Single-Molecule Insights Into the Dynamics of Replicative Helicases.

Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork.

DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies.

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment.

Single-Molecule Fluorescence Methods to Study Protein Exchange Kinetics in Supramolecular Complexes.

Recent single-molecule studies have demonstrated that the composition of multi-protein complexes can strike a balance between stability and dynamics. Proteins can dynamically exchange in and out of the complex depending on their concentration in solution. These exchange dynamics are a key determinant of the molecular pathways available to multi-protein complexes.

Design of DNA rolling-circle templates with controlled fork topology to study mechanisms of DNA replication.

Rolling-circle DNA amplification is a powerful tool employed in biotechnology to produce large from small amounts of DNA. This mode of DNA replication proceeds via a DNA topology that resembles a replication fork, thus also providing experimental access to the molecular mechanisms of DNA replication. However, conventional templates do not allow controlled access to multiple fork topologies, which is an important factor in mechanistic studies.