Enzymology Of DNA Replication Research Articles

Ribonucleotides in DNA: hidden in plain sight.

Mapping of ribonucleotides to single-nucleotide resolution in yeast genomes provides new insight into the enzymology of DNA replication.

Where is the New Biology Taking Us?The Genetic Revolution and Human Rights. Justine Burley

A review of THE GENETIC REVOLUTION AND HUMAN RIGHTS. The Oxford Amnesty Lectures 1998. Edited byJustine Burley. Oxford and New York: Oxford University Press. $14.95 (paper). xxviii + 220 p; index. ISBN: 019-286201-4. 1999. As the twentieth century ends, we have hardly taken stock of what has happened during this century of biology. We began the new century in 1900 (just as we begin, erroneously, the new century and millennium in 2000) with the rediscovery of Gregor Mendel's laws by Hugo De Vries, Carl Correns, and Ernst von Tschermak (the latter despite Curt Stern's efforts to demote him as a rediscoverer). William Bateson quickly took up the cause of Mendelism and named the field of genetics (1906), winning the battle against the biometricians who erroneously (then) thought that Mendelism had nothing to do with either evolution or heredity. In 1909 WilhelmJohannsen gave us a name-the gene-to replace contending verbal monstrosities such as character-unit, unit factor, and other laden terms that misled early investigators. Classical genetics emerged from the fusion of cytology (Walter Sutton's chromosome theory of heredity in 1902) and breeding analysis, especially in the able hands of Edmund Beecher Wilson and Thomas Hunt Morgan at Columbia University. The Drosophila Group (or Fly Lab) established linkage, chromosome maps, X-linked inheritance, the correlation of linkage groups to chromosome number, assorted chromosomal rearrangements, nondisjunction, dosage compensation, complex association of gene and character through chief genes and genetic modifiers, construction of genetic stocks, and a variety of new, wonderful insights and tools for genetic analysis. The studies of plant geneticists (especially by E M East and D FJones) provided the agricultural revolution that liberated, at least for the twentieth century, the world from the gloomy predictions by Malthus of world hunger on a massive scale as human population soared in response to the practical benefits of the germ theory and the reduction of infant mortality. By the 1920s many insights into gene number and function had been established, and mutation of the gene was isolated from other varieties of chromosomal disturbances, allowing geneticists to focus on mutation rates and the search for agents that induce mutations. HermannJoseph Muller was the first to successfully tackle these problems, and his induction of mutations by x-rays in 1927 set off a wave of repetitions in plants, animals, and microbes that created both the field of radiation genetics and the practical application of radiation (later chemical mutagens) to produce desired mutations, including the greatly augmented yields of penicillin from mold. The late 1930s saw the first rumblings of biochemical genetics and the conception of molecular genetics. Virus genetics (Max Delbruick's school), sex in bacteria Joshua Lederberg's findings), and the hints that DNA may be of genetic significance after all (so E B Wilson thought as early as the 1890s) led to the explosion of new knowledge that followed the end of the second World War. DNA was the genetic material (as the Rockefeller and Cold Spring Harbor laboratories reported), its structure (as worked out by Watson and Crick and confirmed by Wilkins and Franklin) was a double helix, and its implications rolled forth: the genetic code, semiconservative replication, molecular basis of mutagenesis, genetic fine structure associated with pseudoallelism and nests of cognate genes, and genetic regulation, all by 1965. In the next thirty-five years came the sequencing of genes, enzymology of DNA replication, the PCR tool of copying genes, identification of introns and exons in eukaryotic genes, directed mutation, and recombinant DNA technology. Genetics suddenly became fused with engineering, in addition to physics, mathematics, physiology, cell biology, embryology, breeding analysis, cytology, chemistry, biochemistry, and evolution. At the same time as genetics shifted in the middle of the century from the classical to molecular approaches, human genetics expanded greatly, purged of eugenics, and found its way into medical schools, infiltrating departments of pathology, obstetrics, and pediatrics.

Ten commandments: lessons from the enzymology of DNA replication.

One of the prevailing fashions in bioscience these days is the application of genomics to eukaryotic gene expression. Largely eclipsed are the approaches of a few decades ago in which enzymes derived from microorganisms blazed the trail to much of our current understanding of macromolecular

DNA replication: enzymology and mechanisms

Research into the enzymology of DNA replication has seen a multitude of highly significant advances during the past year, in both prokaryotic and eukaryotic systems. The scope of this article is limited to chromosomal replicases and origins of initiation. The multiprotein chromosomal replicases of prokaryotes and eukaryotes appear to be strikingly similar in structure and function, although future work may reveal their differences. Recent developments, elaborating the activation of origins in several systems, have begun to uncover mechanisms of regulation. The enzymology of eukaryotic origins has, until now, been limited to viral systems, but over the past few years, enzymology has caught a grip on the cellular origins of yeast.

Evidence for the Presence of Topoisomerase-like Activity in Mitochondria of Saccharomyces cerevisiae

During studies on the enzymology of DNA replication in mitochondria of Saccharomyces cerevisiae , a topoisomerase like activity was detected for the first time. Crude extracts of mitoplast were found to show enzyme activities which could both catenate and relax supercoiled plasmid DNA. Chromatography of the initoplast lysate on a phosphocellulose column, using Tris-HC1 (pH 7.5) buffer containing 0.6 M NaCl as ethent, was found to yield a topoisomerase like activity capable of relaxing supercoiled plasmid DNA (Fraction 1). This fraction was not dependent on ATP for its activity. The other fraction eluting at 1M NaCl showed predominantly catenating activity, which was found to be ATP dependent.

