Richard R. Spinks, Aleksa Lakic, Céline Kelso, Slobodan Jergic, Olga Yurieva, Zhi‐Qiang Xu, Michael E. O’Donnell, Nicholas E. Dixon, Antoine M. van Oijen, Jacob S. Lewis, Lisanne M. Spenkelink
Adenosine triphosphate (ATP) hydrolysis is the main cellular source of energy used to drive biochemical reactions that are otherwise energetically unfavourable. The chemical energy stored in phosphoanhydride bonds is released upon hydrolysis of ATP to ADP and is used to drive mechanical work and conformational change. DNA replication is a canonical process in which the multi-enzyme replisome is thought to rely on ATP hydrolysis for its function. Here we show, through single-molecule visualisation of DNA replication by the Escherichia coli replisome, that the replicative DnaB helicase does not rely on hydrolysis of ATP in the context of the elongating replisome. Even in the presence of physiologically-relevant concentrations of ATP, dTTP is hydrolysed preferably. Finally, we show that the replicative helicases from S. cerevisiae, D. melanogaster, and Homo sapiens can also use dTTP to unwind DNA. Our observations suggest that replicative helicases across domains of life are 'flex-fuel' helicases.