| I have characterized two independent enzymatic activities, unified by their importance in maintenance and organization of the genetic material. First I quantified the ability of yeast Rad51, a protein central to homologous recombination and DNA double-strand break repair, to bypass small sequence heterologies as might be encountered between the two chromosomes of diploid eukaryotes. I found that Rad51 is quite stringent, bypassing heterologous inserts of no more than 9 base pairs. Besides the increased stringency, I found Rad51 to be mechanistically similar to the prokaryotic RecA protein.;After completing the heterology bypass study, I characterized two DNA species that accumulated in heterologous reactions. The two species, which I show are true reaction intermediates, correspond to one or two double-stranded linear substrates reacting with the single-strand circle. I propose a model for the structure and reaction path of the intermediates based on biochemical experiments and direct visualization with electron microscopy. In addition, I defined sites on the substrate that pause branch migration during strand transfer.;My second project focused on condensins, protein complexes required for the condensation of chromosomal DNA before cell division. I show that two in vitro activities previously ascribed only to the condensin complex of Xenopus laevis are conserved in the condensin complex of an unrelated metazoan, the worm Caenorhabditis elegans. The extension of this activity across species validates the assay and suggests that the underlying mechanism is fundamental to in vivo condensation. I also discuss preliminary data suggesting that, of the five subunits in Xenopus condensin, only the structural maintenance of chromosome (SMC) protein dimer is required for the in vitro plasmid knotting activity.;Finally, I describe atomic force microscopy studies of SMC protein and condensin complexes from four species. I found that SMC proteins from prokaryotes through metazoans adopt a "Y" shaped structure suggesting that the coiled-coils associate tightly along half their length. In condensin complexes from three species, non-SMC subunits associated with the paired catalytic domains of the SMC dimer, suggesting that the non-SMC proteins may stabilize a closed dimer conformation and regulate activity through protein-protein interactions. |
