ORCID

http://orcid.org/0000-0003-0464-1641

Date of Award

Spring 5-15-2020

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Non-homologous end joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in G1-phase cells. In particular, developing lymphocytes utilize NHEJ to repair physiologic DSBs generated during the processes of antigen receptor gene assembly and class-switch recombination (CSR). Thus, proper regulation of NHEJ is important not only for immune repertoire diversification, but also for the prevention of chromosomal translocations or deletions that can arise from misrepaired DSBs. The XRCC4-like factor (XLF) has been strongly implicated in promoting NHEJ, presumably by tethering broken DNA ends together in a sleeve-like complex, but its necessity appears to be cell context-dependent. While XLF is required for NHEJ in most cell types, it is dispensable for DSB repair in lymphocytes. Recently, it has been discovered that in lymphoid cells, XLF functions redundantly with other DNA damage response (DDR) proteins that were previously thought to play negligible roles in NHEJ, suggesting that novel NHEJ factors could be revealed in the setting of XLF deficiency. To test this hypothesis, we carried out a genome-wide CRISPR/Cas9 screen in a XLF-deficient pre-B cell-line for proteins that would become essential for DNA end joining during V(D)J recombination in the absence of XLF. We validated two hits from our screen: paralog of XRCC4 and XLF (PAXX), a newly identified NHEJ factor with significant structural homology to XLF, and modulator of retrovirus infection (MRI), an uncharacterized small peptide that been reported to bind to the DSB sensor Ku70/Ku80. We find that the combined genetic deficiency of MRI and XLF in mice leads to an embryonic lethal phenotype that is characteristic of defective NHEJ. Furthermore, we show that MRI is intrinsically disordered and interacts with diverse DDR proteins at both its termini to form large, multimeric complexes. MRI rapidly localizes to DSBs, where it promotes the retention of these DDR proteins on chromatin, and loss of MRI results in increased cellular sensitivity to ionizing radiation (IR). We propose that MRI functions as a multivalent adaptor that enhances the avidity of DDR proteins at DSBs to promote NHEJ.

Language

English (en)

Chair and Committee

Barry P. Sleckman

Committee Members

Gaya K. Amarasinghe, Brian T. Edelson, Takeshi Egawa, Nima Mosammaparast,

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