Position-Specific DNA Base Pair ‘BREATHING’ at the Replication Fork Junction Regulates Helicase Access

Abstract

We previously used near UV circular dichroism and fluorescence spectroscopy of DNA base analogues to measure position-specific DNA ‘breathing’ fluctuations at model replication fork DNA constructs (Jose et al., PNAS, 106, 4231, 2009). Building on that study we have now site-specifically inserted 2-aminopurine bases into such constructs on both sides of the fork junction as spectroscopic probes to monitor specific interactions with an unwinding helicase in ‘real time’. The tight-binding, stable and highly active primosome assembly of the bacteriophage T4 DNA replication system was used as the helicase, and was formed into an active unwinding initiation complex by assembling T4 helicase and primase subunits in a 6:1 subunit ratio in the presence of the non-hydrolyzable GTP-analogue, GTPγS. (The addition of hydrolyzable GTP to this initiation complex results in complete unwinding of the model replication fork.) The binding of this helicase initiation complex at the replication fork, primarily via backbone contacts with the leading strand, traps the first breathing base pair of the fork in an open conformation. The other base and base pair positions were examined to map the interactions of the bound helicase on both sides of the fork. These results suggest that this replication helicase unwinds DNA by a primarily ‘passive’ mechanism, with unwinding depending on DNA breathing.