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Functional role for the [4Fe4S] cluster in human DNA primase as a redox switch using DNA charge transport

O'Brien, Elizabeth and Holt, Marilyn and Thompson, Matthew K. and Salay, Lauren E. and Ehlinger, Aaron C. and Chazin, Walter J. and Barton, Jacqueline K. (2017) Functional role for the [4Fe4S] cluster in human DNA primase as a redox switch using DNA charge transport. In: 254th American Chemical Society National Meeting & Exposition, August 20-24, 2017, Washington, DC. https://resolver.caltech.edu/CaltechAUTHORS:20170911-143259871

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Abstract

DNA-mediated charge transport (DNA CT) provides an avenue for long-range, rapid signaling between redox-active moieties coupled into duplex DNA. Several DNA-processing enzymes have moreover been shown to contain [4Fe4S] clusters, common redox cofactors. Eukaryotic DNA primase, the heterodimeric enzyme responsible for initiating DNA replication, contains a [4Fe4S] cluster in the C-terminal domain of the large subunit (p58C). Primase synthesizes a short RNA primer on a single-stranded DNA template and subsequently hands this template off to DNA polymerase α, another [4Fe4S] protein, through a mechanism which is unclear. Here we show electrochem. evidence that the [4Fe4S] cluster in the p58C domain of human DNA primase performs redox chem. on DNA, cycling reversibly between a tightly DNA-bound, oxidized [4Fe4S]^(3+) state, and a loosely assocd., reduced [4Fe4S]^(2+) state. We demonstrate through structural, biochem., and electrochem. comparisons of wild type and mutant p58C that the redox switch is mediated by a pathway of tyrosine residues between the cluster and bound DNA. Charge transfer pathway mutations in full-length primase, addnl., abrogate initiation of primer synthesis on single-stranded DNA but do not affect nucleotide polymn. We further compare primer elongation on a well-matched and mismatched DNA template, showing that a single-base mismatch in the nascent primer inhibits primase termination. Thus primer termination appears to be gated by mismatch-sensitive DNA charge transport. Based on our exptl. evidence, we propose a model in which electron transfer between [4Fe4S] clusters, gated by DNA-mediated charge transport, regulates DNA binding and substrate handoff between primase and polymerase α to begin replication.


Item Type:Conference or Workshop Item (Paper)
Related URLs:
URLURL TypeDescription
https://www.acs.org/content/acs/en/meetings/fall-2017.htmlOrganizationConference Website
ORCID:
AuthorORCID
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 2017 American Chemical Society.
Record Number:CaltechAUTHORS:20170911-143259871
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170911-143259871
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:81311
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:11 Sep 2017 21:40
Last Modified:03 Oct 2019 18:41

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