| Cre recombinase, from bacteriophage P1, belongs to the lambda integrase family of site-specific recombinases. This family is characterized by a common stepwise strand cleavage and exchange recombination mechanism, including the formation of covalent protein-DNA intermediates and a Holliday junction (HJ) intermediate. Cre catalyzes DNA strand exchange in a precise order at nearly symmetric 34 bp LoxP sites.;I have investigated the modes of regulation of Cre specificity for the LoxP central 8 bp spacer sequence. This spacer sequence is the only asymmetric portion of LoxP, and thus must guide complex assembly and strand cleavage by Cre. Cre makes direct contacts only with the outer two spacer base pairs. I have confirmed that these outermost bases are important in regulating strand cleavage order. The central 6 bp are uncontacted, but are also important in directing the strand cleavage order. My results suggest that Cre utilizes indirect readout of the spacer, recognizing differential bending energies between base steps or the spacer deformability to direct strand cleavage to the LoxP right arm, and thus preferential assembly of the cleaving Cre subunit with the right arm in the initial complex. Further, I analyzed the role of a conserved Cre active site residue, His289, by mutation to Ala or Asn. His289 plays a role during LoxP strand cleavage selection, and may help to activate the right-arm scissile phosphate for cleavage.;Unlike its interaction with the LoxP spacer, Cre makes a large network of contacts with the 13 bp regions that flank the spacer. I have investigated the regulation of Cre reactivity by one set of contacts (Arg259 of Cre with Gua27 of LoxP), where mutations influence both complex assembly and reaction rate. I determined that Cre has a unique mechanism for DNA sequence discrimination that is not observed in other DNA-binding proteins, in that the reaction rate is regulated in part by the isomerization of the HJ intermediate, rather than solely during complex assembly or a chemical cleavage step. This mode of regulation is affinity-dependent, because a Cre variant (non-specifically enhanced for DNA binding) does not exhibit the same HJ isomerization defect. |
