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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

The role of the redox active 4Fe-4S cluster of yeast Dna2

Dna2 is an essential nuclease-helicase conserved throughout eukaryotic organisms with an impressive repertoire of DNA maintenance activity (1). Due to its major roles in Okazaki fragment processing during DNA replication, double strand break repair, telomere maintenance, and mitochondrial DNA maintenance, it is not surprising that both Dna2 upregulation and mutation have been observed in various types of cancer (2,3). Despite the fact that both the helicase and nuclease domains are equally conserved in evolution, the nuclease function of Dna2 dominates on most substrates and the functional switch between helicase and nuclease activity is regulated by an unclear mechanism, which may involve the 4Fe-4S cluster (4). The goal of this work is to elucidate the effect of the redox properties of the 4Fe-4S cluster on the regulation of helicase and nuclease activity in S. cerevisiae Dna2. We have overexpressed wild type S. cerevisiae Dna2 and nuclease-dead Dna2 E675A in E. coli and purified the recombinant enzymes with sufficient yield and purity. Using multiplexed DNA-modified Au electrodes, we have characterized the DNA-bound electron transfer activity of isolated Dna2. Cyclic voltammetry on DNA-modified electrodes showed reversible DNA-bound redox activity centered at ~90 mV vs. NHE. This redox potential is similar to that of other DNA- binding 4Fe-4S enzymes that we have studied and supports a DNA-mediated redox signaling role for the 4Fe-4S cofactor in regulating Dna2 activity. The redox potential of Dna2 unbound to DNA has been determined by protein film electrochemistry to be ~150 mV vs. NHE, 60 mV greater than that of the DNA-bound protein. This positive shift in redox potential suggests that DNA binding activates the 4Fe-4S cluster toward oxidation and is consistent with what has been observed for other DNA-binding proteins containing a 4Fe-4S cluster (5). Bulk oxidation and reduction of Dna2 on DNA-modified electrodes has been performed revealing an increase in signal after oxidation and decrease upon reduction, consistent with an increase in DNA-binding affinity upon oxidation of the 4Fe-4S cluster to the 3+ state. This study illuminates the biochemical role played by the 4Fe-4S cluster and provides insight into the mechanism through which Dna2 promotes genome stability.

References
    1. Budd, M. E. & Campbell, J. L. A yeast gene required for DNA replication encodes a protein with homology to DNA helicases. Proc Natl Acad Sci USA 1995, 92, 7642–7646

    
 2. Lee, S. H., Kim, Y. R., Yoo, N. J. & Lee, S. H. Mutation and Expression of DNA2 Gene in Gastric and Colorectal Carcinomas. Korean J Pathol 2010, 44, 354-359

    
 3. Strauss, C. et al. The DNA2 nuclease/helicase is an estrogen-dependent gene mutated in breast and ovarian cancers. Oncotarget 2014, 5, 9396–9409


    
 4. Pinto, C., Kasaciunaite, K., Seidel, R. & Cejka, P. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases. Elife 2016, 5

    
 5. Bartels, P. L., Zhou, A., Arnold, A. R., Nuñez, N. N., Crespilho, F. N., David, S. S. and Barton, J. K. Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA. Langmuir: the ACS Journal of Surfaces and Colloids 2017, 33(10), 2523–2530

Siobhán Gaustad MacArdle,
Jacqueline Barton

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Siobhan, a doctoral student of Professor Jackie Barton, Caltech, will deliver a short oral from the platform

Department of Chemistry
Chemical Engineering
California Institute of Technology
1200 E. California Blvd.
Pasadena, CA 91126

Email: siobhan@caltech.edu