Title

The RecBCD enzyme of Escherichia coli: The chi effect

Date of Award

2002

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Richard S. Myers, Committee Chair

Abstract

RecBCD has two conflicting roles in E. coli. As ExoV, it is a potent dsDNA exonuclease that destroys linear DNA. As a recombinase it promotes repair of dsDNA breaks and genetic recombination in the vicinity of the DNA sequence chi. These paradoxical roles are accommodated by the chi-dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To understand the role of chi in regulation of RecBCD and the enzymatic activities that are required for initiation of recombination, genetic, physiological and biochemical analyses were performed. Genetic analyses of E. coli recB♣ mutant strains deficient in RecBCD nuclease activity showed that 5' → 3 ' nuclease activity is required for initiation of recombination. This result rules out an attractive model for regulation of RecBCD enzyme activity by chi by demonstrating that helicase activity is not sufficient for recombination. Physiological analyses of recB ♣ cells show that ExoV is required to degrade linear dsDNA that arises during normal growth and to process certain types of DNA damage. Biochemical analysis of RecBCD-chi interaction suggests that RecBCD does not bind specifically to a chi-containing ssDNA oligonucleotide. This result suggests that helicase activity is required for productive chi interaction and the concomitant change in RecBCD enzymatic activity. This change of RecBCD upon chi interaction is not autophosphorylation.

Keywords

Biology, Molecular; Biology, Genetics; Chemistry, Biochemistry

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3071285