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Human RNase H1 Kinetics and Mechanis...

Ponzar, Nathan L.,

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Human RNase H1 Kinetics and Mechanism of Inhibition by α-Hydroxytropolone Inhibitors /

Record Type:	Electronic resources : Monograph/item
Title/Author:	Human RNase H1 Kinetics and Mechanism of Inhibition by α-Hydroxytropolone Inhibitors // Nathan L Ponzar.
Author:	Ponzar, Nathan L.,
Description:	1 electronic resource (134 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 84-03, Section: B.
Contained By:	Dissertations Abstracts International84-03B.
Subject:	Biochemistry. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=29321566
ISBN:	9798845407115

Human RNase H1 Kinetics and Mechanism of Inhibition by α-Hydroxytropolone Inhibitors /
Ponzar, Nathan L.,

Human RNase H1 Kinetics and Mechanism of Inhibition by α-Hydroxytropolone Inhibitors /Nathan L Ponzar. - 1 electronic resource (134 pages)

Source: Dissertations Abstracts International, Volume: 84-03, Section: B.

Ribonucleases H (RNases H) are Mg2+-dependent enzymes that cleave RNA when it is bound to DNA and have vital roles in important cellular processes like replication of the mitochondrial genome and removal of R-loops from the genome. Here, we have optimized conditions for expression and purification of recombinant human RNase H1 and its component domains. The proteins and their interactions with a 12-bp RNA/DNA substrate were characterized using biophysical techniques, RNA/DNA binding assays, and kinetics assays with a fluorogenic RNA/DNA substrate. RNases H are also essential for the replication cycles of HIV and Hepatitis B Virus and are being pursued as drug targets. To better understand how these compounds affect the activity of RNase H proteins, the recombinant RNase H1 proteins were used to evaluate the mechanism of RNase H inhibition by two structurally distinct compounds from the α-Hydroxytropolone class. We determined that turnover rate was reduced by both compounds, but inhibition was not competitive with substrate, despite the inhibitors binding to the active site via the classic two-divalent metal ion coordination mechanism. Since the compounds showed reversible binding to RNase H1, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which αHTs stabilize an inactive enzyme-substrate-inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid development of inhibitors specific for the viral RNases H.

English

ISBN: 9798845407115Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Enzyme inhibition

Human RNase H1 Kinetics and Mechanism of Inhibition by α-Hydroxytropolone Inhibitors /
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245 1 0 $a Human RNase H1 Kinetics and Mechanism of Inhibition by α-Hydroxytropolone Inhibitors / $c Nathan L Ponzar.
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300 $a 1 electronic resource (134 pages)
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500 $a Advisors: Tavis, John Committee members: Antony, Edwin; DiPaolo, Richard; Grandgenett, Duane; Pozzi, Nicola.
502 $b Ph.D. $c Saint Louis University $d 2022.
520 $a Ribonucleases H (RNases H) are Mg2+-dependent enzymes that cleave RNA when it is bound to DNA and have vital roles in important cellular processes like replication of the mitochondrial genome and removal of R-loops from the genome. Here, we have optimized conditions for expression and purification of recombinant human RNase H1 and its component domains. The proteins and their interactions with a 12-bp RNA/DNA substrate were characterized using biophysical techniques, RNA/DNA binding assays, and kinetics assays with a fluorogenic RNA/DNA substrate. RNases H are also essential for the replication cycles of HIV and Hepatitis B Virus and are being pursued as drug targets. To better understand how these compounds affect the activity of RNase H proteins, the recombinant RNase H1 proteins were used to evaluate the mechanism of RNase H inhibition by two structurally distinct compounds from the α-Hydroxytropolone class. We determined that turnover rate was reduced by both compounds, but inhibition was not competitive with substrate, despite the inhibitors binding to the active site via the classic two-divalent metal ion coordination mechanism. Since the compounds showed reversible binding to RNase H1, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which αHTs stabilize an inactive enzyme-substrate-inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid development of inhibitors specific for the viral RNases H.
546 $a English
590 $a School code: 0193
650 4 $a Biochemistry. $3 518028
650 4 $a Molecular biology. $3 517296
650 4 $a Biophysics. $3 518360
653 $a Enzyme inhibition
653 $a Enzyme kinetics
653 $a Inhibition mechanism
653 $a Ribonuclease H
653 $a RNase H1
690 $a 0487
690 $a 0786
690 $a 0307
710 2 $a Saint Louis University. $b Microbiology. $e degree granting institution. $3 3819017
720 1 $a Tavis, John $e degree supervisor.
773 0 $t Dissertations Abstracts International $g 84-03B.
790 $a 0193
791 $a Ph.D.
792 $a 2022
856 4 0 $u https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=29321566