|   Wednesday, July 29, 2026 14h GMT Convert to your time zone   Structural Basis of Bidirectional Fork Initiation by SV40 LTag Helicase |
| Dr. Ammar Danazumi Biomedical Sciences Division King Abdullah University of Science and Technology |

| Abstract   In eukaryotes, DNA replication is initiated when two ring-shaped hexameric helicases load at defined DNA sites, called origins, and engage one another in a head-to-head arrangement. Acting as a double hexamer, they open the origin duplex and set up two divergent replication forks, exposing the single-stranded templates that replicative polymerases require.    To define the structural events underlying this initiation step, we used cryo-electron microscopy (cryo-EM), taking the SV40 Large Tumour Antigen as a tractable AAA+ helicase that recapitulates central features of the eukaryotic replication machinery. We found that each helicase engages and melts its own half of the origin in a manner that is both independent and symmetric. Local melting spans a minimum of five base pairs across the early-palindrome and AT-rich elements of the origin and depends on the stepwise addition of LTag subunits, on ATP binding, and on a reorganisation of the helicase’s DNA-binding loops, which switch from a planar to a staircase-like spiral arrangement as they contact the DNA tracking strand.    Together, these events drive strand separation inside the helicase channel and leave each hexamer poised for ATP-powered translocation. We propose a model in which the duplex is subsequently sheared and the two helicases decouple, yielding bidirectional replication forks. The high conservation of the hexameric helicase core points to a general mechanism that extends from SV40 virus to the replicative helicases of eukaryotes. |