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Title

Repair of DNA Double-strand Breaks in G1-phase Cells

Date of Award

Spring 5-15-2014

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The two main DNA double-strand break (DSB) repair pathways are non-homologous end joining (NHEJ) and homologous recombination (HR). DNA ends are normally resected to form single-stranded overhangs to initiate HR, but resection severely limits repair by NHEJ. In G1-phase cells, NHEJ is the primary DNA DSB repair pathway, so DNA end resection must be restricted in these cells to prevent aberrant repair. Antigen receptor gene assembly occurs in G1-phase lymphocytes, using DNA DSB intermediates to generate a variable exon required for antigen binding. We show that the histone H2A variant, H2AX, which is phosphorylated by ATM kinase to generate gamma-H2AX in chromatin flanking DSBs, prevents the earliest steps of resection at RAG DSBs and other genotoxic DSBs in G1-phase lymphocytes. This gamma-H2AX function relies on its downstream factors MDC1 and RNF8 to concentrate 53BP1 at chromatin flanking the DSB, preventing the activity of resection machinery that can act in G1-phase cells. Resection in G1 is dependent on the protein CtIP, which has been previously shown to promote resection in S-G2 phases of the cell cycle. KAP-1 is a multifunctional adaptor protein that is involved in transcriptional silencing and maintenance of heterochromatin recently been implicated in repair of heterochromatic DNA DSBs. Here, we show that KAP-1 has a role in repair outside of heterochromatin, as it also promotes resection at RAG DSBs, along with CtIP. We have unexpectedly discovered that human KAP-1 blocks resection in mouse G1-phase lymphocytes in a dominant manner. The basis for this difference between human and mouse KAP-1 is a single amino acid polymorphism specific to primates located in a disordered region of the protein. Thus, we have established KAP-1 as part of the resection machinery in G1-phase cells that must be restricted by gamma-H2AX and 53BP1 to prevent resection and aberrant repair of DNA DSBs.

Language

English (en)

Chair and Committee

Barry Sleckman

Committee Members

James B Skeath, Kenneth Murphy, Eugene Oltz, Nima Mosammaparast, Zhongsheng You, Susana Gonzalo

Comments

Permanent URL: https://doi.org/10.7936/K7DN4308

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