Substrate Binding Regulates Redox Signaling in Human DNA Primase.

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.