Analysis of Human Y-family DNA Polymerases and Primpol by Pre-steady-state Kinetic Methods PDF Download
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Author: E. John P. Tokarsky
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages : 121
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Book Description
A specialized primase-polymerase known as PrimPol, was discovered in humans in 2013. PrimPol exhibits similar properties to Y-family polymerases such as displaying relatively low efficiency and fidelity, and for having the ability to bypass certain types of DNA damage. However, based on in vitro experiments, the polymerase and primase activities of PrimPol are differentially regulated based on whether it utilizes manganese (Mn2+) or magnesium (Mg2+) as a divalent metal ion cofactor for catalysis. We sought to determine the effect of divalent metal ions on the polymerase fidelity and sugar selectivity of PrimPol. We found that PrimPol was extremely error-prone (fidelity range 10-1 to 10-2) when utilizing Mn2+, but was ~100-fold more efficient, compared to Mg2+. Finally, we showed that PrimPol could incorporate the nucleoside analogs and anticancer drugs, cytarabine and gemcitabine, as efficiently as normal dCTP in the presence of either Mn2+ or Mg2+.
Author: E. John P. Tokarsky
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages : 121
Get Book Here
Book Description
A specialized primase-polymerase known as PrimPol, was discovered in humans in 2013. PrimPol exhibits similar properties to Y-family polymerases such as displaying relatively low efficiency and fidelity, and for having the ability to bypass certain types of DNA damage. However, based on in vitro experiments, the polymerase and primase activities of PrimPol are differentially regulated based on whether it utilizes manganese (Mn2+) or magnesium (Mg2+) as a divalent metal ion cofactor for catalysis. We sought to determine the effect of divalent metal ions on the polymerase fidelity and sugar selectivity of PrimPol. We found that PrimPol was extremely error-prone (fidelity range 10-1 to 10-2) when utilizing Mn2+, but was ~100-fold more efficient, compared to Mg2+. Finally, we showed that PrimPol could incorporate the nucleoside analogs and anticancer drugs, cytarabine and gemcitabine, as efficiently as normal dCTP in the presence of either Mn2+ or Mg2+.
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
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: Mukunda Krishnaswamy
Publisher:
ISBN:
Category :
Languages : en
Pages : 66
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Book Description
Author: Andrew F. Gardner
Publisher: Frontiers Media SA
ISBN: 2889459276
Category :
Languages : en
Pages : 144
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Book Description
In all organisms, the DNA replication machinery is responsible for accurate and efficient duplication of the chromosome. Inhibitors of replication proteins are commonly used in anti-cancer and anti-viral therapies. This eBook on “The DNA Replication Machinery as Therapeutic Targets” examines the normal functions of replication proteins as well as strategies to target each step during the replication process including DNA unwinding, DNA synthesis, and DNA damage bypass and repair. Articles discuss current strategies to develop drugs targeting DNA replication proteins as well as future outlooks and needs.
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: Costas Demetzos
Publisher: Springer
ISBN: 9811309892
Category : Technology & Engineering
Languages : en
Pages : 480
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Book Description
This book highlights the recent advances of thermodynamics and biophysics in drug delivery nanosystems and in biomedical nanodevices. The up-to-date book provides an in-depth knowledge of bio-inspired nanotechnological systems for pharmaceutical applications. Biophysics and thermodynamics, supported by mathematics, are the locomotive by which the drug transportation and the targeting processes will be achieved under the light of the modern pharmacotherapy. They are considered as scientific tools that promote the understanding of physicochemical and thermotropic functionality and behavior of artificial cell membranes and structures like nanoparticulate systems. Therefore, this book focusses on new aspects of biophysics and thermodynamics as important elements for evaluating biomedical nanosystems, and it correlates their physicochemical, biophysical and thermodynamical behaviour with those of a living organism. In 2018, Prof. Demetzos was honored with an award by the Order of Sciences of the Academy of Athens for his scientific contribution in Pharmaceutical Nanotechnology.
Author: David Mittelman
Publisher: Springer Science & Business Media
ISBN: 1461462800
Category : Medical
Languages : en
Pages : 284
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Book Description
The discovery of stress-induced mutagenesis has changed ideas about mutation and evolution, and revealed mutagenic programs that differ from standard spontaneous mutagenesis in rapidly proliferating cells. The stress-induced mutations occur during growth-limiting stress, and can include adaptive mutations that allow growth in the otherwise growth-limiting environment. The stress responses increase mutagenesis specifically when cells are maladapted to their environments, i.e. are stressed, potentially accelerating evolution then. The mutation mechanism also includes temporary suspension of post-synthesis mismatch repair, resembling mutagenesis characteristic of some cancers. Stress-induced mutation mechanisms may provide important models for genome instability underlying some cancers and genetic diseases, resistance to chemotherapeutic and antibiotic drugs, pathogenicity of microbes, and many other important evolutionary processes. This book covers pathways of stress-induced mutagenesis in all systems. The principle focus is mammalian systems, but much of what is known of these pathways comes from non-mammalian systems.
Author:
Publisher: Academic Press
ISBN: 9780120342693
Category : Science
Languages : en
Pages : 368
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Book Description
DNA Repair and Replication contains an up-to-date review of general principles of DNA replication and an overview of the multiple pathways involved in DNA repair. Specific DNA repair pathways, including base-excision repair, light-dependent direct reversal of UV-damage, nucleotide-excision repair, transcription-coupled repair, double-strand break repair, and mismatch repair, are each discussed in separate chapters. Selected Contents: Base Excision Repair Eukaryotic DNA Mismatch Repair Double Strand Break Repair Functions of DNA Polymerases Somatic Hypermutation: A Mutational Panacea
Author: Ulrich Hubscher
Publisher: World Scientific
ISBN: 9814465453
Category : Medical
Languages : en
Pages : 338
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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, genome sequencing, nucleic acids-based diagnostics and in techniques to analyze ancient and otherwise damaged DNA. 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. We have recently witnessed the discovery of an abundance of novel DNA polymerases in viruses, bacteria, archaea and eukaryotes with specialized properties whose physiological functions are only beginning to be understood. This book summarizes the current knowledge of these fascinating enzymes. It is intended for a wide audience from basic scientists, to diagnostic laboratories and to clinicians who seek a better understanding of these fascinating enzymes.
