Adenosine triphosphate (ATP) hydrolysis is the main cellular energy source to drive biochemical reactions that are otherwise energetically unfavourable. DNA replication is a process that is thought to rely on ATP hydrolysis to promote the chemical and mechanical activities necessary to progress the replication fork. Specifically, the DnaB helicase is known to unwind DNA in an ATP-dependent manner. However, ensemble biochemical assays do not separate helicase loading from replisome activity and thus do not separately test the ATP dependence of elongation.
Using a flow-cell based single-molecule rolling-circle replication assay, we are able to separate helicase loading and replication elongation into discrete steps and test the dependence of replisome-mediated elongation on ATP. In real time, we visualise DNA replication by individual replisomes. Surprisingly, we see efficient DNA replication in the absence of ATP or any other rNTP. This result suggests that DnaB does not require ATP hydrolysis for unwinding in the context of the replisome. Next, we interrogated the replisomal mechanisms that allow the DnaB helicase to function without ATP. We establish that nucleotide incorporation by the leading-strand polymerase, not ATP hydrolysis by the helicase, is the main motor driving the replication process.
Notably, in the absence of DnaB, polymerase mediated unwinding and synthesis occurs at much lower rates, indicating the critical role that DnaB still plays during replication. We propose that DnaB provides the platform that dictates the architecture of the E. coli replisome to facilitate strand separation. While we show that rNTPs are not required during unimpeded replication, it is tempting to speculate that the helicase could use the energy from ATP hydrolysis to sustain replication through secondary structures or during roadblock bypass.