The role of protein degradation in maintaining genome stability through regulation of origin licensing

In eukaryotes cell proliferation requires the formation of a pre-replication complex (preRC) at chromosomal replication origins. PreRCs are constructed from the origin recognition complex (ORC) which recruits Cdc6 and Cdt1, and these three components work together to load the minichromosome maintenance (MCM) DNA helicase at each origin. Once preRC formation is completed the origin is "licensed" and replication of the genome can be initiated. It is important that origin licensing occurs once per cell cycle during G1, and is inhibited outside of G1 and in response to cellular damage. Mechanisms to restrict origin licensing rely on degradation of Cdc6 and Cdt1 and the Cdt1 inhibitor geminin. Failure to properly regulate origin licensing leads to genome instability, contributing to the development of cancer. I describe a novel mechanism for the degradation of Cdc6 in response to ultraviolet (UV) radiation and DNA alkylation by methyl methane sulfonate (MMS). This pathway is independent of previously described pathways for Cdc6 degradation requiring APCCdh1, and p53. Instead, Cdc6 directly binds the HECT-family ubiquitin ligase Huwe1, and degradation of Cdc6 in cells treated with UV and MMS requires Huwe1. My data demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage. Depletion of the Cdt1 inhibitor geminin causes rereplication, a form of endogenous DNA damage. I find that both Cdt1 and Cdc6 are degraded in geminin-depleted cells. Furthermore, I show that Cdt1 degradation in cells that have rereplicated requires the PCNA binding site of Cdt1 and the Cul4DDB1 ubiquitin ligase, and Cdc6 degradation requires the Huwe1 ubiquitin ligase. Moreover, perturbations that disrupt Cdt1 and Cdc6 degradation exacerbate rereplication when combined with geminin depletion. I propose that the degradation of Cdt1 and Cdc6 in rereplicated cells represents an evolutionarily conserved mechanism that minimizes the extent of rereplication. This dissertation researches how Cdc6 and Cdt1 are regulated in response to DNA damage and the consequences when these regulatory pathways are inhibited. The data presented in this dissertation provides insight into the mechanisms that prevent improper origin licensing, thus maintaining genome stability