A single helicase-binding domain of DnaG couples with hexameric helicase DnaB in Bacillus stearothermophilus.

In bacterial DNA replication, helicase DnaB and primase DnaG form the primosome. Helicase DnaB unwinds double-stranded DNA (dsDNA) to provide templates for DNA polymerase, whereas primase DnaG supplies RNA primers to DNA polymerase for the synthesis of Okazaki fragments. How primase DnaG coordinates with helicase DnaB at the DNA replication fork remains unclear. In this study, the interactions between the helicase-binding domain of DnaG (DnaG (HBD)) and DnaB hexamer were studied. A stable ternary complex of DnaB₆/dT₁₆/DnaG(HBD) from Bacillus stearothermophilus was prepared and the homogeneity of the DnaB₆/dT₁₆/DnaG(HBD) complex was verified by dynamic light scattering. The stoichiometry of DnaG(HBD) to process DnaB₆ was investigated by isothermal titration calorimetry. The results show that a single primase DnaG binds to DnaB₆ in the presence of single-stranded DNA. Based on these results, a model is proposed to explain how the primase DnaG couples with the processing DnaB₆ helicase during the Okazaki fragment synthesis cycle. These findings provide valuable insights into the coupling between dsDNA unwinding and RNA primer synthesis in DNA replication.