Insight Into the Fidelity of Two X-family Polymerases PDF Download
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Author: Michelle P. Roettger
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages :
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Book Description
Abstract: DNA polymerase [mu] (Pol [mu]) is a recently discovered X-family DNA polymerase that has been implicated as a potential mutase involved in the somatic hypermutation of immunoglobulin (Ig) genes during antibody affinity maturation. To evaluate the hypothesis which regards Pol [mu] as a mutase in Ig maturation, pre-steady-state kinetic methods were used to measure the fidelity of human Pol [mu] based on all 16 possible deoxynucleotide (dNTP) incorporations and four matched ribonucleotide (rNTP) incorporations into normal DNA primer/template substrates. The overall fidelity of Pol [mu] was estimated to be in the range of 10-3-10-5 for both dNTP and rNTP incorporations. The template-independent polymerization ability of this enzyme was also evaluated, and the potential biological functions of Pol [mu] are discussed on the basis of the pre-steady-state kinetic data. DNA polymerase [beta] (Pol [beta]), another X-family polymerase, plays a role in DNA gap-filling during base excision repair. In pioneering model studies on the mechanism by which polymerase fidelity is achieved, our lab has previously utilized stopped-flow fluorescence to examine the matched dNTP incorporation pathway of Pol [beta]. While monitoring the reaction's progress utilizing a DNA substrate containing a 2-aminopurine fluorescent probe, a biphasic trace is observed. Extensive studies involving a variety of chemical probes indicate that the fast fluorescence transition corresponds to a dNTP-induced subdomain conformational change occurring prior to the rate-limiting chemistry step, while the slow fluorescence transition corresponds to a post-chemistry conformational change, likely subdomain reopening. In this work, stopped-flow fluorescence assays are further utilized: i) to examine the role of R258 in subdomain reopening by mechanism studies on site-specific Pol [beta] mutant, R258A; ii) to investigate the mechanism of Pol [beta] mismatched dNTP incorporation by wild-type and I260Q "mutator" mutant; and iii) to evaluate the contribution of the reverse of the conformational closing step to Pol [beta]'s fidelity. Overall, the results provide first direct evidence that mismatched and matched dNTP incorporations proceed via analogous kinetic pathways, and support our standing hypothesis that the fidelity of Pol [beta] is dictated by the energetic difference between matched and mismatched dNTP incorporation pathways at the transition state of the chemical step.
Author: Michelle P. Roettger
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages :
Get Book Here
Book Description
Abstract: DNA polymerase [mu] (Pol [mu]) is a recently discovered X-family DNA polymerase that has been implicated as a potential mutase involved in the somatic hypermutation of immunoglobulin (Ig) genes during antibody affinity maturation. To evaluate the hypothesis which regards Pol [mu] as a mutase in Ig maturation, pre-steady-state kinetic methods were used to measure the fidelity of human Pol [mu] based on all 16 possible deoxynucleotide (dNTP) incorporations and four matched ribonucleotide (rNTP) incorporations into normal DNA primer/template substrates. The overall fidelity of Pol [mu] was estimated to be in the range of 10-3-10-5 for both dNTP and rNTP incorporations. The template-independent polymerization ability of this enzyme was also evaluated, and the potential biological functions of Pol [mu] are discussed on the basis of the pre-steady-state kinetic data. DNA polymerase [beta] (Pol [beta]), another X-family polymerase, plays a role in DNA gap-filling during base excision repair. In pioneering model studies on the mechanism by which polymerase fidelity is achieved, our lab has previously utilized stopped-flow fluorescence to examine the matched dNTP incorporation pathway of Pol [beta]. While monitoring the reaction's progress utilizing a DNA substrate containing a 2-aminopurine fluorescent probe, a biphasic trace is observed. Extensive studies involving a variety of chemical probes indicate that the fast fluorescence transition corresponds to a dNTP-induced subdomain conformational change occurring prior to the rate-limiting chemistry step, while the slow fluorescence transition corresponds to a post-chemistry conformational change, likely subdomain reopening. In this work, stopped-flow fluorescence assays are further utilized: i) to examine the role of R258 in subdomain reopening by mechanism studies on site-specific Pol [beta] mutant, R258A; ii) to investigate the mechanism of Pol [beta] mismatched dNTP incorporation by wild-type and I260Q "mutator" mutant; and iii) to evaluate the contribution of the reverse of the conformational closing step to Pol [beta]'s fidelity. Overall, the results provide first direct evidence that mismatched and matched dNTP incorporations proceed via analogous kinetic pathways, and support our standing hypothesis that the fidelity of Pol [beta] is dictated by the energetic difference between matched and mismatched dNTP incorporation pathways at the transition state of the chemical step.
