Inhibitory effect of flavonoids on DNA-dependent DNA and RNA polymerases

Summary Flavonoids, (–)-epigallocatechin (1), myricetin (2) and quercetin (3), were investigated for inhibitory effects onE. coli DNA polymerase I and T7 bacteriophage RNA polymerase. In both DNA and RNA synthesis,1 and3 inhibited enzyme reactions by non-competitive and mixed type inhibition respecitively, with regard to template DNAs. Myricetin (2) inhibited DNA and RNA polymerase reactions by mixed type and competitive type inhibition, respectively, with template DNAs. It was suggested that2 interacts with covalently closed/circular DNA.     

Y-family DNA polymerases in mammalian cells

Eukaryotic genomes are replicated with high fidelity to assure the faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and, using their intrinsic exonuclease activities, to excise mistakenly incorporated nucleotides. Cells also possess a variety of specialized DNA polymerases that, by a process called translesion DNA synthesis (TLS), help overcome replication blocks when unrepaired DNA lesions stall the replication machinery. This review considers the properties of the Y-family (a subset of specialized DNA polymerases) and their roles in modulating spontaneous and genotoxic-induced mutations in mammals. We also review recent insights into the molecular mechanisms that regulate PCNA monoubiquitination and DNA polymerase switching during TLS and discuss the potential of using Y-family DNA polymerases as novel targets for cancer prevention and therapy.     

Directed evolution as a tool for understanding and optimizing nucleic acid polymerase function

Polynucleotide polymerases play a crucial role in transmitting genetic information from generation to generation, and they are the most important reagents in biotechnology. Although classical crystal structure analyses as well as biochemical studies have significantly contributed to our understanding of how DNA polymerases function, surprising new insights regarding the importance of certain residues and protein motifs, or of their mutability have been achieved in recent years by evolutionary approaches. Directed evolution has also facilitated the generation of polymerases with tailored substrate repertoires or with stabilities and activities beyond those of their naturally evolved counterparts. Recent new insights in polymerase structure-function relationships and new achievements in the development of tailored polymerases for current methods of nucleic acid synthesis will be summarized in this article. Received 22 April 2005; received after revision 20 July 2005; accepted 27 July 2005     

Base excision DNA repair

DNA repair is a collection of several multienzyme, multistep processes keeping the cellular genome intact against genotoxic insults. One of these processes is base excision repair, which deals with the most ubiquitous lesions in DNA: oxidative base damage, alkylation, deamination, sites of base loss and single-strand breaks, etc. Individual enzymes acting in base excision repair have been identified. The recent years were marked with many advances in understanding of their structure and many interactions that make base excision repair a functional, versatile system. This review describes the current knowledge of structural biology and biochemistry of individual steps of base excision repair, several subpathways of the common base excision repair pathway, and interactions of the repair process with other cellular processes.     

Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACF. An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.     

Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint,DNA repair and apoptosis

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer. Received 10 April 2007; accepted 23 April 2007     

Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis

Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae. Received 21 September 1998; received after revision 23 November 1998; accepted 23 November 1998     

Implications of ancient DNA for phylogenetic studies

The utility of DNA sequence characters from fossil specimens is examined from a phylogenetic perspective. Four ways that fossil characters can alter phylogenetic hypotheses are discussed. Two empirical examples and a third hypothetical example concerning amber-preserved insects are presented to illustrate these phenomena. Fossil DNA sequences as characters will be affected by the problem of missing data and missing taxa. In general, cladogram accuracy will be more greatly affected by missing taxa and cladogram resolution will be affected more acutely by missing data. Due to these points, an examination of the importance of the phylogenetic question being addressed, the utility of the fossil DNA sequences and the rarity of the fossil should be considered before damage of a fossil is undertaken.     

Genetic variation in the New World: Ancient teeth,bone, and tissue as sources of DNA

Examination of ancient and contemporary Native American mtDNA variation via diagnostic restriction sites and the 9-pb Region V deletion suggests a single wave of migration into the New World. This is in contrast to data from Torroni et al.³⁴ which suggested two waves of migration into the New World (the NaDene and Amerind). All four founding lineage types are present in populations in North, Central, and South America suggesting that all four lineages came over together and spead throughout the New World. Ancient Native American DNA shows that all four lineages were present before European contact in North America, and at least two were present in South America. The presence of all four lineages in the NaDene and the Amerinds argues against separate migrations founding these two groups, although admixture between the groups is still a viable explanation for the presence of all four types in the NaDene.     

An improved test for Africanized honeybee mitochondrial DNA

Mitochondrial DNA derived fromApis mellifera scutellata, the ancestor of the Africanized bees of the New World, lacks aBglII restriction site found in other types of honeybee^1,2. We present primers allowing amplification of a 485-bp section of the cytochrome b gene containing this site, using the polymerase chain reaction. Digestion of the amplifiel product withBglII yields contrasting patterns between Africanized and other honeybees.     

Urushiol components as mediators in DNA strand scission

Summary Poison oak urushiol, a mixture of 3-alk(en)ylcatechol derivatives was found to mediate DNA strand scission in the presence of oxygen and with copper(II) chloride as a catalyst. The reactionis believed to occur via activated reduced oxygen produced during oxidation of the catechol into itso-quinone derivative.     

