Activation and Inhibition of Lys63-linked Polyubiquitination via Ubc13 modulation

• Author / Creator

  Hodge, Curtis D

• DNA is a detailed layout of instructions for cellular function. The integrity of human DNA is constantly challenged by a barrage of exogenous and endogenous sources. A regime of genome stability maintenance has evolved to repair the many different types of DNA damage. A form of communication involved in the majority of cellular functions is ubiquitination. This entails a process whereby a small protein called ubiquitin is tagged singularly or successively to other proteins to confer a specific signal that is recognized by a host of different ubiquitin binding domains. The functional unit used to perform ubiquitin chain building is an E2 conjugating enzyme and E3 ligase pair (E2:E3). In particular, really interesting new gene (RING)/U-box E3s are known to provide a target for ubiquitination and stimulate the E2-conjugating activity of E2 enzymes that catalyze ubiquitin conjugation. One very important functional unit is the E2 Ubc13 and RING E3 RNF8 pair that interact to build ubiquitin chains with a specific lysine 63 (Lys63) linkage. E3s are traditionally thought to provide a target for E2 enzymes. Lys63 chains recruit DNA repair enzymes to the sites of DNA double strand breaks (DSBs). Ubc13 has recently been shown to participate in the pathogenesis of a number of different cancers. Here, we investigate two small-molecule inhibitors of Ubc13, through structural, biochemical and cellular methods. We developed a Ubc13 mutant which resists inhibition by one of these compounds and, using this mutant, we show that the inhibition of cellular DNA damage and NF-kB signaling is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors. Using X-ray crystallography, SAXS, and cell biology, we also separate E3-dependent E2-targeting from stimulation and demonstrate that the DNA damage response critically depends on the stimulatory activity for proper recruitment of DNA repair factors. Overall we reveal important insights into the function of the critical Ubc13:RNF8, E2:E3 ubiquitination pair.

• Subjects / Keywords

  • Cancer
  • DNA damage response
  • Ubiquitination

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3TM72C1D

• 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.