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Control of Directionality in Mycobacteriophage L5 Integrase-mediated Site-specific Recombination

Lewis, John August (2002) Control of Directionality in Mycobacteriophage L5 Integrase-mediated Site-specific Recombination. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Control of directionality in integrase-mediated site-specific recombination reactions is achieved by an architectural change fashioned by a class of accessory proteins know as recombination directionality factors (RDFs). In the mycobacteriophage L5 system, no RDF had been previously identified. In the course of this work, the gene, 36, was identified using in vivo screens and shown to play this role. The protein was over expressed using an E. coli expression system and then used to create an in vitro excision reaction assay. Initial work was done in characterizing the excision reaction including supercoiling and host factor requirements. Further analysis has shown that the protein binds specifically to a region within the left side of attP and attR. Once bound, complexes are formed that inhibit integration in the case of attP and stimulate excision when attR is present. Additional work was done to increase the utility of the L5 based integrating vector system, which has been commonly used to create stably integrated single copy transformants. The system lacked an effective means to recover DNA for high copy replication or curing of strains containing inserted DNA. With the identification of the L5 xis gene, we have been able to develop systems that allow the recovery of DNA into E. coli and curing strains of the integrated DNA. The third part of this work involved the characterization of the RDF class of proteins. Since this class of proteins is composed of a diverse group of small proteins, no previous attempt had been made to characterize them. Extensive data mining yielded a collection of 63 putative or known RDFs. Further analysis of sequence data, chemical characteristics and other known properties argues that this class of proteins has evolved from multiple ancestral origins.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Lewis, John Augustrugby@pitt.edurugby@pitt.edu
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairHatfull, Grahamgfh@pitt.edugfh@pitt.edu
Committee MemberPipas, Jamespipas@pitt.edupipas@pitt.edu
Committee MemberBrodsky, Jeffjbrodsky@pitt.edujbrodsky@pitt.edu
Committee MemberHendrix, Rogerrhx@pitt.edurhx@pitt.edu
Committee MemberKhan, Saleemkhan@pitt.edukhan@pitt.edu
Date: 11 March 2002
Date Type: Completion
Defense Date: 15 October 2001
Approval Date: 11 March 2002
Submission Date: 14 December 2001
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: excise; excision; excisionase; integrase; integration; L5; mycobacteria; mycobacteriophage; mycobacterium; RDF; recombination directionality factors; site-specific recombination
Other ID: http://etd.library.pitt.edu:80/ETD/available/etd-12142001-024248/, etd-12142001-024248
Date Deposited: 10 Nov 2011 20:10
Last Modified: 29 May 2012 15:45
URI: http://d-scholarship.pitt.edu/id/eprint/10364

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