Genetics and enzymology of DNA replication in Escherichia coli.

PERSPECTIVE AND INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447 INITIATION OF REPLICATION AT SPECIFIC ORIGINS . . . . . . . . . . . . . . . . 45 1 Initiation at oriC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 Initiation at oriA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454 Initiation at oriP] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456 Initiation of ColEI and pBR322 replication . . . . . . . . . . . . . . . . . . . . . . . . 458 Summary of Initiation Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461 REPLICATION FORK MOVEMENT, PRIMING AND DNA SYNTHESIS . . . . . . 462 The Core Replication Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 Replication Fork Movement and Interactions at the Fork . . . . . . . . _ _ . . . . . _ 466

Genetics and Enzymology of DNA Replication in Escherichia coli

Genetics and Enzymology of DNA Replication in Escherichia coli

DNA-dependent adenosinetriphosphatase A is the eukaryotic analog of the bacteriophage T4 gene 44 protein: immunological identity of DNA replication-associated ATPases

We report the construction of three stable murine hybridomas that secrete monoclonal antibodies which recognize calf thymus DNA-dependent adenosinetriphosphatase A. All three of the antibodies react specifically with calf thymus ATPase A and the gene 44 protein from the bacteriophage T4 DNA-dependent ATPase. Each of the three anti-ATPase A antibodies appears to recognize a different epitope and none of the antibodies inhibit DNA-dependent ATP hydrolysis by ATPase A. Furthermore, one of the antibodies has been shown to react with two different preparations of HeLa cell DNA-dependent ATPases and a yeast DNA-dependent ATPase, all of which have been implicated in the enzymology of DNA replication. These findings provide strong evidence for the role of ATPase A in DNA replication. These observations lead us to conclude that, apart from the nucleotide binding sites, there are at least three epitopes common to both the bacteriophage and eukaryotic DNA-dependent ATPases that we have examined and that the different preparations of the eukaryotic ATPases contain the same DNA-dependent ATPase.

Mutational specificity of animal cell DNA polymerases.

Since DNA polymerases are involved in DNA replication, recombination, and repair, the frequency with which these enzymes commit errors during synthesis is likely to be an important factor in controlling mutation rates in cells. The fidelity of DNA polymerases was originally studied by following misincorporation using synthetic nucleic acid templates containing only one or two bases. Later, by assaying for reversion of an amber codon after copying phi X174 single-stranded DNA molecules, the base substitution accuracy of in vitro DNA synthesis on natural DNA was determined. Most recently, a forward mutation assay has been developed that uses gap-filling synthesis on an M13mp2 DNA template, thus permitting the detection of a variety of different errors during DNA synthesis on natural DNA templates. Detailed mutational spectra for animal cell polymerases-alpha, beta, and gamma have been determined and demonstrate that a variety of errors can be generated by these purified enzymes. The frequencies of base mispairs, base additions, and deletion errors by DNA polymerases vary widely and depend on both the DNA sequence and the enzyme used. An understanding of the mechanisms by which DNA polymerases avoid or generate various mutations depends on the definition of the parameters that influence the frequency and specificity of particular errors. Future experiments will combine the use of the methods available to measure fidelity with advances in DNA replication enzymology and should lead to exciting new insights into the mechanisms of spontaneous mutagenesis.

The replication of adenovirus DNA with purified proteins

This year marks the 30th anniversary of the discovery of adenoviruses and the publication of the structure of the double helix of DNA, with its implications for the mechanism of DNA replication . Although the structure of DNA suggested that its duplication might be simple, the elegant studies on the replication of procaryotic DNA in vitro have shown that the enzymology of DNA replication is complex . Relatively little is known about eucaryotic cell DNA replication, but adenoviruses have proved to be valuable tools for determining how mammalian cells work and may provide valuable insight into some aspects of cell DNA replication as well . A landmark in this endeavor was the development by Challberg and Kelly (PNAS 76, 655-659, 1979) of a soluble, cell free extract capable of initiation of adenovirus DNA replication on exogenous template DNA . Recent characterization of this cell free system has led to the purification of enzymes that replicate adenovirus DNA, as well as the identification of DNA template requirements for initiation of DNA synthesis . These studies have helped to expound the protein-priming mechanism for the initiation of DNA synthesis at the replication origin . The human adenoviruses contain a double-strand linear genome of -36,000 bp, which contains a protein of 55,000 daltons (terminal protein, TP) covalently attached via a serine-dCMP phosphodiester bond to each 5' terminus (reviewed by Challberg and Kelly, Ann . Rev . Biochem. 51, 901-934, 1982) . Another feature of the termini of adenovirus DNA is an inverted, repeated sequence ranging from 102 to 162 by in various human adenoviruses . This inverted sequence, which contains the origin of DNA replication, allows DNA synthesis to begin at either end of the molecule . Synthesis then proceeds via type I

Why work on the enzymology of DNA replication?

Why work on the enzymology of DNA replication?