Genome Maintenance and Quantitative Proteomics
Cells use proteins to do many tasks, including the replication and segregation of their genome during each cell division. No single protein is capable of these tasks alone - dozens of proteins must assemble into increasingly larger protein complexes to replicate and segregate chromosomes. To study these processes, our work falls into three areas. First, the regulatory mechanisms that control DNA replication and DNA damage response. Second, the controls of kinetochore assembly towards accurate chromosome segregation. Third, the development of Concatemer-Assisted Stoichiometry Analysis (CASA) for quantitative analysis of protein complexes and their modifications.
Although how individual MCM is loaded and activated to form the CMG helicase are now well understood, their cellular controls are not. We have a long-standing interest in the role of DNA damage checkpoint kinases in preventing genome rearrangements. More recently, we have focused on the function of SUMO, a small ubiquitin-like protein, in preventing genome rearrangements through site-specific MCM sumoylation (Albuquerque 2013 and 2016, Liang 2018, Suhandynata 2021, and Quan 2022). These findings uncovered an unprecedented role of SUMO is controlling MCM loading, which we are investigating further.
Kinetochores control chromosome segregation by connecting chromosomal centromeres to the spindle microtubules and scaffolding many signaling enzymes to prevent mis-segregation. We are using a combination of genetic, biochemical, structural, and proteomic approaches to study how kinetochores are made and to define the roles of signaling molecules, including SUMO and phosphorylation by protein kinases. Recent studies focused on the role of the Ulp2 desumoylase in regulating the inner kinetochore, and the role of protein kinases in controlled Mif2-dependent inner kinetochore assembly (Suhandynata 2019, Quan 2021, Hinshaw 2023, and Deng 2023). We continue to investigate how cells assemble kinetochores to control accurate chromosome segregation.
Concatemer-Assisted Stoichiometry Analysis (CASA)
Mass spectrometry (MS) has played a key role in the identification of hundreds of protein complexes in cells, and it has also found numerous applications in translational and clinical research. In most instances, data-dependent-acquisition has been used to identify as many proteins as possible. Increasingly, the focus is to obtain quantitative information about protein complexes and their modifications. To facilitate this transition, we, in collaboration with Suhandynata lab, have developed CASA and the use of targeted MS methods (Parallel-Reaction Monitoring, etc.) for precise, sensitive, accurate and robust quantification of proteins and their modifications. A resource and education center dedicated to sharing this technology, distributing reagents, and fostering collaborations is under construction. Stay tuned.