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UvrC Coordinates an O₂-Sensitive [4Fe4S] Cofactor

Silva, Rebekah M. B. and Grodick, Michael A. and Barton, Jacqueline K. (2020) UvrC Coordinates an O₂-Sensitive [4Fe4S] Cofactor. Journal of the American Chemical Society, 142 (25). pp. 10964-10977. ISSN 0002-7863. PMCID PMC7392197. https://resolver.caltech.edu/CaltechAUTHORS:20200615-113627458

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Abstract

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV–visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60–70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/jacs.0c01671DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392197PubMed CentralArticle
ORCID:
AuthorORCID
Silva, Rebekah M. B.0000-0002-9144-4939
Grodick, Michael A.0000-0001-6618-6731
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 2020 American Chemical Society. Received: February 11, 2020; Published: May 29, 2020. We are grateful to the NIH (RM35GM126904 to J.K.B.) and The Gordon and Betty Moore Foundation (to the Caltech Center for the Chemistry of Cellular Signaling) for their financial support. R.M.B.S. recognizes the National Science Foundation (NSF) for support through a Graduate Research Fellowship and the Center for Environmental Microbial Interactions (CEMI, Caltech) through a Pilot Grant. M.A.G. was an NIH predoctoral trainee (NIH/NRSA 5T32GM07616) and also a recipient of a CEMI Pilot Grant. The authors are especially grateful to the Rees group at Caltech, particularly Ailiena Maggiolo, for use of their vacuum manifold for anaerobic purification of UvrC. The authors also thank undergraduate researchers, Jenny He and Sirus Han, who contributed to early studies with cysteine mutants. This work was also facilitated by use of the Caltech EPR facility which is supported by the NSF (NSF-1531940) and the Dow Next Generation Educator Fund. In addition, we thank A. Boal for her early insights regarding UvrC and a possible association with a [4Fe4S] cluster. The authors declare no competing financial interest.
Group:Caltech Center for Environmental Microbial Interactions (CEMI)
Funders:
Funding AgencyGrant Number
NIHRM35GM126904
Gordon and Betty Moore FoundationUNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
NIH Predoctoral Fellowship5T32GM07616
NSFCHE-1531940
Dow Next Generation Educator FundUNSPECIFIED
Issue or Number:25
PubMed Central ID:PMC7392197
Record Number:CaltechAUTHORS:20200615-113627458
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200615-113627458
Official Citation:UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor. Rebekah M. B. Silva, Michael A. Grodick, and Jacqueline K. Barton. Journal of the American Chemical Society 2020 142 (25), 10964-10977 DOI: 10.1021/jacs.0c01671
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:103920
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 Jun 2020 18:48
Last Modified:31 Jul 2020 17:48

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