Unlike a photocopier, the protein complex that helps initiate DNA replication lacks an owner’s manual. Consequently, this complex, which is called the origin recognition complex (ORC), has features that are still mysterious. Since scientists lack anything as convenient as a “quick start” guide, they have to muddle through as best they can. Instead of pressing buttons and checking the paper tray, scientists crystallize proteins, zap them with X-rays, and extract structural information from the resulting X-ray diffraction patterns.

In the case of ORC, scientists at Johns Hopkins University determined a crystal structure to a high degree of resolution, on the scale of billionths of an inch. The structure highlights a domain-swapped organization for ORC and captures the complex in an unanticipated, autoinhibited conformation. Surprisingly, ORC is not always “on.” This discovery is rather like pressing the copy button on a copier and finding that nothing will start—the power-save feature is engaged.

The extra level of control over the initiation of DNA copying was described in an article published March 11 in Nature. The article, entitled “Crystal structure of the eukaryotic origin recognition complex,” shows that a subunit of the six-piece ORC complex may shift its position, facilitating DNA copying, or not.

It was previously known that five of the six ORC subunits form a slightly opened ring and that the sixth subunit, Orc6, forms a tail. Mistakes in Orc6 cause assembly problems, which affect the function of the whole machine and contribute to a dwarfism disorder called Meier-Gorlin syndrome.

It has also been known that ORC interacts with the mini-chromosome maintenance 2–7 (MCM2–7) complex, which unwinds paired DNA strands, allowing them to be accessed and copied. MCM is a closed protein ring that must be opened up before it can encircle the long strands of DNA. The ring formed by MCM is cracked open by ORC so that MCM can fit around DNA and unwind it.

What’s new is that the recently determined crystal structure for ORC shows exactly where Orc6 connects to the ring of ORC.

“Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2–7 (MCM2–7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain [ATPases associated with a variety of cellular activities] disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel,” wrote the authors of the Nature article. “Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.”

In other words, the three-dimensional ORC model shows the existence of an unexpected regulatory mechanism. It was previously thought that ORC was always “on,” just not always present in the nucleus where it does its work. The model shows that it can exist in an inactive state, raising the question: How does it turn on and off?

“In hindsight, it’s not surprising that there is another level of regulation for ORC,” said James Berger, Ph.D., professor of biophysics and biophysical chemistry at Johns Hopkins. To explain, he added, “As soon as an egg cell is fertilized, it has to jump into action to create the embryo through multiple rounds of cell division, which first requires DNA replication. This inactive state might allow egg cells to stockpile ORC inside the nucleus so it’s available when needed.” Dr. Berger’s team plans to test this idea soon.

Previous articleWuXi Chooses Philadelphia for New Cell Therapy Manufacturing Facility
Next article23andMe Forms Therapeutics Group Led by Genentech Veteran