The effect of DDT on DNA replication in the larval salivary gland cells ofDrosophila melanogaster

Summary The inhibitory effect of DDT on the initial stage of the DNA replication process in polytene chromosomes of larval salivary gland cells ofDrosophila melanogaster was investigated and possible mechanisms for the inhibition are discussed.     

Towards progress on DNA vaccines for cancer

Cancer immunotherapy faces many obstacles that include eliciting immune reactions to self antigens as well as overcoming tumor-derived immunosuppressive networks and evasion tactics. Within the vaccine arsenal for inhibiting cancer proliferation, plasmid DNA represents a novel immunization strategy that is capable of eliciting both humoral and cellular arms of the immune response in addition to being safely administered and easily engineered and manufactured. Unfortunately, while DNA vaccines have performed well in preventing and treating malignancies in animal models, their overall application in human clinical trials has not impacted cancer regression to date. Since the establishment of these early trials, progress has been made in terms of increasing DNA vaccine immunogenicity and subverting the suppressive properties of tumor cells. Therefore, the success of future plasmid DNA use in cancer patients will depend on combinatorial strategies that enhance and direct the DNA vaccine immune response while also targeting tumor evasion mechanisms. Received 2 April 2007; received after revision 14 May 2007; accepted 21 May 2007     

Peptides as DNA mimics: cross-reactivity and mimicry in systemic autoimmune diseases

No Abstract. Received 21 February 2002; received after revision 16 May 2002; accepted 14 June 2002     

Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants

Many virus and transposon DNAs can integrate into the host genome. In this review, techniques, including inverse polymerase chain reaction (IPCR), novel Alu-PCR and vectorette- or splinkerette-PCR are introduced as possible strategies for cloning flanking DNA regions of the integrants. Targeted gene-walking PCR, restriction-site PCR, capture PCR, and panhandle PCR and boomerang DNA amplification are also described. The principles, advantages and limitations of each approach are discussed. Received 9 July 1998; received after revision 2 October 1998; accepted 7 October 1998     

Epifluorescence microscopy ofAnabaena sp.: Nucleoid configurations and evidence for inclusion-associated DNA

Vital staining of the nucleoid inAnabaena sp. PCC7118 was performed using the double stranded DNA-specific fluorochrome DAPI. In the unicellular mutant, the central epifluorescent zone had a dense skein configuration, while in the filamentous parent strain protrusions, lobes, and distinct isolated elements of the nucleoid were visible. Both variants contrasted with the mainly peripheral, partitioned structure typical of plastids and prochlorophytes. Blue-white emittance from the DNA-DAPI complex was maximum in dividing cells, suggesting that DNA configuration is linked to the cell cycle events. In stationary cultures, epifluorescent cell inclusions were conspicuous: based on this observation, we argue that DNA is associated with carboxysomes in situ.     

The search for the right partner: Homologous pairing and DNA strand exchange proteins in eukaryotes

Finding the right partner is a central problem in homologous recombination. Common to all models for general recombination is a homologous pairing and DNA strand exchange step. In prokaryotes this process has mainly been studied with the RecA protein ofEscherichia coli. Two approaches have been used to find homologous pairing and DNA strand exchange proteins in eukaryotes. A biochemical approach has resulted in numerous proteins from various organisms. Almost all of these proteins are biochemically fundamentally different from RecA. The in vivo role of these proteins is largely not understood. A molecular-genetical approach has identified structural homologs to theE. coli RecA protein in the yeastSaccharomyces cerevisiae and subsequently in other organisms including other fungi, mammals, birds, and plants. The biochemistry of the eukaryotic RecA homologs is largely unsolved. For the fungal RecA homologs (S. cerevisiae RAD51, RAD55, RAD57, DMC1; Schizosaccharomyces pombe rad51; Neurospora crassa mei3) a role in homologous recombination and recombinational repair is evident. Besides recombination, homologous pairing proteins might be involved in other cellular processes like chromosome pairing or gene inactivation.     

A simple test using restricted PCR-amplified mitochondrial DNA to study the genetic structure ofApis mellifera L

The COI-COII intergenic region ofApis mellifera mitochondrial DNA contains an important length polymorphism based on a variable number of copies of a 192–196 bp sequence (Q) and the completer or partial deletion of 67 pb sequence (Po). This length variability has been combined with a restriction site polymorphism to produce a rapid and simple test for the characterization of mtDNA haplotypes. This test included the amplification by the polymerase chain reaction of the COI-COII region followed by aDraI restriction of the amplified fragment. In a survey of 302 colonies belonging to 12 subspecies, 21 different haplotypes have been found which have been unambiguously allocated to one of the 3 mtDNA lineages of the species. Although all colonies of lineage C exhibit the same pattern (C1), each one of lineages A and M presents up to 10 different haplotypes, opening the way to studies on the genetic structure and the evolution of a large fraction of the species. This test also differentiates southern Spanish and South African colonies, which can be of great interest for the Africanized bee problem.