Control of Directionality in Mycobacteriophage L5 Integrase-mediated Site-specific Recombination
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.
Advisor:Graham Hatfull; Dr. Jeff Brodsky; Dr. Roger Hendrix; Dr. Saleem Khan; Dr. James Pipas
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication:03/11/2002