DNA replication is a conserved process that must be highly regulated. DNA replication involves a complex known as the replisome that interacts with proteins to unwind and incorporate new problems to the cells, therefore cells have specialized machinery that replace the replisome to prevent stalling of the replicative fork. It is predicted that YoaA is a component of this machinery that functions as a putative helicase to proofread and unwind DNA. YoaA complex is comprised of single stranded binding proteins (SSB) and two clamp loader components HolC and HolD and it has been shown to interact with each of these elements. Our studies utilize the Yeast-Two-Hybrid system and protein pull-down assays to assess the interactions between YoaA and the associated DNA proteins. Pertaining to the Yeast Two Hybrid system, we are curious how HolC Wild Type and specific HolC mutants will interact between previously stated DNA associated proteins. We hypothesize that interactions with HolC mutants and the SSB will be maintained and interactions with the HolC mutants and YoaA and HolD will be disrupted. In the pull down assays we utilized mutant YoaA with a single residue mutation in a predicted binding site with HolC and expected a disrupted association. We have found all wild type interactions between HolC-HolD, HolC-YoaA, and HolC-SSB are strong. Interactions in HolC mutants were eliminated in YoaA, whereas some interaction between HolC with HolD and SSB were maintained. However, we found mutant YoaA to have only slightly reduced binding with HolC whereas the wild type binding was unaffected.
- Characterization of the Repair Protein Complex: Putative Helicase YoaA and its Associated DNA Proteins
- Kirsten Brand
- Susan Thomas Lovett (Advisor)Vincent A. Sutera (Advisor)
- Brandeis University, Graduate School of Arts and Sciences; Master of Science (MS)
- Master of Science (MS), Brandeis University, Graduate School of Arts and Sciences
- Brandeis University
- 10192/36554; 9923879946801921
- Copyright by Kirsten Brand 2019
- Department of Biology
- English
- Thesis