The integrity of our genome is especially at risk while it is being replicated. During DNA replication, the DNA must be “unzipped”, giving rise to structures known as replication forks. While traversing the genome, replication forks often encounter obstacles to their progression, including DNA lesions, hard-to-replicate sequences, transcription intermediates, or protein-DNA complexes. These encounters are potential sources of DNA breaks, chromosomal rearrangements and aneuploidy, all of which are hallmarks and drivers of cancer. Proper control of replication fork progression is therefore essential for genome integrity and cancer avoidance. Paradoxically, numerous anti-cancer agents such as topoisomerase inhibitors, DNA crosslinkers and DNA alkylators, kill cancer cells by impairing the regulated progression of the replication machinery and inducing replication stress. Our laboratory studies how cells sense, signal and prevent DNA replication stress to ensure faithful genome replication. We seek a deep mechanistic and integrated understanding of the replicative stress response and its implications for tumorigenesis and improved cancer treatment.
A phosphorylation code for the control of the replication stress response. We are investigating how the actions of kinases are translated into a coordinated cellular response that ensures proper completion of genome replication. As shown in the figure below, our recent work using budding yeast as a model organism has uncovered how phosphorylation events mediated by CDK and checkpoint kinases form a “code” of combinatorial protein interactions that lead to distinct functional outputs, therefore allowing timely coordination of DNA replication, cell cycle and DNA repair (1-3). Central to this combinatorial mode of regulation is the multi-BRCT domain scaffold Dpb11, capable of “reading” a range of phosphorylation events and physically coupling distinct protein complexes. We are currently investigating how TOPBP1, the human ortholog of Dpb11, coordinates DNA damage responses via a similar phosphorylation-mediated code.
Phosphoproteomics. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Understanding the molecular basis of kinase action and function requires knowledge of the kinase substrates, as well as comprehensive characterization of the dynamics and role of the phosphorylation events. Because many kinases are active in a cell and thousands of proteins are phosphorylated, the study of phosphorylation-mediated signaling pathways is challenging and powerful technologies are needed. We have developed and applied quantitative mass spectrometry technologies for the phosphorylation analysis of protein complexes and for global screens of in vivo kinase substrates (4). We are now expanding the use of these technologies to quantitatively characterize signaling dynamics and regulation at a proteome-wide scale.
1. Cussiol, J.R., et al. EMBO J 34, 1704-17 (2015).
2. Ohouo, P.Y. et al. Nature 493, 120-4 (2013).
3. Ohouo, P.Y. et al. Mol Cell 39, 300-6 (2010).
4. Bastos-de-Oliveira, F., Kim, D. et al. Mol Cell 57, 1-9 (2015).