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Yeast require redox switching in DNA primase

O’Brien, Elizabeth and Salay, Lauren E. and Epum, Esther A. and Friedman, Katherine L. and Chazin, Walter J. and Barton, Jacqueline K. (2018) Yeast require redox switching in DNA primase. Proceedings of the National Academy of Sciences of the United States of America, 115 (52). pp. 13186-13191. ISSN 0027-8424. PMCID PMC6310810. http://resolver.caltech.edu/CaltechAUTHORS:20181213-083058525

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Abstract

Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S]^+ species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.1810715115DOIArticle
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1810715115/-/DCSupplementalPublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310810PubMed CentralArticle
ORCID:
AuthorORCID
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 2018 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved November 5, 2018 (received for review June 22, 2018). Published ahead of print December 12, 2018. We are grateful to the NIH for Operating Grants R01 GM126904 (to J.K.B.), R35 GM40120 (to W.J.C.), and R01 GM123292 (to K.L.F.), and Training Grants T32 GM07616 (to E.O.) and T32 GM08230 (to L.E.S.). We received additional support from the Moore Foundation (J.K.B.) and a Ralph M. Parsons Fellowship (to E.O.). Diffraction data were collected at the Advanced Photon Source, operated by the US Department of Energy (Contract DE-AC02-06CH11357). Author contributions: E.O., L.E.S., K.L.F., W.J.C., and J.K.B. designed research; E.O., L.E.S., and E.A.E. performed research; E.O., L.E.S., E.A.E., K.L.F., W.J.C., and J.K.B. analyzed data; and E.O. and J.K.B. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.rcsb.org [PDB ID codes: WT p58C (Saccharomyces cerevisiae), 6DI6; Y395F (S. cerevisiae), 6DTV; Y395L (S. cerevisiae), 6DU0; Y397F (S. cerevisiae), 6DTZ; and Y397L (S. cerevisiae), 6DI2]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1810715115/-/DCSupplemental.
Funders:
Funding AgencyGrant Number
NIHR01 GM126904
NIHR35 GM40120
NIHR01 GM123292
NIH Predoctoral FellowshipT32 GM07616
NIH Predoctoral FellowshipT32 GM08230
Gordon and Betty Moore FoundationUNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
Department of Energy (DOE)DE-AC02-06CH11357
Subject Keywords:DNA replication; DNA charge transport; iron–sulfur proteins
PubMed Central ID:PMC6310810
Record Number:CaltechAUTHORS:20181213-083058525
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20181213-083058525
Official Citation:Yeast require redox switching in DNA primase. Elizabeth O’Brien, Lauren E. Salay, Esther A. Epum, Katherine L. Friedman, Walter J. Chazin, Jacqueline K. Barton. Proceedings of the National Academy of Sciences Dec 2018, 115 (52) 13186-13191; DOI: 10.1073/pnas.1810715115
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:91752
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:13 Dec 2018 18:16
Last Modified:08 Jan 2019 19:47

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