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Substrate Binding Regulates Redox Signaling in Human DNA Primase

O'Brien, Elizabeth and Holt, Marilyn E. and Salay, Lauren E. and Chazin, Walter J. and Barton, Jacqueline K. (2018) Substrate Binding Regulates Redox Signaling in Human DNA Primase. Journal of the American Chemical Society, 140 (49). pp. 17153-17162. ISSN 0002-7863. http://resolver.caltech.edu/CaltechAUTHORS:20181115-142248448

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Abstract

Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/jacs.8b09914DOIArticle
https://pubs.acs.org/doi/suppl/10.1021/jacs.8b09914PublisherSupporting Information
ORCID:
AuthorORCID
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 2018 American Chemical Society. Received: September 12, 2018; Published: November 15, 2018. This research was supported by National Institutes of Health grants R01 GM126904 (J.K.B.), R35 GM118089 (W.J.C.), T32 GM80320 (L.E.S. and M.E.H.) and T32GM07616 (E.O.B.) with additional support from the Moore Foundation (J.K.B.) and a Ralph M. Parsons fellowship (E.O.B.). The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
NIHR01 GM126904
NIHR35 GM118089
NIH Predoctoral FellowshipT32 GM80320
NIH Predoctoral FellowshipT32 GM07616
Gordon and Betty Moore FoundationUNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
Record Number:CaltechAUTHORS:20181115-142248448
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20181115-142248448
Official Citation:Substrate Binding Regulates Redox Signaling in Human DNA Primase. Elizabeth O’Brien, Marilyn E. Holt, Lauren E. Salay, Walter J. Chazin, and Jacqueline K. Barton. Journal of the American Chemical Society 2018 140 (49), 17153-17162. DOI: 10.1021/jacs.8b09914
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:90927
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:15 Nov 2018 22:52
Last Modified:13 Dec 2018 17:19

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