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001 AAI13806441
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006 m o d
007 cr mn ---uuuuu
008 201105s2019 miu sbm 000 0 eng d
020 9781085777407
035 (MiAaPQ)AAI13806441
040 MiAaPQ|beng|cMiAaPQ|dNTU
100 1 East, Kyle William
245 14 The Role of Protein-Nucleic Acid Interactions in the
Function and Fidelity of DNA Polymerase β and CRISPR Cas9
Monitored by NMR
264 0 |c2019
300 1 online resource (148 pages)
336 text|btxt|2rdacontent
337 computer|bc|2rdamedia
338 online resource|bcr|2rdacarrier
500 Source: Dissertations Abstracts International, Volume: 81-
03, Section: B
500 Advisor: Loria, J. Patrick
502 Thesis (Ph.D.)--Yale University, 2019
504 Includes bibliographical references
520 Protein-nucleic acid interactions are one of the most
important processes required for life on earth. These
interactions make up the core of the central dogma of
molecular biology first published by Francis Crick in
1958. For over six decades, the interactions of proteins
with DNA and RNA have been heavily studied in order to
better understand how the proteome interacts with the
genome and nucleic acid polymers. The complexity of these
interactions is best highlighted by the thousands of
proteins that interact with genomic DNA and various RNAs.
These protein-nucleic acid interactions are required for
the integrity of the genome and the stability of the
processes required by the central dogma. The integrity of
genomic DNA requires the fidelity of the DNA polymerases
that copy and repair the millions of DNA base pairs per
cell. One polymerase that repair DNA in humans is DNA
polymerase β. Studies of DNA polymerase β (pol β) have
shown that the interactions of pol β with the target DNA
are tightly regulated to maximize the fidelity of the
enzyme. The wild type enzyme was monitored using NMR
spectroscopy in order to study the effects of the four
template nucleotides (adenosine, guanosine, thymine, and
cytosine) have on the structure and dynamics of the
enzyme. The wild type enzyme was shown to bind
independently of the nucleotide; however, the steric
effects of the template nucleotide and the incoming
nucleotide triphosphate show that the mechanism for
selecting the appropriate nucleotide is heavily dependent
on the steric effects of the base pair. To further these
studies, several cancer-associated mutants of pol β have
been identified that are still active polymerases but with
reduced fidelity. One of these cancer-associated mutants,
E288K, has a reduced fidelity only when the templating
nucleotide is adenosine. This reduced fidelity allowed for
the study of how the mutation of E288K altered the
interaction of pol β with the target DNA.The CRISPR Cas9
endonuclease is an important molecular system for genome
editing and the future of gene therapy. NMR studies of the
entire Cas9 enzyme are hindered by the large size of the
enzyme-RNA complex. Therefore, a construct of the HNH
endonuclease domain of Cas9 was designed to allow for the
assignment and study of part of Cas9. The HNH construct
was assigned to 85%, allowing for the characterization of
the internal structure and dynamics of the enzyme in
solution by NMR. The HNH construct is structurally
indistinguishable from the HNH domain of the full length
Cas9 enzyme by x-ray crystallography. The measurement of
dynamics on multiple time scales by NMR adds further
evidence to the suggestion that the HNH domain is
necessary for the communication between the PAM
recognition domain and the second endonuclease domain,
RuvC. This communication pathway is of great interest in
the intelligent design of Cas9 enzymes for future gene
therapy techniques
533 Electronic reproduction.|bAnn Arbor, Mich. :|cProQuest,
|d2020
538 Mode of access: World Wide Web
650 4 Chemistry
650 4 Biophysics
653 Crispr-cas9
653 Dna polymerase beta
653 Nmr
655 7 Electronic books.|2local
690 0485
690 0786
710 2 ProQuest Information and Learning Co
710 2 Yale University.|bChemistry
773 0 |tDissertations Abstracts International|g81-03B
856 40 |uhttp://pqdd.sinica.edu.tw/twdaoapp/servlet/
advanced?query=13806441|zclick for full text (PQDT)
912 PQDT
