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Functional and structural similarity of human DNA primase [4Fe4S] cluster domain constructs

Holt, Marilyn E. and Salay, Lauren E. and O’Brien, Elizabeth and Barton, Jacqueline K. and Chazin, Walter J. (2018) Functional and structural similarity of human DNA primase [4Fe4S] cluster domain constructs. PLoS ONE, 13 (12). Art. No. e0209345. ISSN 1932-6203. PMCID PMC6298731. http://resolver.caltech.edu/CaltechAUTHORS:20190102-135257451

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Abstract

The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C construct revealed that its affinity for DNA is sensitive to the redox state of the [4Fe4S] cluster. Concerns about the validity of this conclusion have been raised, based in part on differences in X-ray crystal structures of the p58C_(272-464) construct used for that study and that of a N-terminally shifted p58C_(266-456) construct and consequently, an assumption that p58C_(272-464) has abnormal physical and functional properties. To address this controversy, a new p58C_(266-464) construct containing all residues was crystallized under the conditions previously used for crystallizing p58C_(272-464), and the solution structures of both constructs were assessed using circular dichroism and NMR spectroscopy. In the new crystal structure, p58C_(266-464) exhibits the same elements of secondary structure near the DNA binding site as observed in the crystal structure of p58C_(272-464). Moreover, in solution, circular dichroism and ^(15)N,^1H-heteronuclear single quantum coherence (HSQC) NMR spectra show there are no significant differences in the distribution of secondary structures or in the tertiary structure or the two constructs. To validate that the two constructs have the same functional properties, binding of a primed DNA template was measured using a fluorescence-based DNA binding assay, and the affinities for this substrate were the same (3.4 ± 0.5 μM and 2.7 ± 0.3 μM, respectively). The electrochemical properties of p58C_(266-464) were also measured and this p58C construct was able to engage in redox switching on DNA with the same efficiency as p58C_(272-464). Together, these results show that although p58C can be stabilized in different conformations in the crystalline state, in solution there is effectively no difference in the structure and functional properties of p58C constructs of different lengths.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1371/journal.pone.0209345DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298731/PubMed CentralArticle
ORCID:
AuthorORCID
Holt, Marilyn E.0000-0002-3164-869X
Barton, Jacqueline K.0000-0001-9883-1600
Chazin, Walter J.0000-0002-2180-0790
Additional Information:© 2018 Holt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: May 25, 2018; Accepted: November 14, 2018; Published: December 18, 2018. We thank Dr. Nicholas Reiter and Dr. William Martin for their guidance in generating and purifying the triphosphorylated RNA oligos. Software used in this project was curated by SBGrid [29]. This research was supported by National Institutes of Health (https://www.nih.gov/) grants R35 GM118089 (W.J.C.), R01 GM126904 (J.K.B.), T32 GM80320 (M.E.H, L.E.S.) and T32 GM07616 (E.O.B.) with additional support from the Moore Foundation (https://www.moore.org/) (J.K.B.) and a Ralph M. Parsons fellowship (http://rmpf.org/) (E.O.B.). 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 No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). NMR data collection was supported in part by instrumentation grants from the National Science Foundation (https://www.nsf.gov/) (0922862) and National Institutes of Health (S10 RR025677), and matching funds from Vanderbilt University (https://www.vanderbilt.edu/). Fluorescence data collection was supported in part by an instrumentation grant from the National Institutes of Health (S10 OD021483). Molecular graphics and analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: All crystallography files are available from the Protein Data Bank archive (accession number 6DHW). All other relevant data are within the paper and its Supporting Information files. The authors have declared that no competing interests exist.
Funders:
Funding AgencyGrant Number
NIHR35 GM118089
NIHR01 GM126904
NIH Predoctoral FellowshipT32 GM80320
NIH Predoctoral FellowshipT32 GM07616
Gordon and Betty Moore FoundationUNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
Department of Energy (DOE)DE-AC02-06CH11357
Michigan Economic Development CorporationUNSPECIFIED
Michigan Technology Tri-Corridor085P1000817
NSFDBI-0922862
NIHS10 RR025677
Vanderbilt UniversityUNSPECIFIED
NIHS10 OD021483
NIHP41 GM103311
PubMed Central ID:PMC6298731
Record Number:CaltechAUTHORS:20190102-135257451
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20190102-135257451
Official Citation:Holt, M. E., Salay, L. E., O'Brien, E., Barton, J. K., & Chazin, W. J. (2018). Functional and structural similarity of human DNA primase [4Fe4S] cluster domain constructs. PloS one, 13(12), e0209345. doi:10.1371/journal.pone.0209345
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:91997
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:03 Jan 2019 15:55
Last Modified:03 Jan 2019 15:55

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