Scaffolding for the DNA building site

Molecular engineers home in on DNA replication.

DNA lends itself to a supremely elegant replication mechanism but there are quite a few devils in the detail. The two most significant problems are the helicity and the fact that the two DNA strands have chemical polarities running in opposite directions. These complications require a lot of cutting and pasting during DNA synthesis. To guarantee that the components of the DNA synthesis apparatus stay in place during those operations, all types of organisms have a sliding clamp, a doughnut-shaped protein complex, which serves as a builder?s scaffold.

Another protein complex, known as the clamp loader, helps the cell get the DNA through the sliding clamp. US and Japanese researchers have now revealed detailed snapshots of the process by which the sliding clamp is loaded onto the DNA primed for replication.

Kosuke Morikawa?s group at the Biomolecular Engineering Institute in Osaka studied the clamp loading complex of the archaebacterium Pyrococcus furiosus, consisting of the DNA, the clamp (known as PCNA, for proliferating cell nuclear antigen), and the clamp loader (RFC, for replication factor C). Because the clamp-loading process consumes ATP, the researchers created a stalled version of the complex by offering a non-hydrolysable analogue of ATP and analysing the structure of this assembly by electron microscopy.

Meanwhile, John Kuriyan?s team at the University of California solved a crystal structure of the RFC?PCNA complex from yeast, and fitted the DNA by molecular modelling.

Although the systems studied come from fundamentally different domains of life, both structures share the essential features, both displaying a horse shoe (the clamp loader) stacked on top of a doughnut (the clamp).

The understanding emerging from these images is that the clamp loader prises open the ring of the clamp and allows the DNA to thread through the hole. This is apparently independent of energy supplies, as the ATP is only used for the release of the clamp loader.

Michael Gross