Author ORCID Identifier

orcid.org/0000-0001-8739-0571

Date of Graduation

5-2017

Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Jessica Tyler, Ph.D.

Committee Member

Bin Wang, Ph.D.

Committee Member

Grzegorz Ira, Ph.D.

Committee Member

Pierre McCrea, Ph.D.

Committee Member

Xiaobing Shi, Ph.D.

Abstract

The access-repair-restore model for the role of chromatin in DNA repair infers that chromatin is a mere obstacle to DNA repair. However, here we show that blocking chromatin assembly of newly-synthesized histones, via knockdown of the histone chaperones ASF1A, CAF-1 or a mutation that specifically prevents ASF1 binding to histones, hinders loading of Rad51 onto ssDNA during homologous recombination, as a consequence of reduced recruitment of the Rad51 loader MMS22L/TONSL to ssDNA, resulting in persistent RPA foci, extensive DNA end-resection, and persistent activation of the ATR-Chk1 pathway. By contrast, ASF1 and CAF-1 render the rapid inactivation of ATM Chk2 pathway via facilitating histone acetylation to allow the recruitment of ATM to DSBs for efficient repair by non homologous end joining. Furthermore, in response to DSB damage, ASF1A is specifically phosphorylated by DNA-PKcs and potentially other kinases, enhancing its interaction with histones H3.1/H4 and CAF-1, and promoting the recruitment of MMS22L/TONSL to the damaged DNA to form the Rad51 nucleofilament during homologous recombination. We propose a model whereby DSB-induced phosphorylation of ASF1A promotes transient assembly of newly-synthesized histones onto single-stranded resected DNA, which serves to recruit TONSL-MMS22L to efficiently form the Rad51 vii nucleofilament for strand invasion. As such, we have uncovered an unexpected role for chromatin assembly in an intermediate stage of homologous recombination, revealing that chromatin assembly plays an active role in DNA repair per se.

Keywords

Chromatin assembly, ASF1, CAF-1, homologous recombinational repair, Rad51, DNA damage, ssDNA, MMS22L/TONSL, Histone, Chk1

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