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Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase

Salay, Lauren E. and Blee, Alexandra M. and Raza, Md Kausar and Gallagher, Kaitlyn S. and Chen, Huiqing and Dorfeuille, Andrew J. and Barton, Jacqueline K. and Chazin, Walter J. (2022) Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase. Biochemistry, 61 (11). pp. 1113-1123. ISSN 0006-2960. doi:10.1021/acs.biochem.2c00100. https://resolver.caltech.edu/CaltechAUTHORS:20220629-707785000

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Abstract

DNA synthesis during replication begins with the generation of an ∼10-nucleotide primer by DNA primase. Primase contains a redox-active 4Fe-4S cluster in the C-terminal domain of the p58 subunit (p58C). The redox state of this 4Fe-4S cluster can be modulated via the transport of charge through the protein and the DNA substrate (redox switching); changes in the redox state of the cluster alter the ability of p58C to associate with its substrate. The efficiency of redox switching in p58C can be altered by mutating tyrosine residues that bridge the 4Fe-4S cluster and the nucleic acid binding site. Here, we report the effects of mutating bridging tyrosines to phenylalanines in yeast p58C. High-resolution crystal structures show that these mutations, even with six tyrosines simultaneously mutated, do not perturb the three-dimensional structure of the protein. In contrast, measurements of the electrochemical properties on DNA-modified electrodes of p58C containing multiple tyrosine to phenylalanine mutations reveal deficiencies in their ability to engage in DNA charge transport. Significantly, this loss of electrochemical activity correlates with decreased primase activity. While single-site mutants showed modest decreases in activity compared to that of the wild-type primase, the protein containing six mutations exhibited a 10-fold or greater decrease. Thus, many possible tyrosine-mediated pathways for charge transport in yeast p58C exist, but inhibiting these pathways together diminishes the ability of yeast primase to generate primers. These results support a model in which redox switching is essential for primase activity.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/acs.biochem.2c00100DOIArticle
http://doi.org/10.2210/pdb7TL2/pdbDOIData
http://doi.org/10.2210/pdb7TL3/pdbDOIData
http://doi.org/10.2210/pdb7TL4/pdbDOIData
ORCID:
AuthorORCID
Salay, Lauren E.0000-0001-8513-1891
Blee, Alexandra M.0000-0003-4959-5466
Raza, Md Kausar0000-0002-9175-198X
Barton, Jacqueline K.0000-0001-9883-1600
Chazin, Walter J.0000-0002-2180-0790
Additional Information:© 2022 American Chemical Society. Received: February 18, 2022; Revised: May 12, 2022; Published: May 26, 2022. Accession Codes: Crystal structures are deposited as PDB entries 7TL2, 7TL3, and 7TL4. DNA and protein sequences of the yeast PRI1 and PRI2 genes can be accessed as UniProt entries P10363 and P20457, respectively. The authors thank Professor Jason Slinker for providing multiplexed chips. The authors also acknowledge Professor Mike Hill, Dr. Rebekah Silva, and Dr. Adela Nano for help with initial electrochemistry training and valuable discussions. The authors also thank Dr. Paula H. Oyala for performing EPR data collection and for valuable discussions. The authors acknowledge the LS-CAT beamline scientists and staff. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corp. and the Michigan Technology Tri-Corridor (Grant 085P1000817). Author Contributions: L.E.S., A.M.B., and M.K.R. contributed equally to this work. This research was supported by National Institutes of Health Grants R35 GM118089 (W.J.C.), R01 GM126904 (J.K.B.), T32 GM80320 (L.E.S.), and T32 CA009582 (A.M.B.). Further funding was provided by the Vanderbilt University Leadership Alliance FYRE program to A.J.D. and the Jiangsu University Study-Abroad Program to H.C. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
Department of Energy (DOE)DE-AC02-06CH11357
Michigan Technology Tri-Corridor085P1000817
NIHR35 GM118089
NIHR01 GM126904
NIH Predoctoral FellowshipT32 GM80320
NIH Predoctoral FellowshipT32 CA009582
Vanderbilt UniversityUNSPECIFIED
Jiangsu UniversityUNSPECIFIED
Subject Keywords:Cluster chemistry, Fungi, Genetics, Monomers, Peptides and proteins
Issue or Number:11
DOI:10.1021/acs.biochem.2c00100
Record Number:CaltechAUTHORS:20220629-707785000
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20220629-707785000
Official Citation:Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase. Lauren E. Salay, Alexandra M. Blee, Md Kausar Raza, Kaitlyn S. Gallagher, Huiqing Chen, Andrew J. Dorfeuille, Jacqueline K. Barton, and Walter J. Chazin. Biochemistry 2022 61 (11), 1113-1123; DOI: 10.1021/acs.biochem.2c00100
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:115301
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:30 Jun 2022 15:51
Last Modified:30 Jun 2022 15:51

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