Novel Allosteric Properties Exhibited by SgrAI, A Restriction Endonulease Native to Streptomyces Griseus

Persistent Link:
http://hdl.handle.net/10150/146903
Title:
Novel Allosteric Properties Exhibited by SgrAI, A Restriction Endonulease Native to Streptomyces Griseus
Author:
Ghare, Imran Riaz
Issue Date:
May-2010
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
An 8-bp recognition sequence, secondary activity, and allosteric activity for the primary sequence make SgrAI a rather peculiar endonuclease. Recent evidence suggests that SgrAI exists as a dimer in solution in the absence of DNA and is able to bind to one duplex of DNA. However, the molecule mechanisms of secondary site and allosteric activity are poorly understood. Through a series of kinetic and binding assays we have reason to believe that the rate of DNA cleavage by SgrAI is significantly accelerated through the formation of HMWS (high molecular weight species), an aggregate of SgrAI dimers bound to DNA. Native gel electrophoresis suggests that increasing concentrations of PCP (precut primary site), in the presence of excess SgrAI enzyme, increases formation of the HMWS. Furthermore, our kinetic assays show acceleration of DNA cleavage by the addition of PCP in the presence of excess SgrAI. As such, it appears that the HMWS is an activated form of the enzyme. Crystal structures show that the tetramer has an N-terminal region which is domain swapped with another dimer to form a tetramer, which may be a building block of the HMWS. In order to further test this hypothesis, we measured HMWS formation and the stimulation of DNA cleavage by mutated enzymes, P27G/W SgrAI. Both mutants enzymes were designed to disrupt the domain swapped tetramer. Native gel electrophoresis suggests that P27W/G SgrAI enzymes do not form HMWS to the same extent as wild-type SgrAI (wtSgrAI). Additionally, P27W/G SgrAI do not exhibit the same acceleration of DNA cleavge as wtSgrAI, which leads us to believe that formation of the tetramer is necessary for formation of the HMWS. It also further supports the hypothesis that the active conformation of the enzyme can be found in the HMWS. This work is supported by Howard Hughes Medical Institute and NIH, General Medical Sciences.
Type:
text; Electronic Thesis
Degree Name:
B.S.
Degree Level:
bachelors
Degree Program:
Honors College; Biochemistry and Molecular Biophysics
Degree Grantor:
University of Arizona

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleNovel Allosteric Properties Exhibited by SgrAI, A Restriction Endonulease Native to Streptomyces Griseusen_US
dc.creatorGhare, Imran Riazen_US
dc.contributor.authorGhare, Imran Riazen_US
dc.date.issued2010-05-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractAn 8-bp recognition sequence, secondary activity, and allosteric activity for the primary sequence make SgrAI a rather peculiar endonuclease. Recent evidence suggests that SgrAI exists as a dimer in solution in the absence of DNA and is able to bind to one duplex of DNA. However, the molecule mechanisms of secondary site and allosteric activity are poorly understood. Through a series of kinetic and binding assays we have reason to believe that the rate of DNA cleavage by SgrAI is significantly accelerated through the formation of HMWS (high molecular weight species), an aggregate of SgrAI dimers bound to DNA. Native gel electrophoresis suggests that increasing concentrations of PCP (precut primary site), in the presence of excess SgrAI enzyme, increases formation of the HMWS. Furthermore, our kinetic assays show acceleration of DNA cleavage by the addition of PCP in the presence of excess SgrAI. As such, it appears that the HMWS is an activated form of the enzyme. Crystal structures show that the tetramer has an N-terminal region which is domain swapped with another dimer to form a tetramer, which may be a building block of the HMWS. In order to further test this hypothesis, we measured HMWS formation and the stimulation of DNA cleavage by mutated enzymes, P27G/W SgrAI. Both mutants enzymes were designed to disrupt the domain swapped tetramer. Native gel electrophoresis suggests that P27W/G SgrAI enzymes do not form HMWS to the same extent as wild-type SgrAI (wtSgrAI). Additionally, P27W/G SgrAI do not exhibit the same acceleration of DNA cleavge as wtSgrAI, which leads us to believe that formation of the tetramer is necessary for formation of the HMWS. It also further supports the hypothesis that the active conformation of the enzyme can be found in the HMWS. This work is supported by Howard Hughes Medical Institute and NIH, General Medical Sciences.en_US
dc.typetexten_US
dc.typeElectronic Thesisen_US
thesis.degree.nameB.S.en_US
thesis.degree.levelbachelorsen_US
thesis.degree.disciplineHonors Collegeen_US
thesis.degree.disciplineBiochemistry and Molecular Biophysicsen_US
thesis.degree.grantorUniversity of Arizonaen_US
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