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Published September 13, 2017 | Accepted Version + Supplemental Material
Journal Article Open

The Oxidation State of [4Fe4S] Clusters Modulates the DNA-Binding Affinity of DNA Repair Proteins


A central question important to understanding DNA repair is how certain proteins are able to search for, detect, and fix DNA damage on a biologically relevant time scale. A feature of many base excision repair proteins is that they contain [4Fe4S] clusters that may aid their search for lesions. In this paper, we establish the importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process. We utilize DNA-modified electrodes to generate repair proteins with [4Fe4S] clusters in the 2+ and 3+ states by bulk electrolysis under an O_2-free atmosphere. Anaerobic microscale thermophoresis results indicate that proteins carrying [4Fe4S]^(3+) clusters bind to DNA 550 times more tightly than those with [4Fe4S]^(2+) clusters. The measured increase in DNA-binding affinity matches the calculated affinity change associated with the redox potential shift observed for [4Fe4S] cluster proteins upon binding to DNA. We further devise an electrostatic model that shows this change in DNA-binding affinity of these proteins can be fully explained by the differences in electrostatic interactions between DNA and the [4Fe4S] cluster in the reduced versus oxidized state. We then utilize atomic force microscopy (AFM) to demonstrate that the redox state of the [4Fe4S] clusters regulates the ability of two DNA repair proteins, Endonuclease III and DinG, to bind preferentially to DNA duplexes containing a single site of DNA damage (here a base mismatch) which inhibits DNA charge transport. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process.

Additional Information

© 2017 American Chemical Society. Received: July 13, 2017; Published: August 17, 2017. We gratefully recognize the NIH (GM61077) for financial support. E.C.M.T. appreciates the Croucher Foundation for a postdoctoral fellowship. T.J.Z. is an NSF fellow (DGE-1144469). E.C.M.T. also thanks Zhou for assisting in the protein purification process and Dr. Deng for providing Au working electrodes. We are also grateful to the Caltech Center for the Chemistry of Cellular Signaling for instrumentation. The authors declare no competing financial interest.

Attached Files

Accepted Version - nihms957091.pdf

Supplemental Material - ja7b07230_si_001.pdf


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August 19, 2023
October 17, 2023