Substrate Binding Regulates Redox Signaling in Human DNA Primase.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• DNA replication requires DNA primase to synthesize RNA primers.
• Primase is a heterodimer (p48/p58) with distinct functional domains.
• The [4Fe4S] cluster in p58 is crucial for enzyme activity.

Purpose of the Study:

• To investigate the redox switching properties of the [4Fe4S] cluster in the intact DNA primase heterodimer.
• To understand how DNA and NTP binding influence the cluster's electrochemical signaling.
• To explore the relationship between redox properties and primase catalytic activity.

Main Methods:

• DNA electrochemistry was employed to study the full p48/p58 primase heterodimer.
• Electrochemical measurements were performed to analyze the redox potential of the [4Fe4S] cluster.
• Enzyme activity assays were conducted to assess the impact of NTP binding on catalysis.

Main Results:

• Robust redox signaling of the [4Fe4S] cluster required simultaneous binding of both DNA and NTPs.
• NTP binding shifted the cluster's redox potential to a physiological range (~160 mV vs NHE).
• Preloading primase with NTPs enhanced catalytic activity, indicating a more active conformation.

Conclusions:

• Substrate binding (DNA and NTPs) and conformational changes regulate the [4Fe4S] cluster's redox signaling.
• Electrostatic interactions and subunit alignment influence the cluster's environment and signaling.
• These findings are relevant for understanding signaling in multisubunit DNA-processing enzymes with [4Fe4S] clusters.