段可作为探针用于检测人型、牛型结核杆菌并鉴别结核杆菌与其它分枝杆菌.     

Inhibition of DNA replication in vitro by pefloxacin

Pefloxacin (a novel quinolone antibiotic) is demonstrated to be a drug inhibiting DNA replication 10-times more efficiently than oxolinic acid measured either in toluene-treated E. coli or in an in vitro replication system for oriC plasmids [6]. DNA repair synthesis is not inhibited by the drug.     

Fast detection of quadruplex structure in DNA by the intrinsic fluorescence of a single-stranded DNA binding protein

Single-stranded guanine-rich (G-rich) DNA can fold into a four-stranded G-quadruplex structure and such structures are implicated in important biological processes and therapeutic applications. So far, bioinformatic analysis has identified up to several hundred thousand of putative quadruplex sequences in the genome of human and other animal. Given such a large number of sequences, a fast assay would be desired to experimentally verify the structure of these sequences. Here we describe a method that identifies the quadruplex structure by a single-stranded DNA binding protein from a thermoautotrophic archaeon. This protein binds single-stranded DNA in the unfolded, but not in the folded form. Upon binding to DNA, its fluorescence can be quenched by up to 70%. Formation of quadruplex greatly reduces fluorescence quenching in a K+-dependent manner. This structure-dependent quenching provides simple and fast detection of quadruplex in DNA at low concentration without DNA labelling.     

Dissociation in a Polymerization Model of Homochirality

A fully self-contained model of homochirality is presented that contains the effects of both polymerization and dissociation. The dissociation fragments are assumed to replenish the substrate from which new monomers can grow and undergo new polymerization. The mean length of isotactic polymers is found to grow slowly with the normalized total number of corresponding building blocks. Alternatively, if one assumes that the dissociation fragments themselves can polymerize further, then this corresponds to a strong source of short polymers, and an unrealistically short average length of only 3. By contrast, without dissociation, isotactic polymers becomes infinitely long.     

Mechanics of DNA flexibility visualized by selective 2'-amine acylation at nucleotide bulges

We used selective acylation of 2'-amine-substituted nucleotides to visualize local backbone conformations that occur preferentially at bulged sites in DNA duplexes. 2'-Amine acylation reports local nucleotide flexibility because unconstrained 2'-amino nucleotides more readily reach a reactive conformation in which the amide-forming transition state is stabilized by interactions between the amine nucleophile and the adjacent 3'-phosphodiester group. Bulged 2'-amine-substituted cytidine nucleotides react approximately 20-fold more rapidly than nucleotides constrained by base-pairing at 35 degrees C. In contrast, base-paired 2'-amine-substituted nucleotides flanked by a 5' or 3' bulge react two- or six-fold more rapidly, respectively, than the perfectly paired duplex. The relative lack of 2'-amine reactivity for nucleotides adjacent to a DNA bulge emphasizes, first, that structural perturbations do not extend significantly into the flanking duplex structure. Second, the exquisite sensitivity towards very local perturbations in nucleic acid structure suggests that 2'-amine acylation can be used to chemically interrogate deletion mutations in DNA. Finally, these data support the mechanical interpretation that the reactive ribose conformation for 2'-amine acylation requires that the base lies out of the helix and in the major groove, a mechanistic insight useful for designing 2'-amine-based sensors.     

Assessment of Genetic Diversity in Thai Isolates of Pyricularia grisea by Random Amplification of Polymorphic DNA

One hundred and seventy‐four isolates of Pyricularia grisea were collected from various hosts such as barley, rice, weed and wild rice in Thailand. Seven arbitrary decamer primers from the set of University of British Columbia were employed and nine lineages were classified. Lineages B, C and H were predominant, contributing up to 70% of total pathogens in this study. Analysis showed that the distribution of each lineage differs from the predominant lineages across Thailand in such that other lineages were restricted in particular area. For instance, lineage A was limited only in southern Thailand, whereas wide distribution of lineages B and C reflected an influence of both biological and physical effects on pathogen variation. Principal component analysis resulted in a total of four groups of blast pathogen with small distinctions between barley‐, rice‐, weed‐ and wild rice‐infected blast. Bridging relationships occurred among border isolates of weed and rice blast suggesting a chance of migrations between hosts. Higher diversity was observed in northern, north‐eastern and central Thailand while eastern and southern parts were rather low. Genetic diversity indices elucidated an abundance of pathogen lineages existing in northern Thailand suggesting that it should be the centre of diversity.     

Genotypic diversity in a localized population of Ralstonia solanacearum as revealed by random amplified polymorphic DNA markers

AIMS: To assess genotypic diversity within Ralstonia solanacearum isolates of a single field. METHODS AND RESULTS: A total of 44 field isolates and 22 in vitro generated clones of R. solanacearum were studied for genotypic diversity by random amplified polymorphic DNA (RAPD) technique. Genomic DNA of these isolates and clones was extracted by proteinase-K-SDS lysis mini-prep method. RAPD analysis was done with 30 decamer primers. The data were analysed using NTSYSpc 2.02h software. Forty-two out of 44 field isolates and all the clonal isolates were identified as distinct genotypes at 70% similarity level. CONCLUSION: Very high level of genome variability was observed within the field and clonal isolates of R. solanacearum. This might be a reason for the wide host range of this bacterium and for quick breakdown of wilt resistance in host plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that it would be difficult to design specific diagnostic protocol for R. solanacearum even for a localized population and to breed cultivars with broad-spectrum resistance.