A genetic analysis of RecA-LexA protein interactions in Escherichia coli

Persistent Link:
http://hdl.handle.net/10150/282594
Title:
A genetic analysis of RecA-LexA protein interactions in Escherichia coli
Author:
Mustard, Julie Ann
Issue Date:
1998
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:
The RecA protein of Escherichia coli is a small protein involved in many important functions including homologous recombination, mutagenic repair, regulation of the SOS system, and prophage induction. In normally growing cells, RecA is in an inactive form. However, when cells are subjected to DNA damage, RecA forms a helical filament with single stranded DNA and ATP. This ternary complex is the activated form of RecA. A key step in the regulation of several of these processes is the RecA mediated cleavage of different proteins. However, RecA is not a classic protease, but instead causes these proteins to undergo autodigestion. The main goal of this research was to investigate the role that RecA plays in cleavage by determining what residues in RecA interact with the cleavage substrates LexA, UmuD and λ CI. A possible model for the binding of the cleavage substrates in the cleft formed between two adjacent RecA monomers in an activated filament has been proposed. Site-directed mutagenesis was used to change residues in RecA that map to the cleft. An analysis of previously characterized recA mutants also suggested other regions of RecA that might interact with the cleavage substrates. Site-directed mutagenesis was used to also change these residues. Candidate residues were changed to alanine in order to reduce side chain contacts while minimizing perturbation of protein folding. The RecA mutant proteins were then characterized for ability to do recombination and DNA repair, and were examined for ability to mediate the cleavage of LexA, UmuD and λ CI. Several mutants showed some defects in cleavage of LexA or λ CI, while being proficient for other RecA functions. The fact that these mutants are selectively defective for cleavage of one protein, but not the others, suggests they can form activated filaments. Most of the mutations that differentially affected cleavage of LexA or λ CI mapped outside of the cleft region. This result suggests that the cleavage substrates do not bind in the cleft, but instead that the cleavable proteins may bind in the groove formed between turns of the RecA filament.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Chemistry, Biochemistry.; Chemistry, Biochemistry.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Little, John

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleA genetic analysis of RecA-LexA protein interactions in Escherichia colien_US
dc.creatorMustard, Julie Annen_US
dc.contributor.authorMustard, Julie Annen_US
dc.date.issued1998en_US
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.abstractThe RecA protein of Escherichia coli is a small protein involved in many important functions including homologous recombination, mutagenic repair, regulation of the SOS system, and prophage induction. In normally growing cells, RecA is in an inactive form. However, when cells are subjected to DNA damage, RecA forms a helical filament with single stranded DNA and ATP. This ternary complex is the activated form of RecA. A key step in the regulation of several of these processes is the RecA mediated cleavage of different proteins. However, RecA is not a classic protease, but instead causes these proteins to undergo autodigestion. The main goal of this research was to investigate the role that RecA plays in cleavage by determining what residues in RecA interact with the cleavage substrates LexA, UmuD and λ CI. A possible model for the binding of the cleavage substrates in the cleft formed between two adjacent RecA monomers in an activated filament has been proposed. Site-directed mutagenesis was used to change residues in RecA that map to the cleft. An analysis of previously characterized recA mutants also suggested other regions of RecA that might interact with the cleavage substrates. Site-directed mutagenesis was used to also change these residues. Candidate residues were changed to alanine in order to reduce side chain contacts while minimizing perturbation of protein folding. The RecA mutant proteins were then characterized for ability to do recombination and DNA repair, and were examined for ability to mediate the cleavage of LexA, UmuD and λ CI. Several mutants showed some defects in cleavage of LexA or λ CI, while being proficient for other RecA functions. The fact that these mutants are selectively defective for cleavage of one protein, but not the others, suggests they can form activated filaments. Most of the mutations that differentially affected cleavage of LexA or λ CI mapped outside of the cleft region. This result suggests that the cleavage substrates do not bind in the cleft, but instead that the cleavable proteins may bind in the groove formed between turns of the RecA filament.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectChemistry, Biochemistry.en_US
dc.subjectChemistry, Biochemistry.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLittle, Johnen_US
dc.identifier.proquest9829320en_US
dc.identifier.bibrecord.b3855169xen_US
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