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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.

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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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      Item Type: University of Pittsburgh ETD
      ETD Committee:
      ETD Committee TypeCommittee MemberEmailORCID
      Committee ChairHatfull, Grahamgfh@pitt.edu
      Committee MemberPipas, Jamespipas@pitt.edu
      Committee MemberBrodsky, Jeffjbrodsky@pitt.edu
      Committee MemberHendrix, Rogerrhx@pitt.edu
      Committee MemberKhan, Saleemkhan@pitt.edu
      Title: Control of Directionality in Mycobacteriophage L5 Integrase-mediated Site-specific Recombination
      Status: Unpublished
      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.
      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; The work is available for access worldwide immediately.
      Patent pending: No
      Institution: University of Pittsburgh
      Thesis Type: Doctoral Dissertation
      Refereed: Yes
      Degree: PhD - Doctor of Philosophy
      URN: etd-12142001-024248
      Uncontrolled Keywords: excise; excision; excisionase; integrase; integration; L5; mycobacteria; mycobacteriophage; mycobacterium; RDF; recombination directionality factors; site-specific recombination
      Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
      Date Deposited: 10 Nov 2011 15:10
      Last Modified: 29 May 2012 11:45
      Other ID: http://etd.library.pitt.edu:80/ETD/available/etd-12142001-024248/, etd-12142001-024248

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