Runtong
Tan
Sponsor: Wolf Heyer, Ph.D. Microbiology & Molec Genetics The BRCA2 protein functions as a tumor suppressor, regulating genome stability by facilitating assembly of RAD51-ssDNA filaments central to homologous recombination for DNA repair and replication processes. Loss of the functional BRCA2 results in accumulation of mutations from defective DNA repair causing cancer. The protein can be divided into the N terminus, the middle domain, and C terminus. It is presently unclear whether the middle domain, composed of 8 BRC repeats, contains another DNA binding element and what the functional significance of the helical and tower domains located in the C terminus are. This project aims to define the DNA binding regions in BRCA2 and how they function within the context of the full-length protein. Towards this end, we have created three plasmid constructs-full length BRCA2 open reading frame, mutant BRCA2 genes with a deletion of the helical domain or tower domain. We have transfected the plasmids into human HeLa cells to express the desired proteins. We will then compare the purified mutant proteins against the wild type to investigate the function of the targeted domains. DNA binding is a fundamental property of BRCA2, and our analysis will define the DNA binding properties of the various elements involved. Expression and Purification of the RAD54 Motor and C-terminal Domains to Study Their Interaction with RAD54B Protein in Homologous Recombination
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Runtong Tan
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DNA double-strand breaks (DSBs) are a lethal form of DNA damage, and their repair is crucial for maintaining genomic integrity. RAD54 and its paralog RAD54B are double-stranded DNA(dsDNA)-dependent ATPase that play a crucial and partially overlapping role in homologous recombination (HR). In HR, they facilitate RAD51-mediated strand invasion and the subsequent removal of RAD51 from dsDNA by their remodeling activity. Our laboratory recently identified a physical interaction between the two paralogs. However, the interaction sites and molecular implications governing the interaction remain unclear. In this study, we aim to uncover the interaction dynamics of the RAD54 motor domain and the adjacent C-terminal domain with RAD54B protein. To achieve this, we generated a recombinant baculovirus expressing 2MBP-RAD54 (Motor domain or C-terminal domain) fused with Flag and His?? tags at C-terminus end to facilitate purification and detection. Using the insect cell expression system, we generated viral generations (P0-P4) to achieve high- titer viral stocks and optimized protein expression. The expression levels were evaluated through Western blotting across different generations of viruses to determine the most effective titer and stability of target proteins. Following expression optimization, the tagged RAD54-domains will be purified through affinity chromatography and analyzed for their affinity interacting with RAD54B. Book Reading Versus Toy Playing: How Parents Talk to Children Differently Makoa Tandy
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UC Davis / Microbiology & Molec Genetics / 2026
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Runtong Tan