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Substrate Binding Regulates Redox Signaling in Human DNA Primase.

O'Brien E, Holt ME, Salay LE, Chazin WJ, Barton JK
J Am Chem Soc. 2018 140 (49): 17153-17162

PMID: 30433774 · PMCID: PMC6470046 · DOI:10.1021/jacs.8b09914

Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.

MeSH Terms (11)

DNA DNA Primase Electrochemical Techniques Humans Iron-Sulfur Proteins Nucleotides Oxidation-Reduction Protein Binding Protein Domains Transcription Elongation, Genetic Transcription Initiation, Genetic

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