Tubulin-Like Proteins in Prokaryotic DNA Positioning.

A family of tubulin-related proteins (TubZs) has been identified in prokaryotes as being important for the inheritance of virulence plasmids of several pathogenic Bacilli and also being implicated in the lysogenic life cycle of several bacteriophages. Cell biological studies and reconstitution experiments revealed that TubZs function as prokaryotic cytomotive filaments, providing one-dimensional motive forces. Plasmid-borne TubZ filaments most likely transport plasmid centromeric complexes by depolymerisation, pulling on the plasmid DNA, in vitro. In contrast, phage-borne TubZ (PhuZ) pushes bacteriophage particles (virions) to mid cell by filament growth. Structural studies by both crystallography and electron cryo-microscopy of multiple proteins, both from the plasmid partitioning sub-group and the bacteriophage virion centring group of TubZ homologues, allow a detailed consideration of the structural phylogeny of the group as a whole, while complete structures of both crystallographic protofilaments at high resolution and fully polymerised filaments at intermediate resolution by cryo-EM have revealed details of the polymerisation behaviour of both TubZ sub-groups.

Helicase mediated vectorial folding of telomere G-quadruplex.

The G-rich single-stranded telomere overhang can self-fold into G-quadruplex (G4) structure both in vivo and in vitro. In somatic cells, telomeres shorten progressively due to the end-replication. In stem cells, however, telomeres are replenished by a special enzyme, telomerase which synthesizes single-stranded telomere overhang. The active extension by the telomerase releases G-rich overhang segmentally in 5' to 3' direction as the overhang folds into G4 structure after successive elongation. To replicate such vectorial G4 folding process, we employed a superhelicase, Rep-X to release the G-rich sequence gradually. Using single-molecule assay we demonstrated that the folded conformation achieved by the vectorial folding is inherently different from the post-folding where the entire overhang is allowed to fold at once. In addition, the vectorially folded overhangs are less stable and more accessible to a complementary C-rich strand and the telomere binding protein, POT1 compared to the post-folded state. The higher accessibility may have implications for the facile loading of shelterin proteins after DNA replication.