53BP1/Shieldin Counteract DSB Resection Through Fill-In Synthesis

Author

Zachary Kenneth Mirman

Date of Award

2022

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

de Lange Laboratory

Abstract

53BP1 is a DNA damage response (DDR) factor that gained notoriety because it determines the efficacy of PARP1 inhibitors (PARPi) in BRCA1-deficient cancers. Additionally, 53BP1 promotes end-to-end fusions of telomeres lacking end protection from the shelterin component TRF2, and facilitates end-joining at programmed breaks generated during class switch recombination in the immune system. The role of 53BP1 in double strand break (DSB) repair outside of these three well-studied contexts is less clear, though it has been proposed that 53BP1 acts as a master regulator of so-called “DSB repair pathway choice,” promoting classical non-homologous end-joining (cNHEJ) at the expense of homology-directed repair (HDR). In the introductory chapter to the thesis, we first discuss our current understanding of the literature regarding 53BP1 and then present an alternate view: that the features of 53BP1 have evolved to promote high-fidelity DNA repair and avoid mutagenesis. This notion is supported by the data presented in the subsequent two chapters. We demonstrate that 53BP1 and its downstream effectors RIF1 and the shieldin complex limit the generation of long 3’ overhangs at DSBs by recruiting CST/Polα to counteract resection, challenging the long-standing model of a block to resection by this pathway. Like loss of 53BP1/RIF1/shieldin, disruption of CST/Polα/primase reverses the hallmarks of BRCA1- deficiency. CST/Polα localize to DSBs, and we directly detect fill-in synthesis by observing shieldin/CST/Polα/primase-dependent nucleotide incorporation. We also demonstrate that tethering CST to DSBs can bypass the need for recruitment by 53BP1/shieldin. A novel shieldin mutant which fails to recruit CST was non-functional in BRCA1-deficient cells, but its function could be fully restored by chemical-induced dimerization with CST. These results indicated that in BRCA1-deficient cells, CST/Polα/primase is the major effector of 53BP1/shieldin function. After an interlude (Chapter 4) on the role of CRL4/DDB1/WDR70 in regulation of the DDR, I discuss and contextualize the new results and revisit the model of 53BP1 as a DSB escort which controls DSB processing and promotes high-fidelity repair.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Mirman, Zachary Kenneth, "53BP1/Shieldin Counteract DSB Resection Through Fill-In Synthesis" (2022). Student Theses and Dissertations. 650.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/650