Separation and functional analysis of eukaryotic DNA topoisomerases by chromatography and electrophoresis

DNA topoisomerases are enzymes that control DNA topology by cleaving and rejoining DNA strands and passing other DNA strands through the transient gaps. Consequently, these enzymes play a crucial role in the regulation of the physiological function of the genome. Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers. In this review we summarize current protocols for extracting and purifying DNA topoisomerases, and for separating subtypes and isoforms of these enzymes. Furthermore, we discuss methods for measuring the catalytic activity of topoisomerases and for monitoring the molecular effects of topoisomerase-directed antitumor drugs in cell-free assays.     

Evidence of the existence of a high molecular weight form of DNA polymerase alpha in sea urchin eggs

DNA polymerase alpha combined with the endoplasmic reticulum (ER) was isolated from unfertilized sea urchin eggs. NaCl treatment of this fraction released DNA polymerase alpha from the ER. The molecular size (the S value) of the ER-free DNA polymerase alpha changed with the concentration of NaCl used; being 23 S, 11-15 S and 6-8 S in the presence of 0.05-0.12 M, 0.12-0.24 M and more than 0.24 M NaCl. DNA polymerase alpha activity decreased concomitantly with the reduction in molecular size. The 6-8 S form of DNA polymerase alpha did not aggregate by itself nor with other cellular components nonspecifically, when the 23 S form was present. These results are evidence of the presence of 6-8 S DNA polymerase alpha as a high molecular weight form (23 S-form) in sea urchin eggs.     

The essential DNA polymerases delta and epsilon are involved in repair of UV-damaged DNA in the yeast Saccharomyces cerevisiae.

We have studied the ability of yeast DNA polymerases to carry out repair of lesions caused by UV irradiation in Saccharomyces cerevisiae. By the analysis of postirradiation relative molecular mass changes in cellular DNA of different DNA polymerases mutant strains, it was established that mutations in DNA polymerases delta and epsilon showed accumulation of single-strand breaks indicating defective repair. Mutations in other DNA polymerase genes exhibited no defects in DNA repair. Thus, the data obtained suggest that DNA polymerases delta and epsilon are both necessary for DNA replication and for repair of lesions caused by UV irradiation. The results are discussed in the light of current concepts concerning the specificity of DNA polymerases in DNA repair.     

Replication of chromosomal and episomal DNA in X-ray-damaged human cells: a cis- or trans-acting mechanism?

Episomal plasmids and viruses in mammalian cells present small targets for X-ray-induced DNA damage. At doses up to 100 Gy, DNA strand breaks or endonuclease III-sensitive sites were not discernible in 10.3-kb Epstein-Barr virus-based plasmid DNA or in 4.9-kb defective simian virus 40 DNA. DNA replication in these small molecules, however, was inhibited strongly by X-ray doses of greater than or equal to 20 Gy, decreasing to only 20 to 40% of control values. Inhibition was relieved slightly by growth in caffeine but was increased by growth in 3-aminobenzamide. Inhibition of DNA replication in episomal DNA molecules that are too small to sustain significant damage directly to their DNA may be due to either (a) a trans-acting diffusible factor that transfers the consequences of DNA breakage to episomes and to other replicating molecules, (b) a cis-acting mechanism in which episomes are structurally linked to genomic chromatin, and replication of both episomal and chromosomal replicons is under common control, or (c) radiation damage on other cellular structures unrelated to DNA. The resolution of these cellular mechanisms may shed light on the X-ray-resistant replication in ataxia-telangiectasia and may suggest strategies for molecular characterization of potential trans- or cis-acting factors.     

Cis and trans mechanisms of DNA repair.

DNA repair is essential for genetic stability and variability. Remarkable advances in the understanding of DNA repair by the molecular analysis of the substrate (gene repair) or the enzyme (repair genes), emphasize evolutionary conservation. Recent progress also stresses the interaction(s) between DNA repair and numerous other cellular metabolic processes, including non-nuclear and/or non-genetic responses.     

