Investigation into synaptic and post-synaptic events of RAD51 mediated Homologous Recombination

Homologous Recombination (HR) is the only means by which the deadliest form of DNA damage (Double Strand Break) can be repaired without error. Thus, I set out to better understand HR. Specifically, I wanted to address two holes in the field which were: (1) how are the template DNA strands found (synapsis) during HR; and (2) what happens afterwards. To address these two questions, I purified the main mammalian recombinase (RAD51) and other key proteins involved in HR to study in vitro. Through my studies, I have found that synapsis occurs through RAD51-nucleoprotein complexes which we term Homology-Search-Networks. This network has the following characteristics: (1) it consists of ssDNA, dsDNA, and RAD51; (2) it is essential to strand-exchange; (3) it is organized ; and (4) it is based upon multiple weak contacts. Furthermore, I found that in the post-synaptic phase, RAD51 dissociates D-Loops by unwinding dsDNA. Moreover, this process can be controlled by BRCA2 through BRC4 interaction. The result is a fine-tuning mechanism at the site of DNA damage to control strand-exchange and thereby HR. The implication is that the old notions of co-aggregates during synapsis can be updated and the current findings fit nicely with new single-molecule studies by Kowalczykowski, Ha, and Greene. Furthermore, my post-synaptic findings provide a solid molecular mechanism of the RAD51-BRCA2 homeostasis that controls Homologous Recombination. Together, my work will impact the fields of DNA repair, cancer biology, and genetic engineering.