Analyses of Site-Specific Recombination

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DESCRIPTION (provided by applicant): Tyrosine recombinases perform site-specific recombination reactions in a variety of cellular processes. Site-specific recombination is a term used to describe genetic rearrangements of DNA segments that require only limited regions of homology. This work is centered around a conjugative transposon called CTnDOT that encodes a recombinase, called IntDOT, that performs a site-specific recombination reaction. CTnDOT resides in the chromosomes of clinically important Bacteroides strains. It carries genes encoding resistances to the antibiotics tetracycline and erythromycin. CTnDOT also encodes a variety of accessory proteins called Orf2c, Orf2d and Exc that are required for excision. During excision, IntDOT, the accessory proteins and a host factor(s) promote the excision of the element to form a circular copy of the element. A single strand of CTnDOT is transferred to a recipient strain by conjugation. After the DNA is circularized and replicated a copy of CTnDOT is integrated into the recipient chromosome by a reaction promoted by IntDOT and a host factor. This works focuses on the mechanism of catalysis by IntDOT and the mechanism of the excision reaction. We will perform a structural analysis of IntDOT. The wild type and mutant proteins will be crystallized in the presence of DNA substrates. This work will help understand the differences in the catalytic sites of IntDOT and other tyrosine recombinases. A second aim is to analyze the mechanism of the excision reaction. We will purify the accessory proteins and study their interactions with DNA and with IntDOT. We will also analyze the formation and resolution of Holliday Junction (HJ) intermediates formed during the IntDOT reaction. We will analyze the interactions of accessory proteins with HJs and the effects of accessory proteins on the resolution of HJs to substrates or products. The final aim is to analyze mutant IntDOT proteins. We have isolated a large set of mutants that produce IntDOT proteins that are defective in recombination in vivo. We will characterize the mutants for defects in the recombination pathway. These studies will complement results obtained from previous biochemical studies and provide information for interpreting physical studies. PUBLIC HEALTH RELEVANCE: Increasing antibiotic resistance in opportunistic pathogens and prominent microbiota components such as Bacteroides spp. is becoming a serious clinical problem. The proposed work will provide new insights into the mechanisms of integration and excision of conjugative transposons, which are major contributors to the spread of resistance genes among Bacteroides and other important pathogens such as the gram positive cocci.