Faithful duplication of the genome is crucial for proper cell division and thus the genetic integrity of all organisms. Errors in DNA replication can lead to genomic instability, a characteristic feature of most cancer cells. A key component of the replication machinery (replisome) is the
DNA helicase, which separates the double helix into its component strands so they can be copied.In this talk, I will discuss our efforts to decipher the molecular mechanisms of two helicases: the MCM2-7 complex that acts as the replicative helicase in eukaryotes, and large T antigen (T-ag), the replicative helicase of Simian Virus 40 that serves as a paradigm for eukaryotic replication. Despite decades of study, the basic mechanisms by which these helicases unwind DNA at the replication fork have been controversial. Both enzymes were proposed to function as a physically coupled pair of hexamers that pumps double-stranded DNA through the helicase central channel, splitting the duplex apart when it emerges from the protein complex. Others challenged this view and proposed that they function as single hexamers that encircle and translocate along a single strand and unwind DNA by excluding the other strand from the central channel (“steric exclusion”). Determining which model is correct has profound implications for the spatial organization and inner workings of the replisome. However, due to limitations inherent to conventional biochemical approaches, it has been difficult to answer which model applies to MCM2-7 and T-ag. I developed novel methodologies that combine single-molecule imaging and DNA nanomanipulation in extract-based systems. These tools, together with conventional bulk assays, not only differentiated between different models of unwinding by MCM2-7 and T-ag, but also led to a new model regarding how T-ag deals with replication barriers. In the future, I will study eukaryotic DNA replication at the single-molecule level that will lend important new insights into the mechanism and dynamics of the complex molecular machines that carry out genome duplication.