Auto-regulation of the real-time kinetics of the human mitochondrial replicative helicase

Abstract

The human mitochondrial replicative helicase, Twinkle, is essential for the replication and integrity of mitochondrial DNA (mtDNA). Therefore, investigating the real-time kinetics of Twinkles activities and their regulation is crucial for understanding mtDNA maintenance. Here, we combine biochemical and single-molecule manipulation and visualization techniques to investigate the loading of Twinkle onto the DNA fork, its real-time DNA unwinding and rewinding kinetics, and the regulation of these processes by the amino- (N) and carboxyl- (C) terminal ends of the helicase and the mitochondrial SSB protein (mtSSB). We observed that Twinkle rapidly diffuses along dsDNA scanning for the DNA fork, where it establishes specific interactions that halt further diffusion. Our results show that during DNA unwinding, the real-time kinetics of the helicase are downregulated by interactions of its N-terminal Zinc-binding domain (ZBD) with DNA and the control of the ATPase activity by the C-terminal tail. We found that binding of mtSSB to DNA likely outcompetes the ZBD-DNA interactions, alleviating the down regulatory effects of this domain. Furthermore, we show that ZBD-DNA interactions, together with the ATP binding, also control the real-time kinetics of the DNA rewinding events that follow helicase stalling. Our findings reveal that ZBD and C-terminal tail play a major role in regulation of Twinkles real-time kinetics. Their interplay constitutes an auto-regulatory mechanism that may be crucial for coordination of the mtDNA maintenance activities of the helicase.