This project aims to elucidate tightly controlled mechanisms that allow eukaryotic cells to faithfully reproduce their genetic material despite the presence of DNA damage. DNA lesions can block replication, triggering a cascade of events leading to cell death. To prevent this, cells utilize translesion synthesis (TLS) DNA polymerases that can copy over the DNA lesions while temporarily leaving them unrepaired. These TLS enzymes are central to cell survival after DNA damage; however, they are also error-prone and often introduce mutations in genomic DNA. The project will employ an interdisciplinary approach that combines structural biology, biochemical methods and in vivo assays to decipher key protein-protein interactions (PPIs) underlying mutagenic TLS in a yeast model system. This research will advance our understanding of the structural organization of the multi-protein TLS complexes and also provide new insights into mechanistic questions about TLS: How do mutagenic TLS enzymes gain access to DNA replication forks? How do active multi-polymerase complexes assemble? What molecular events drive TLS DNA polymerase switching? These questions are central to our understanding of events leading to mutagenesis in eukaryotes and the ability of cells to cope with DNA damage and faithfully transfer genetic information from one generation to the next. This project creates a solid platform for integration of research and education at multiple levels, from advanced post-PhD training to K-12 education. The project will provide plentiful material for graduate and undergraduate teaching, including advanced training courses such as the Connecticut NMR Workshop, laboratory projects for graduate students, and mini-projects for the diverse undergraduate students from the Undergraduate Summer Research Internship program. Finally, it will also engage students from the nearby Farmington High School in "real world" scientific research under the umbrella of the Cutting Edge Bioresearch Internship program.
In S. cerevisiae, replicative bypass of most DNA lesions requires the coordinated action of TLS polymerases Rev1, pol eta and pol zeta that replace replicative polymerases at replication forks stalled by DNA damage or fill damage-containing single-stranded DNA gaps left after replication. This process is initiated by mono-ubiquitination of the processivity factor PCNA, which plays a key role in the assembly of the multi-polymerase TLS complex at DNA damage sites. The mechanism by which this TLS complex, often called the Rev1/pol zeta mutasome, is assembled on ubiquitinated PCNA is poorly understood. Important questions remain unanswered about how TLS enzymes gain access to sites of DNA damage and how DNA polymerases switch during TLS. The main goal of this project is to understand the mechanisms by which the TLS mutasome achieves efficient lesion bypass by analyzing its organization at a structural level. Our central hypothesis is that mutasome activity is regulated by protein-protein interactions (PPIs) mediated by accessory domains and regulatory subunits of the TLS DNA polymerases. We will test this hypothesis by using a structural analysis to precisely map PPIs that influence the assembly of the S. cerevisiae Rev1/pol zeta mutasome, combined with in vivo assays to probe the significance of these PPIs for mutagenic TLS. To date, the structure and PPIs of accessory modules of yeast TLS enzymes remain largely uncharacterized. To gain insights into the structural organization and regulation of the yeast TLS machinery, we will undertake the following specific aims. In Aim 1, we will examine key PPIs that control the Rev1/pol zeta mutasome interactions with the sliding clamp PCNA. In Aim 2, we will determine structures and probe PPIs of the essential accessory modules that mediate assembly of the Rev1/pol zeta complex. In Aim 3, we will explore additional PPIs that stabilize multi-subunit Rev1/pol zeta assembly. The project will provide new insights into how individual components of the TLS mutasome work together to achieve efficient lesion bypass.
The project is funded jointly by the Genetic Mechanisms Cluster and the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.
