Elucidation of the Mechanism of Microhomology-Mediated End Joining by DNA Polymerase Theta

PROJECT SUMMARY Double-stranded DNA breaks (DSBs) are a severe form of DNA damage that can give rise to devastating chromosomal aberrations. While DSBs are usually repaired with high fidelity through homologous recombination (HR), cells bearing mutations in HR-associated genes such as breast cancer related genes (BRCA)1/2, pivot to

a non-classical DSB repair process called microhomology-mediated end joining (MMEJ). Unlike HR, MMEJ is highly error-prone and can lead to genomic instability and carcinogenesis. A key player in MMEJ is DNA polymerase theta (PolΘ), which is recruited to DSBs to carry out the DNA microhomology annealing, strand

separation, and gap-filling processes via a unique DNA DSB repair mechanism that remains poorly understood. Many cancers, such as BRCA1/2 deficient breast, ovarian, prostate, and pancreatic cancers, exhibit HR pathway deficiencies and rely heavily on MMEJ, making PolΘ a promising therapeutic target due to its fundamental

involvement in this DNA repair pathway. Successful targeting and inhibition of PolΘ to treat HR-deficient cancers requires a mechanistic understanding of how PolΘ coordinates MMEJ. To gain new biomedically impactful insights into these unknown mechanisms, I will combine established biochemical and cell-based assays with

innovative gene editing and structural biology approaches to elucidate the molecular mechanism of MMEJ and identify intramolecular and intermolecular interactions that are indispensable for efficient MMEJ by PolΘ. During my first year in the Lander lab, I determined the first DNA-bound structure of the PolΘ helicase domain using a

DNA substrate I identified with a native PAGE screen. This structure reveals the isolated PolΘ helicase domain performing microhomology annealing, which is the primary initiation step of MMEJ. This structure also reveals how DNA is threaded through the helicase during MMEJ, a shift in helicase quaternary structure from tetramer

to dimer, and a hinged rotation of both helicase C-terminal domains to accommodate DNA microhomology annealing between the protomers. These observations provide novel insights into the role of the PolΘ helicase domain in MMEJ. These preliminary data establish a solid foundation for my studies aimed at (Aim 1) defining

the atomistic relationship between PolΘ and MMEJ DNA substrates and illuminating novel strategies for development of PolΘ-targeted therapeutics to treat HR-deficient cancers, and (Aim 2) elucidating the cellular mechanism of MMEJ and investigating cellular consequences of my structural and biochemical insights. These

combined biological and technical studies will address a critical unmet need in BRCA1/2-mutant breast and ovarian cancers by revealing new strategies for development of PolΘ inhibitors to be investigated as potential therapeutics for treating HR-deficient myopathies.