Hexameric helicase G40P unwinds DNA in single base pair steps

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Replicative helicases are crucial hexameric motors for DNA unwinding.
• The precise chemo-mechanical mechanisms of helicase translocation and DNA unwinding remain incompletely understood.

Purpose of the Study:

• To investigate the DNA unwinding mechanism of the DnaB-family helicase G40P at single-molecule resolution.
• To elucidate the role of ATP hydrolysis and subunit coordination in G40P helicase activity.
• To determine the effect of the primase DnaG on helicase translocation and stability.

Main Methods:

• Utilized a single-molecule fluorescence assay with single base pair resolution to monitor G40P DNA unwinding.
• Employed ATP analogs (ATPγS) to probe the coordination of ATP hydrolysis among subunits.
• Investigated the interaction between G40P and DnaG during DNA unwinding.

Main Results:

• G40P exhibits weak helicase activity, stalling at GC base pairs and unwinding DNA with a single base pair step size.
• ATPγS binding indicated highly coordinated ATP hydrolysis across the six subunits, capable of stalling unwinding.
• Frequent helicase slippage was observed, which was completely abolished by the presence of DnaG.

Conclusions:

• G40P's DNA unwinding mechanism is characterized by single base pair steps and susceptibility to slippage.
• Coordinated ATP hydrolysis is essential for the function of this hexameric helicase.
• DnaG plays a critical role in stabilizing G40P translocation and preventing slippage, suggesting intricate regulation in DNA replication.