The Regulation of End Resection in the Repair of DNA Double-Strand Breaks

• Author / Creator

  Locke, Andrew J

• DNA double-strand breaks (DSBs) are considered the most deleterious DNA lesions. Unrepaired or incorrectly repaired DSBs can lead to cell cycle arrest, apoptosis, or over time, the accumulation of mutations and chromosomal abnormalities that give rise to cancer. The repair of DSBs by homologous recombination (HR) requires the overall 5’ to 3’ enzymatic trimming of DNA away from the DSB, a process known as DNA end resection. Using a variety of biochemical and cell biological techniques on cultured human cell lines, the studies herein investigate how DNA end resection is regulated. This work places emphasis on the impact that post-translational modification (PTM) events, such as the conjugation of phosphoryl, ubiquitin, and SUMO moieties onto target proteins, have on the function of proteins involved in end resection. The work described in Chapter 3 reveals that BMI-1, a transcriptional repressor belonging to the Polycomb group of proteins, promotes DNA end resection and thus HR. Mechanistically, BMI-1 promotes end resection through inhibiting RNA polymerase II-dependent transcription, while also facilitating the deposition of ubiquitin onto residue K119 of histone H2A (H2AK119ub). Depletion of BMI-1 impairs the recruitment of the end resection-promoting factor CtIP to DSBs, and the H2AK119ub mark may mediate CtIP’s accrual on chromatin, as CtIP can bind to ubiquitin directly. In Chapter 2, we uncover that CtIP itself is a target for modification by SUMO-2, particularly during the S phase of the cell cycle. CtIP SUMOylation is dependent on the E3 SUMO ligase PIAS4, and occurs primarily on residue K578. Relative to cells expressing wildtype CtIP, cells expressing CtIP with the K578R substitution mutation are impaired in DNA end resection and HR, are more sensitive to killing by the DSB-inducing drug camptothecin, and defective in protecting stalled replication forks from nucleolytic degradation. Lastly, in Chapter 4, we demonstrate that the E3 ubiquitin ligase and pro-resection factor RNF138 is modified by ubiquitylation (at residue K158) and cyclin-dependent kinase-dependent phosphorylation (at residue T27). Preventing the occurrence of either PTM, by arginine and alanine substitution mutations, respectively, strongly reduces the ability for RNF138 to promote end resection and HR. Together, these findings uncover novel intricacies in the tightly orchestrated process of DNA end resection for the repair of DSBs.

• Subjects / Keywords

  • DNA repair
  • genome integrity
  • post-translational modification
  • phosphorylation
  • ubiquitin
  • ubiquitinylation
  • ubiquitylation
  • transcription
  • double-strand break
  • end resection
  • genome instability
  • chromatin
  • SUMO
  • SUMOylation
  • RNF138
  • BMI-1
  • CtIP
  • DNA
  • repair
  • DNA damage
  • homologous recombination
  • non-homologous end joining
  • HR
  • NHEJ
  • Bmi1
  • transcriptional repression
  • cancer
  • DSB
  • PTM
  • RNF2
  • Polycomb
  • cell cycle
  • signaling
  • chromosome
  • homology
  • post translational modification
  • transcription
  • CDK
  • PIAS
  • replication
  • replication stress
  • nuclease
  • post translational modifications
  • post-translational modifications
  • double strand break
  • genome stability
  • cyclin
  • genome
  • nucleus
  • homologous
  • recombination
  • kinase
  • transcriptional silencing
  • signalling

• Graduation date

  Spring 2024

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-axc4-vm88

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.