Study reveals how protein machinery binds and wraps DNA to start replication

March 6, 2012, Brookhaven National Laboratory
The DNA replication origin recognition complex (ORC) is a six-protein machine with a slightly twisted half-ring structure (yellow). ORC is proposed to wrap around and bend approximately 70 base pairs of double stranded DNA (red and blue). When a replication initiator Cdc6 (green) joins ORC, the partial ring is now complete and ready to load another protein onto the DNA. This last protein (not shown) is the enzyme that unwinds the double stranded DNA so each strand can be replicated.

( -- Before any cell - healthy or cancerous - can divide, it has to replicate its DNA. So scientists who want to know how normal cells work - and perhaps how to stop abnormal ones - are keen to understand this process. As a step toward that goal, scientists at the U.S. Department of Energy's Brookhaven National Laboratory and collaborators have deciphered molecular-level details of the complex choreography by which intricate cellular proteins recognize and bind to DNA to start the replication process. The study is published in the March 7, 2012, issue of the journal Structure.

"Every cell starts to replicate its genome at defined DNA sites called 'origins of ,'" said Huilin Li, a biologist at Brookhaven Lab and Stony Brook University, who led the study. "A cell finds those origins in its vast genome with a protein 'machine' called the 'origin recognition complex,' or ORC."

In a typical , comprised of several million base pairs - the "letters" of the - there is only one such origin. However, in more complex eukaryotic organisms, such as humans with a genome of 3.4 billion base pairs, there may be tens of thousands of replication origins so that can be carried out simultaneously at these sites to duplicate the genome in a reasonable time.

The goal of the current effort was to understand the first steps of the enormously complex task of duplicating a eukaryotic genome: how the ORC recognizes and binds to the origin DNA, and how the origin-bound ORC enables the attachment of additional protein machinery that unwinds the DNA double helix into two single strands in preparation for DNA copying.

"This level of detail on the shape of the origin recognition complex and its interaction with DNA provides insight into a key cellular process, the initiation of DNA replication," said Daniel Janes, who oversees DNA replication grants at the National Institutes of Health's National Institute of General Medical Sciences, which partially supported the work. "Because DNA replication is closely tied to cell division, a thorough understanding of the process may lead to new ways to fight the uncontrolled cell division that characterizes cancer."

Previous studies have looked at similar but simpler protein machines in bacteria and other prokaryotes. In , which have more complex cellular structure, the proteins themselves are more complicated - and larger - making them harder to study.

Some studies have looked at the eukaryotic proteins in relatively low resolution and in isolation. But none has taken a more detailed look and included how they bind with DNA - until now.

Jingchuan (Jim) Sun, a Brookhaven biologist who works with Li, used an imaging method known as cryo-electron microscopy to make higher resolution images of the eukaryotic ORC, in isolation, as it binds to DNA, and one step further in the process, when another protein unit binds to activate the entire structure. The research team used proteins from baker's yeast, which is a model system for eukaryotes.

The cryo-EM images produced a map, or outline, of the entire ORC structure as it changes during the activation process.

To explore the details of these changes, the scientists then turned to atomic-level x-ray crystal structures of small protein subunits that had been produced by other scientists. These subunit structures were made from prokaryotic cells known as archaea, which are evolutionarily related to eukaryotes, and so could serve as "stand-ins" for the eukaryotic subunit structures, which are still unknown.

By fitting these subunits into the cryo-EM maps, the scientists were able to propose a detailed structure and mechanism for how the ORC may work: In simplest form, it starts as a two-lobed, crescent-shaped protein complex that wraps around and bends the origin DNA along the interior curve of the crescent. Sequential binding of a "replication initiator" known as Cdc6 (for cycle 6) then induces a significant conformational change in the origin-bound ORC structure.

This structural conformation, the scientists say, is likely what opens the way for the attachment of the next piece of protein machinery essential to the DNA-replication process - the one that unwinds the two strands of the so that each can be copied.

"Our study is at a very basic level, trying to answer the fundamental biological questions about how this process works," said Li. "But our work has strong implications for health and disease, because unregulated or disregulated chromosomal duplication and uncontrolled cellular proliferation are the hallmarks of cancer. So understanding details of the mechanisms of DNA replication could potentially lead to new ways to fight cancer," he said.

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1 / 5 (5) Mar 06, 2012
These subunit structures were made from prokaryotic cells known as archaea, which are evolutionarily related to eukaryotes,

So they just could not leave out the reference to evolution.

Will someone please explain exactly how such an incredibly complex operation as replication can arise spontaneously from nothing? Remember that this is the process of life itself, way before such events as mutation and selection can even begin to work.
Whoever is able to demonstrate convincingly with peer approval just how such a process can arise from nothing more than unordered physical material, will receive a Nobel prize.
5 / 5 (3) Mar 06, 2012
Remember that this is the process of life itself, way before such events as mutation and selection can even begin to work.

Here's where you're absolutely wrong. Self replicating RNA is subject to mutation and natural selection and has sprung up as a promising hypothetical segway into life as we know it.


and you have self-replicating RNA-based protocells. This is precisely what I'm planning to write a 20-30 page grant proposal about next year.

Now, kevin, kindly fuck off.

Edit: the complex operations did not spontaneously arise from nothing, you moron. They arose from less complex steps, which were also subject to natural selection. Get your shitty blind watchmaker fallacy out of here. You've used it more than enough.
5 / 5 (1) Mar 06, 2012
Not really sure why it keeps getting rid of my other links... Anyway,


combine self-replicating RNA with naturally occurring self-replicating liposomes-


and you have self-replicating RNA-based protocells.
5 / 5 (1) Mar 06, 2012
@kevinrts: Read 'What is Life' (1944) by Erwin Schrödinger. Herein the great man engages in a discussion of negentropy and the concept of a complex molecule, specifically an aperiodic crystal, with the genetic code for living organisms. He then derives, from first principles of statistical & quantum mechanics, all the basic properties of the heredity molecule of life.

Coincidentally, both James Watson and Francis Crick report that they were heavily inspired by Schrödinger's speculations to actually research the gene. The rest, as you may already know, is history.
not rated yet Mar 06, 2012
kevinrts: in case you're thinking of bringing up Hoyle's "tornado in a junkyard" analogy, don't. Hoyle did not take into account the fact that various atoms and molecules have a *tendency* to link up. A tendency in a system is a bias -- like loaded dice. And when the dice are loaded, ordinary probabilistic computations are essentially useless.
"Aha, but where did this tendency come from?!" you might be thinking.
Good question. Maybe, if there is a Creator, *there* is the Divine Fingerprint -- "Let there be a Big Bang, bringing forth a cosmos with a certain set of basic constants, with the capability of generating life-bearing planets." ...And it came to pass.
Think about it.

In the meantime, don't bother bringing non-physical and non-mathematical arguments to a discussion that is based in physics and mathematics.

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