Author:
Publisher:
ISBN:
Category : Dissertations, Academic
Languages : en
Pages : 810
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Book Description
Author: Tamar Schlick
Publisher: Royal Society of Chemistry
ISBN: 1849734623
Category : Science
Languages : en
Pages : 355
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Book Description
This two volume set describes innovations in biomolecular modeling and simulation, in both the algorithmic and application fronts.
Author: Whitney Yin
Publisher: Frontiers Media SA
ISBN: 2832503829
Category : Science
Languages : en
Pages : 118
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Book Description
olymerases are the nucleotidyl transferases that are responsible for synthesizing DNA and RNA. They are crucial for essential cellular processes including cellular and viral genome replication, DNA repair and damage tolerance, and transcription. Consistent with their vital roles, polymerases are found in all domains of life. The overall chemistry employed by these enzymes is conserved but there are variations among the different groups of polymerases that confer different substrate specificities and nucleotide incorporation fidelities that allow them to be involved in a wide array of cellular activities. Since polymerases were first isolated more than six decades ago, we have made great progress in understanding how different polymerases have adapted to their specific roles. In this Research Topic we will focus on the enzymatic mechanisms of these enzymes and the relationships between polymerase structure and mechanism, to highlight common themes and unique adaptations.
Author: Errol C. Friedberg
Publisher: American Society for Microbiology Press
ISBN: 1555813194
Category : Science
Languages : en
Pages : 2587
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Book Description
An essential resource for all scientists researching cellular responses to DNA damage. • Introduces important new material reflective of the major changes and developments that have occurred in the field over the last decade. • Discussed the field within a strong historical framework, and all aspects of biological responses to DNA damage are detailed. • Provides information on covering sources and consequences of DNA damage; correcting altered bases in DNA: DNA repair; DNA damage tolerance and mutagenesis; regulatory responses to DNA damage in eukaryotes; and disease states associated with defective biological responses to DNA damage.
Author: Thomas A Steitz
Publisher: World Scientific
ISBN: 9811215871
Category : Science
Languages : en
Pages : 640
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Book Description
Several years ago, Thomas Steitz agreed to contribute a volume to the 'World Scientific Series in Structural Biology' that would deal with the contributions he and his coworkers have made to structural biology during his remarkable career. Sadly, Tom died in the fall of 2018 before he had had time to do more than produce an outline for this book, and a list of the reprints he wanted it to contain.Fortunately, Tom's colleagues and coworkers responded enthusiastically when they were informed later that fall that if they were willing to help out, a volume would be published to commemorate his career. It fell to Anders Liljas, Peggy Eatherton, Tom's longtime administrative assistant, and Peter Moore, a close colleague, to oversee their efforts. Thomas Steitz is best known for the work he and his coworkers did to elucidate the biochemical basis of gene expression. The structures of a large number of the macromolecules involved in transcription and translation emerged from his laboratory over the course of his career. This book includes reprints of the most important papers he had published, grouped according to the structures they relate to, and commentaries written by the scientists who collaborated with him to solve each of them. It thus summarizes the achievements of one of the most distinguished biochemists of the second half of the 20th century.
Author: Zvi Kelman
Publisher: Frontiers Media SA
ISBN: 2889194558
Category : Biotechnology
Languages : en
Pages : 147
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Book Description
DNA polymerases are core tools for molecular biology including PCR, whole genome amplification, DNA sequencing and genotyping. Research has focused on discovery of novel DNA polymerases, characterization of DNA polymerase biochemistry and development of new replication assays. These studies have accelerated DNA polymerase engineering for biotechnology. For example, DNA polymerases have been engineered for increased speed and fidelity in PCR while lowering amplification sequence bias. Inhibitor resistant DNA polymerase variants enable PCR directly from tissue (i.e. blood). Design of DNA polymerases that efficiently incorporate modified nucleotide have been critical for development of next generation DNA sequencing, synthetic biology and other labeling and detection technologies. The Frontiers in Microbiology Research Topic on DNA polymerases in Biotechnology aims to capture current research on DNA polymerases and their use in emerging technologies.
