Abstract:
DNA replication in eukaryotes begins with the assembly of two hexameric helicases in a head-to-head orientation at specific DNA locations known as origins. These double hexameric helicases unwind the origin DNA and establish two opposing replication forks, providing single-stranded templates for replicative DNA polymerases. To explore the structural basis of this process, we used Cryo-EM to study a model AAA+ helicase, the SV40 LTag, which mimics key aspects of eukaryotic replication machinery. Our evidence suggests that the helicases bind and locally melt the two halves of the origin independently and symmetrically. The melting process involves at least five base pairs at the EP- and AT-rich regions of the origin and is facilitated by sequential assembly of LTag subunits, ATP binding, and a rearrangement of the DNA-binding loops within the helicase chamber from a planar to a staircase configuration induced by their interaction with the DNA tracking strand. These factors aid in strand separation within the helicase core, enabling each hexamer to adopt a configuration primed for ATP-driven translocation. Based on these findings, we propose a mechanism for subsequent DNA shearing and helicase uncoupling, leading to the formation of bidirectional replication forks. The high structural conservation of core helicase regions implies that the mechanism uncovered is applicable to hexameric helicases across various domains of life.

Bio:
Ammar Dan Azumi is a PhD candidate from Bioscience program at KAUST under Prof. Alfredo De Biasio. His research employs cryo-electron microscopy to understand how eukaryotic DNA replication initiates, with a focus on the SV40 Large T antigen helicase and its coordination with host polymerases.