Mechanism of subunit coordination of an AAA+ hexameric molecular nanomachine

Simian virus 40 large tumor antigen (LT) is both a potent oncogenic protein and an efficient hexameric nanomachine that harnesses the energy from ATP binding/hydrolysis to melt origin DNA and unwind replication forks. However, how the six subunits of the helicase motor coordinate during ATP hydrolysis and DNA unwinding/translocation is unresolved. Here we investigated the subunit coordination mechanisms "binomial distribution mutant doping" experiments in the presence of various DNA substrates. For ATP hydrolysis, we observed multiple coordination modes, ranging from random and semi-random, and semi-coordinated modes, depending on which type of DNA is present. For DNA unwinding, however, the results indicated a fully-coordinated mode for the natural origin-containing duplex DNA, but a semi-coordinated mode for a pre-existing fork-DNA, providing direct evidence for LT to use potentially different mechanisms to unwind the two types of substrates. The results of this study provide insights into DNA translocation and unwinding mechanisms for LT hexameric biomotor. From the clinical editor: The study describes the subunit coordination of simian virus 40 large tumor antigen (LT) showing that multiple mechanisms exist that handle the specific needs of different stages of DNA replication.