First Steps in Human DNA Replication Dance Captured at Atomic Resolution

We’d never be able to get it right. The process is so complex, calling for the orchestration of over a hundred highly specialized proteins, each of which must play its part at precisely the right moment and in the proper spatial orientation. It has often been compared to an exquisitely choreographed molecular dance. The smallest errors, left uncorrected, can have deadly consequences. It is essential that the genome replicates once and only once during each cell-division cycle.

In the journal eLife, a team of biologists co-led by Cold Spring Harbor Laboratory (CSHL) Professor and HHMI Investigator Leemor Joshua-Tor and CSHL President and Professor Bruce Stillman has published pictures at atomic resolution of the multi-part protein complex that performs the very first step in the genome-replication dance. The images of the human version of this complex, called ORC—for origin recognition complex—show it in its active mode.

A view of the human ORC protein, showing 4 of 5 ORC subunits. A missing subunit makes it possible to see how DNA (grey) fits through the “ring” as a bolt fits snugly through the center of a nut
 Credit:  Cold Spring Harbor Laboratory

ORC complexes self-assemble in the cell nucleus and bind at specific spots called start sites or origins along the double helix in chromosomes. In human cells, ORC assembles at literally thousands of origin sites across the entire genome, to form an initial configuration called the pre-replication complex, or pre-RC. Once assembled, these pre-RCs are like highly prepared Olympic swimmers standing on the starting block, waiting for the signal to start the race.

Like fast swimmers, each complex needs fuel — in ORC, to recruit its “motor” that opens the two strands of the double helix. ORC’s fuel is ATP, or adenosine triphosphate. In ORC’s active phase, the researchers showed that a subassembly containing ORC subunits 1,2,3,4 and 5 engages multiple ATP molecules and forms a partial ring-shaped complex. ATP is also used to recruit another protein component called CDC6, transforming the open ring into a closed ring. By this time, the multi-part assembly has engaged and bound to the double helix, which passes through the center of the ring like a bolt through the center of a nut. The ring is designed to fit the DNA snugly.

ORC was discovered in 1991 in Stillman’s lab. “Bruce made his initial discovery of ORC in yeast,” notes Joshua-Tor, “and we’ve known for many years that there are great structural similarities in the ORC complex across vastly different organisms, from yeast to flies to mammals. Our new pictures help account for what appeared to be differences in shape between ORC in fruit flies and in humans.”

The human ORC complex when fully assembled is ring-shaped, as shown in these images at atomic resolution, secured via x-ray crystallography and cryo-electron microscopy.