Repair of DNA damage is crucial to prevent accumulation of mutations that can cause human disease, such as cancer. Many proteins are important for DNA repair including Sgsl, a protein that when mutated in human cells leads to many devasting diseases (i.e. Bloom, Werner, Rothmund-Thomson syndromes), which are all fundamentally characterized by cancer predisposition. Sgs1 genetically interacts with a group of proteins collectively called the SHU complex. Although Sgsl has been extensively analyzed, the molecular mechanism of how it functions to repair DNA damage and its relationship to the SHU complex has remained elusive, largely because its deletion leads to many pleiotropic phenotypes. During the K99 phase of this proposal, I will utilize a separation-of-function allele of Sgs1 that delineates its role during DNA repair from DNA replication. My preliminary results suggest that an alternative pathway is used to repair DNA replication errors that is distinct from the homologous recombination machinery. The experiments proposed here will use genetic and cell biological approaches to characterize the proteins involved in this novel pathway and determine how utilization of this pathway is differentially regulated. The second part of the K99 phase will use flourescent microscopy to place the Sgs1/Top3/Rmi1 proteins in the order of protein assembly utilized during DNA repair and determine if the genetic requirements for Sgsl foci formation differ depending upon the type of DNA damage. During the R00 phase, I will focus on the SHU complex and first analyze the role of one SHU component, Shu1, in rDNA repair and rDNA chromatin structure.
My second aim will elucidate the mechanistic role of the SHU complex during DNA repair and replication through its physical interaction with Srs2. Finally, I will determine if the SHU proteins have unique cellular functions despite forming a complex and address the significance of complex formation. The training that I receive during the K99 portion of the fellowship will enable me to develop the skills necessary to begin my own laboratory where my ultimate career goal is to be a tenured professor at a research institution. Relevance: Repair of broken DNA is one of the most fundamental of cellular processes. When DNA repair is inhibited, cells can accumulate genetic mutations and rearrangements that are hallmarks of cancer. Functional analysis of proteins required for DNA repair is crucial for understanding the molecular basis of tumorigenesis.