Synthesis of Avian Oncornavirus DNA in Infected Chicken Cells

The intracellular synthesis and integration of viral DNA (vDNA) into the host cell genome was studied in cultured chicken embryo fibroblasts infected with avian sarcoma or leukemia viruses. The newly synthesized vDNA was detected by hybridization with 70S viral RNA. Extraction of infected cell DNA by the selective procedure of Hirt resulted in the enrichment of newly synthesized vDNA in the low molecular weight supernatant fraction while leaving the bulk of cellular DNA containing integrated vDNA in the high molecular weight pellet fraction. This approach led to detection of intracellular vDNA synthesis within 1 h after infection and to vDNA integration into cellular DNA within 24 h. There was a several-fold increase in the vDNA content of infected cells during the initial phase of virus infection. But only a part of this newly synthesized vDNA appeared to become covalently linked with high molecular weight cellular DNA. Most of the remaining unintegrated vDNA gradually disappeared. The sedimentation profiles of minimally sheared cellular DNA in alkaline sucrose velocity gradients suggest that vDNA is synthesized as free linear molecules of approximately 3 x 10(6) daltons which subsequently are covalently linked to host cell DNA.     

Depletion of cellular pre-replication complex factors results in increased human cytomegalovirus DNA replication

Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recognition. Consequently, we have asked whether cellular pre-replication (pre-RC) factors that are either initially associated with cellular origin of replication (e.g. ORC2), those which recruit other replication factors (e.g. Cdt1 or Cdc6) or those which are subsequently recruited (e.g. MCMs) play any role in the HCMV DNA replication. We show that whilst RNAi-mediated knock-down of these factors in the cell affects cellular DNA replication, as predicted, it results in concomitant increases in viral DNA replication. These data show that cellular factors which initiate cellular DNA synthesis are not required for the initiation of replication of viral DNA and suggest that inhibition of cellular DNA synthesis, in itself, fosters conditions which are conducive to viral DNA replication.     

Phage-exclusion enzymes: a bonanza of biochemical and cell biology reagents?

Many parasitic DNA elements including prophages and plasmids synthesize proteins that kill the cell after infection by other phages, thereby blocking the multiplication of the infecting phages and their spread to other nearby cells. The only known function of these proteins is to exclude the infecting phage, and therefore to protect their hosts, and thereby the DNA elements themselves, against phage contagion. Many of these exclusions have been studied extensively and some have long been used in molecular genetics, but their molecular basis was unknown. The most famous of the phage exclusions are those caused by the Rex proteins of λ prophage. The Rex exclusions are still not completely understood, but recent evidence has begun to lead to more specific models for their action. One of the Rex proteins, RexA, may be activated by a DNA-protein complex, perhaps a recombination or replication intermediate, produced after phage infection. In the activated state, RexA may activate RexB, which has been proposed to be a membrane ion channel that allows the passage of monovalent cations, destroying the cellular membrane potential, and killing the cell. We now understand two other phage exclusions at the molecular level which use strategies that are remarkably similar to each other. The parasitic DNA elements responsible for the exclusions both constitutively synthesize enzymes that are inactive as synthesized by the DNA element but are activated after phage infection by a short peptide determinant encoded by the infecting phage. In the activated state, the enzymes cleave evolutionarily conserved components of the translation apparatus, in one case EF-Tu, and in the other case tRNA^Lys. Translation is blocked and development of the phage is arrested. A myriad of different phage-exclusion systems are known to exist and many of these may also be specific for highly conserved cellular components, furnishing generally useful enzymes for biochemical and biomedical research.     

Why Are There So Many Diverse Replication Machineries?

The replicon model has initiated a major research line in molecular biology: the study of DNA replication mechanisms. Until now, the majority of studies have focused on a limited set of model organisms, mainly from Bacteria or Opisthokont eukaryotes (human, yeasts) and a few viral systems. However, molecular evolutionists have shown that the living world is more complex and diverse than believed when the operon model was proposed. Comparison of DNA replication proteins in the three domains, Archaea, Bacteria, and Eukarya, have surprisingly revealed the existence of two distinct sets of non-homologous cellular DNA replication proteins, one in Bacteria and the other in Archaea and Eukarya, suggesting that the last universal common ancestor possibly still had an RNA genome. A major puzzle is the presence in eukaryotes of the unfaithful DNA polymerase alpha (Pol α) to prime Okazaki fragments. Interestingly, Pol α is specifically involved in telomere biosynthesis, and its absence in Archaea correlates with the absence of telomeres. The recent discovery of telomere-like GC quartets in eukaryotic replication origins suggests a link between Pol α and the overall organization of the eukaryotic chromosome. As previously proposed by Takemura, Pol α might have originated from a mobile element of viral origin that played a critical role in the emergence of the complex eukaryotic genomes. Notably, most large DNA viruses encode DNA replication proteins very divergent from their cellular counterparts. The diversity of viral replication machineries compared to cellular ones suggests that DNA and DNA replication mechanisms first originated and diversified in the ancient virosphere, possibly explaining why they are so many different types of replication machinerie.     

