Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
Faithful replication of the genome during cell division is essential for the avoidance of disease-promoting mutations. Until recently, accurate quantification of replication factor alterations in response to cellular stress primarily relied on low sensitivity assays such as cell staining and nuclear extraction assays. In the past few years, the Cortez, Groth, and Santocanale laboratories have developed procedures to retrieve and analyze proteins associated with actively replicating DNA (iPOND, NCC, and Dm-Chp, respectively). Herein, we report improvements to iPOND that increase protein yield and quantitative sensitivity, as well as permit better statistical evaluation of candidate factors (iPOND2). These improvements were achieved by employment of sucrose based density gradient fractionation of samples prior to EdU-biotin retrievals. The use of iPOND2 increased the dynamic range of protein quantification by Mass Spec by more than 40-fold compared to recent iPOND. We investigated the replisome component response to stress and assessed the role of p97-mediated degradation in protein turnover at the fork with or without cell cycle checkpoint protein, ATR. Furthermore, increased replisome component yields permitted the detection of ubiquitylated peptides without secondary affinity-based retrievals. Due to the increased yield of iPOND2 we were able to combine iPOND2 with other purification methods such as K-ɛ-GG IP to gain further utility from the addition of sucrose fractionation to iPOND. For example, we have further improved our ability to analyze ubiquitin sites on the replisome in a high-throughput way and potentially developed a method capable of assessing terminated fork or origin composition across a variety of treatment conditions. In summary, iPOND2 exhibits greatly improved replisome retrieval specificity, yield, quantitative dynamic range, and statistical power to detect changes in replication-associated factors in response to stress conditions. iPOND2 can be used alone, in conjunction with a variety of MS analytical methods, and combined with secondary affinity purifications to improve our understanding of replisome dynamics following stress.
Rivard, Rebecca, "Ipond2: The Next Generation The Development And Application Of Improved Methods For Assessment Of Replisome Protein Dynamics" (2017). Publicly Accessible Penn Dissertations. 2552.