Author: Lynne S Cox
Publisher: Royal Society of Chemistry
ISBN: 1847559859
Category : Science
Languages : en
Pages : 467
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Book Description
DNA replication, the process of copying one double stranded DNA molecule to form two identical copies, is highly conserved at the mechanistic level across evolution. Interesting in its own right as a fascinating feat of biochemical regulation and coordination, DNA replication is at the heart of modern advances in molecular biology. An understanding of the process at both the biological and chemical level is essential to developing new techniques in molecular biology. Insights into the process at the molecular level provide opportunities to modulate and intervene in replication. Rapidly dividing cells need to replicate their DNA prior to division, and targeting components of the replication process is a potentially powerful strategy in cancer treatment. Conversely, ageing may be associated with loss of replication activity and restoring it to cells may moderate some of the diseases associated with old age. Replication is, therefore, fundamental to a huge range of molecular biological and biochemical applications, and provides many potential targets for drug design. The fast pace of replication research, particularly in providing new structural insights, has outdated the majority of available texts. This learned, yet accessible, book contains the latest research written by those conducting it. It examines conserved themes providing a biological background for biochemical, chemical and pharmaceutical studies of this huge and exciting field. Rather than simply "itemising" the replication steps and the proteins involved, replication is tackled from a novel perspective. The book provides logical groupings of processes based upon biochemical similarities. The emphasis on mechanisms and the relationship between structure and function targets the chapters towards biochemists and biological chemists as well as molecular and cell biologists. The book highlights new insights into the replication process, from the assembly of pre-replication complexes, through polymerisation mechanisms, to considering replication in the context of chromatin and chromosomes. It also covers mitochondrial DNA replication, and includes archaeal paradigms, which are proving increasingly relevant to the study of replication in higher eukaryotes. Exciting potential drug targets in DNA replication are discussed, particularly in the context of treating malaria and cancer.
Author: Giovanni Maga
Publisher: World Scientific
ISBN: 9813226420
Category : Science
Languages : en
Pages : 398
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Book Description
Maintenance of the information embedded in the genomic DNA sequence is essential for life. DNA polymerases play pivotal roles in the complex processes that maintain genetic integrity. Besides their tasks in vivo, DNA polymerases are the workhorses in numerous biotechnology applications such as the polymerase chain reaction (PCR), cDNA cloning, next generation sequencing, nucleic acids based diagnostics and in techniques to analyze ancient and otherwise damaged DNA (e.g. for forensic applications). Moreover, some diseases are related to DNA polymerase defects and chemotherapy through inhibition of DNA polymerases is used to fight HIV, Herpes and Hepatitis B and C infections. This book focuses on (i) biology of DNA polymerases, (ii) medical aspects of DNA polymerases and (iii) biotechnological applications of DNA polymerases. It is intended for a wide audience from basic scientists, to diagnostic laboratories, to companies and to clinicians, who seek a better understanding and the practical use of these fascinating enzymes.
Author: Katsuhiko S. Murakami
Publisher: Springer Science & Business Media
ISBN: 3642397964
Category : Science
Languages : en
Pages : 342
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Book Description
This book provides a review of the multitude of nucleic acid polymerases, including DNA and RNA polymerases from Archea, Bacteria and Eukaryota, mitochondrial and viral polymerases, and other specialized polymerases such as telomerase, template-independent terminal nucleotidyl transferase and RNA self-replication ribozyme. Although many books cover several different types of polymerases, no book so far has attempted to catalog all nucleic acid polymerases. The goal of this book is to be the top reference work for postgraduate students, postdocs, and principle investigators who study polymerases of all varieties. In other words, this book is for polymerase fans by polymerase fans. Nucleic acid polymerases play a fundamental role in genome replication, maintenance, gene expression and regulation. Throughout evolution these enzymes have been pivotal in transforming life towards RNA self-replicating systems as well as into more stable DNA genomes. These enzymes are generally extremely efficient and accurate in RNA transcription and DNA replication and share common kinetic and structural features. How catalysis can be so amazingly fast without loss of specificity is a question that has intrigued researchers for over 60 years. Certain specialized polymerases that play a critical role in cellular metabolism are used for diverse biotechnological applications and are therefore an essential tool for research.