DNA replication and cell cycle in plants: learning from geminiviruses

Plant cell growth and development depend on continuous cell proliferation which is restricted to small regions of the plant called meristems. Infection by geminiviruses, small DNA viruses whose replicative cycle relies on host cell factors, is excluded from those proliferating areas. Since most of the replicative factors are present, almost exclusively, in proliferating cells, geminivirus infection is believed to induce a cellular state permissive for viral DNA replication, e.g. S-phase or, at least, some specific S-phase functions. The molecular basis for this effect seems to be the interference that certain geminivirus proteins exert on the retinoblastoma-related (RBR) pathway, which analogously to that of animal cells, regulates plant cell cycle activation and G(1)-S transition. In some cases, geminiviruses induce cell proliferation and abnormal growth. Mechanisms other than sequestering plant RBR probably contribute to the multiple effects of geminivirus proteins on cellular gene expression, cell growth control and cellular DNA replication. Current efforts to understand the coupling of geminivirus DNA replication to cell cycle and growth control as well as the directions in which future research is aiming are reviewed.     

Viral manipulation of DNA repair and cell cycle checkpoints

Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation.     

Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.     

Eukaryotic DNA repair: glimpses through the yeast Saccharomyces cerevisiae.

Eukaryotic cells are able to mount several genetically complex cellular responses to DNA damage. The yeast Saccharomyces cerevisiae is a genetically well characterized organism that is also amenable to molecular and biochemical studies. Hence, this organism has provided a useful and informative model for dissecting the biochemistry and molecular biology of DNA repair in eukaryotes.     

Analysis of telomeric DNA: Current approaches and methods

Terminal parts of linear chromosomes in eukaryotes are represented by telomeric DNA and associated proteins. Length, sequence and structure are the major parameters of telomeric DNA. The present review describes a set of molecular, cellular and genetic methods used to study the parameters of telomeric DNA.     

Mitochondrial DNA nucleoids determine mitochondrial genetics and dysfunction

Mitochondrial DNA plays a crucial role in cellular homeostasis; however, the molecular mechanisms underlying mitochondrial DNA inheritance and propagation are only beginning to be understood. To ensure the distribution and propagation of the mitochondrial genome, mitochondrial DNA is packaged into macromolecular assemblies called nucleoids, composed of one or more copies of mitochondrial DNA and associated proteins. We review current research on the mitochondrial nucleoid, including nucleoid-associated proteins, nucleoid dynamics within the cell, potential mechanisms to ensure proper distribution of nucleoids, and the impact of nucleoid organization on mitochondrial dysfunction. The nucleoid is the molecular organizing unit of mitochondrial genetics, and is the site of interactions that ultimately determine the bioenergetic state of the cell as a whole. Current and future research will provide essential insights into the molecular and cellular interactions that cause bioenergetic crisis, and yield clues for therapeutic rescue of mitochondrial dysfunction.     

病毒感染对细胞周期调控影响的研究进展

大量研究表明,病毒感染细胞时,病毒编码的蛋白或DNA可以扰乱细胞周期通路:促进细胞向S期转化或者使细胞静息于G2/M期。在细胞内,细胞周期的调控机制十分复杂,其包含了由DNA损伤导致的细胞通路活化及其他方式。关于病毒对细胞周期的调控方式及细胞周期的改变对于病毒感染的研究已取得一定进展。对于病毒的此类研究可以揭示细胞活动中的关键调控因子及细胞周期检查点的具体分子机理。对病毒调控宿主细胞周期以达到自身最大化复制的机理进行综述。