Defining the molecular mechanism and functions of PCNA ubiquitination in the DNA damage response

Biological change comes in the form of genetic mutations. On the organismal level, mutations provide the genetic diversity that is the driving force behind evolution. On the cellular level however, mutations can be beneficial under conditions where genetic diversity is advantageous, such as somatic hypermutation and antibody generation, or lethal when they disrupt basic cellular processes and cause uncontrolled cell growth and cancer. Mutations arise from inaccurate processing of lesions generated by endogenous and exogenous DNA damaging agents, and the genome is particularly vulnerable to such damage during S phase. In this phase of the cell cycle, lesions in the DNA template block replication, resulting in replication fork stalling. Removal of these lesions would result in a double-strand DNA break, while prolonged stalling of replication forks can lead to fork collapse, DNA breaks and genetic instability.;In order to avoid these pernicious hazards, eukaryotic cells mount an active response to DNA damage by undergoing cell cycle arrest, activating DNA repair pathways, stabilizing the damaged DNA and in some cases, initiating apoptotic pathways. The ATR-dependent DNA damage checkpoint pathway plays a crucial role in mediating these responses, however these lesions must be still be repaired or bypassed in order to complete DNA replication. Lesion bypass is carried out by a set of error-prone and error-free processes collectively referred to as DNA damage tolerance mechanisms. Mutagenic bypass occurs when specialized low-fidelity TLS polymerases replicate over the lesion in a process known as translesion synthesis. In template switching modes of lesion bypass, the lesion is avoided altogether by using the undamaged sister chromatid as an alternate template.;Proper regulation of DNA damage tolerance is essential for maintaining genetic fidelity and genome stability. One protein show to have a crucial role in coordinating DNA damage tolerance processes is PCNA. During normal replication, PCNA functions as a processivity factor for replicative DNA polymerases. In response to DNA damage, PCNA can be either mono- or polyubiquitinated to promote translesion synthesis and template switching, respectively. While genetic studies in yeast have been instrumental in identifying the key enzymes involved in PCNA ubiquitination, what triggers PCNA ubiquitination and how these modifications direct DNA damage tolerance processes remain unknown.;This dissertation describes two major contributions: (1) Biochemical characterization of the structural and molecular requirements required for PCNA ubiquitination. Using the Xenopus egg extract system, we show that primed single-stranded DNA generated in response to replication fork stalling is minimally required for PCNA ubiquitination. Previous work by Byun and coworkers (Byun et al. Genes Dev. 2005) demonstrated that different types of DNA damage are processed into a common intermediate through a functional dissociation of the replicative helicase and DNA polymerase activities. Here, we show that ubiquitination of PCNA does not require ATR function, and that both pathways are simultaneously but independently activated in response to the same molecular signal. (2) Identification of Rad18, the E3 ubiquitin ligase responsible for monoubiquitination of PCNA, as a novel substrate of the DNA damage checkpoint pathway. Human Rad18 knockout cell lines display marked hypersensitivity to camptothecin-induced DNA damage, indicating that Rad18 plays a critical role in the DNA damage response. Human Rad18 is phosphorylated by the ATM/ATR checkpoint kinases at serine 403. This phosphorylated form of Rad18 is not required for Rad18's role in DNA damage tolerance; phosphorylation of Rad18 at S403 is dispensable for PCNA ubiquitination, Rad18 binding to Rad6, and Rad18 binding to TLS polymerase eta in human cells.