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Cracking Open a Molecular Calculator: DNA Charge Transport and Primase

Holt, Marilyn E. and O'Brien, Elizabeth and Salay, Lauren and Thompson, Matthew and Ehlinger, Aaron and Barton, Jacqueline and Chazin, Walter (2016) Cracking Open a Molecular Calculator: DNA Charge Transport and Primase. Biophysical Journal, 110 (3). 21A. ISSN 0006-3495. doi:10.1016/j.bpj.2015.11.172.

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De novo initiation of DNA synthesis by DNA polymerases requires formation of an initial “primer” on the template. In eukaryotes, this function is provided by a distinct DNA primase. Primase, a DNA-dependent RNA polymerase, generates short (8-12 nt) RNA primers on ssDNA templates. These initial RNA primers are handed off to DNA polymerase α (pol α), which extends the primer by ∼20 nts. This primed RNA/DNA substrate is then handed off to processive polymerases ε or δ, which synthesize the vast majority of new DNA on the leading and lagging strands. X-ray crystal structures of human primase have been used to propose models for the catalytic mechanism. However, the molecular basis for primer counting and handoff remains unclear. Primase contains a 4Fe-4S cluster in the p58C domain critical to pol-prim loading and activity, but its specific function is not known. We are investigating the hypothesis that the function of the cluster is to drive primer length counting. Well-stacked duplex DNA has been shown capable of transporting charge over long distances, and it has been proposed this phenomenon enables communication between Fe-S cluster-containing proteins. In this study, we show the 4Fe-4S cluster in wild-type p58C is capable of mediating charge transport (CT) with DNA. DNA-modified electrodes were used to characterize the electrochemistry of p58C. Based on previously determined WT structures, we identified a possible pathway from the cluster to the DNA binding site in p58C and designed mutants of key residues in this pathway. Mutation of Tyr345 to Phe greatly diminishes DNA CT. Primase activity assays with WT and mutant proteins revealed substantial differences in product length distribution. These data suggest charge transport is involved in primer length counting and may signal for primer template handoff to pol α.

Item Type:Article
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URLURL TypeDescription
Holt, Marilyn E.0000-0002-3164-869X
Barton, Jacqueline0000-0001-9883-1600
Chazin, Walter0000-0002-2180-0790
Additional Information:© 2016 Biophysical Society. Published by Elsevier Inc.
Issue or Number:3
Record Number:CaltechAUTHORS:20160603-153447332
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Official Citation:Marilyn E. Holt, Elizabeth O'Brien, Lauren Salay, Matthew Thompson, Aaron Ehlinger, Jacqueline Barton, Walter Chazin, Cracking Open a Molecular Calculator: DNA Charge Transport and Primase, Biophysical Journal, Volume 110, Issue 3, Supplement 1, 16 February 2016, Page 21a, ISSN 0006-3495, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67660
Deposited By: Tony Diaz
Deposited On:04 Jun 2016 17:41
Last Modified:11 Nov 2021 03:52

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