Kinetic Characterization of Human DNA Polymerase E PDF Download
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Author: Walter J. Zahurancik
Publisher:
ISBN:
Category : Biochemistry
Languages : en
Pages :
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The results of my work lay the foundation for future studies exploring the effects of other DNA replication enzymes on the polymerization and fidelity of hPole. Furthermore, my work provides the basis for investigating the mechanism of mutagenesis in tumors containing hPole exonuclease domain mutants. Overall, this study represents an important advance toward our comprehensive understanding of human DNA replication.
Author: Walter J. Zahurancik
Publisher:
ISBN:
Category : Biochemistry
Languages : en
Pages :
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The results of my work lay the foundation for future studies exploring the effects of other DNA replication enzymes on the polymerization and fidelity of hPole. Furthermore, my work provides the basis for investigating the mechanism of mutagenesis in tumors containing hPole exonuclease domain mutants. Overall, this study represents an important advance toward our comprehensive understanding of human DNA replication.
Author: Jill Olivia Fuss
Publisher:
ISBN:
Category :
Languages : en
Pages : 298
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Author: Nikunj Bhatt
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages : 96
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Abstract: Since 1999, the human genome project has led to the discovery of several novel DNA polymerases. Among these new polymerases were human DNA polymerase ?. (Pol?) and Sulfolobus solfataricus DNA polymerase IV (Dpo4). PoP, an X family polymerase, displays both 5'-2-deoxyribose-5-phosphate lyase (dRPase) activity and polymerase activity and efficiently incorporates nucleotides into short-gapped primer- primer/templates, the natural substrate for short-patch base excision repair (BER). Crystal studies of the ternary structure of truncated PoIA (tPolX) reveal that a 5'- phosphate of a downstream primer interacts with a positively-charged pocket in the dRPase domain. In this study, we constructed three substrates: (i) a 21-19/41-mer single nucleotide gapped DNA with a 5'-phosphate on the terminal end of the downstream 19- mer; (ii) a 21-19/41-mer single nucleotide gapped DNA with a 5'-OH on the downstream 19-mer; and (iii) a 21/41-mer with no downstream primer. Pre-steady state kinetics revealed that Pol[lamda]'s incorporation efficiency for the DNA substrate with the 5'-phosphate moiety on the downstream 1 9-mer primer is 11-fold more efficient at incorporating a correct nucleotide compared to the DNA substrate with the 5'-OH on the terminal end of the downstream 19-mer, and 160-fold more efficient than the DNA substrate with no downstream primer. Another aim of this thesis was to characterize Po1[lamda]' s preference for deoxynucleotides over ribonucleotides via pre-steady state kinetics. A previous study with polymerase u (Polu) revealed that the glycine433 residue mutated to tyrosine dramatically increased sugar selectivity for that enzyme. Sequence alignment of the M a-helix of Po1[lamda] and three other X family polymerases, Polu, terminal deoxynucleotidyl transferase (TDT), and polymerase [beta] (Polf[beta]), suggested that its tyrosine505 may be involved in determining sugar selectivity, while the crystal structure of the tPo[lamda]. ternary complex suggested that the backbone of the M a-helix blocks the 2'-OH of ribonucleotides forming a "stearic gate". In this thesis, the tyrosine505 mutated to glycine actually increased sugar selectivity indirectly supporting the stearic gate hypothesis. The second enzyme studied, Dpo4, a Y family polymerase that bypasses lesions and exhibits low fidelity, consists of an extra little finger domain in addition to the standard catalytic core, consisting of the finger, thumb, and palm domains. The little finger, which has been shown to be important to polymerase activity, is tethered to the thumb domain via 14 amino acid linker (P1 = 10), known as the little finger linker. Using 1, 4, and 6 glycine additions and 1, 4, and 6 deletions in the center of the little finger linker, a fluorescent titration assay revealed a significant decrease in binding affinity as the size of the little finger linker was increased and decreased. This suggests nature has optimized the length of the little finger linker. In addition, a kink in the little finger linker was removed by a proline236 substitution to alanine which did not significantly effect DNA binding to Dpo4. Finally, an optimized procedure for the cisplatination of an 1 8-mer DNA substrate was prepared and described in this thesis.
Author: Alexander Rodney Abbas
Publisher:
ISBN:
Category :
Languages : en
Pages : 316
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Author: Nayef Ali Mazloum
Publisher:
ISBN:
Category :
Languages : en
Pages : 162
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Author: Renee Ann Beardslee
Publisher:
ISBN:
Category :
Languages : en
Pages : 145
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![Human DNA Polymerase [epsilon] Associated Proteins PDF](https://books.google.com/books/content/images/frontcover/k_1OJgAACAAJ?fife=w80-h100)
Author: Minna Mäkiniemi
Publisher:
ISBN: 9789514259685
Category :
Languages : en
Pages : 68
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Author: E. John P. Tokarsky
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages : 121
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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: 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:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 1422
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