Interrogation and engineering of RAD51:BRC repeat interactions
The maintenance of genetic information is a fundamental function of every organism. The DNA in every human cell endures thousands of lesions per day, and eukaryotes have developed a sophisticated response to tackle this damage. Double strand breaks (DSBs) are the most toxic type of DNA damage, potentially leading to chromosomal rearrangements and tumorigenesis. Homologous recombination (HR) is a repair pathway that uses a homologous template to faithfully repair the DSBs. Central to HR is a recombinase protein RAD51, which forms a nucleoprotein filament with the broken ends, and allows efficient homology search. RAD51 is a promising therapeutic target in oncology. BRCA2 is a crucial mediator of RAD51 function and interacts with RAD51 through a set of 8 BRC repeats. A BRC repeat consists of two modules, an FxxA and an LFDE module, each containing hot-spot residues that mediate binding. In this work, I set out to utilise these for the pharmacological targeting of human RAD51. First, using biophysical methods, I investigated a novel, high-affinity chimeric repeat BRC8-2 composed of FxxA and LFDE modules from different natural repeats. I determined the X-ray crystallographic structure of its complex, which allowed me to uncover the determinants of high affinity binding. I applied these findings to the design of novel macrocyclic peptide inhibitors of RAD51. A method for cysteine stapling of recombinantly produced peptides was optimised to yield the correct product of high purity. The optimised stapling methodology provides a promising general approach for recombinant production and evaluation of cysteine-stapled peptides. Peptides were rationally designed in a structure-guided manner, and a variety of stapling architectures were evaluated for binding. The resulting purified molecules exhibit high potency and are stable in serum. Further crystallographic studies shed light on how the stapling moiety affects the peptide binding mode. The derived peptides serve as novel modalities for targeting Rad51 protein-protein interactions and can inform subsequent development of therapeutic drugs. In addition to the human proteins, I investigated the BRC:RAD51 interactions in orthologs from Leishmania infantum, the causative agent of leishmaniasis. Using crystallography and biophysical methods, I uncovered novel features of BRC repeat binding. The presented work expands our understanding of the structural determinants of homologous recombination.