There are many complex steps to the dance of DNA replication. And scientists must learn to sway along in order to understand how both healthy and cancerous cells divide.
Scientists at Brookhaven National Laboratory have begun to learn how to follow the complex molecular choreography by which intricate cellular proteins recognize and bind to DNA to start the replication process.
The replication process starts off the same way in every cell. In the cell's DNA, there are defined sites called the "origin of replication." The cell in which the DNA is housed uses a protein called the "origin recognition complex," or ORC, to begin replication. Unlike the bacterial genome, which has only one origin in its genome of several million base pairs, more complex eukaryotic organisms, such as humans, with a genome of 3.4 billion base pairs, have tens of thousands of replication origins so that DNA replication can be carried out simultaneously at these sites to duplicate the genome quickly.
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.
The Brookhaven scientists' goal in this study was to understand the first "moves" of eukaryotic genome replication. Specifically, how the ORC recognizes and binds to the origin DNA, and how the origin-bound ORC enables the attachment of additional protein machinery that unfurls the DNA double helix into two single strands in preparation for DNA copying. The work has strong implications for health and disease, because unregulated or disregulated chromosomal duplication and uncontrolled cellular proliferation are the hallmarks of cancer.
Whereas previous studies have approached these topics in simpler, prokaryotic organisms, this study is examining the eukaryotic protein's complex processes in detail.
Scientists used an imaging method known as cryo-electron microscopy to make high-resolution images of ORC in yeast, a model eukaryote, in isolation, as it binds to DNA, and later in the process when another protein unit binds to activate the entire structure.
This imaging produced a map of the entire ORC structure as it changes during the activation process.
The scientists then turned to atomic-level x-ray crystal structures of small protein subunits that had been produced by other scientists to explore the details of ORC's behavior. Basically, ORC is a two-lobed, crescent-shaped protein complex that wraps around and dips the DNA strand along the interior curve of the crescent. Sequential binding of a "replication initiator" then induces a significant shape change in the origin-bound ORC structure.
This structural alteration 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 DNA double helix so that each can be copied...so the replication tango can begin again.
This guest post was written by Natalie Crnosija, a science-writing intern at Brookhaven National Laboratory, and Karen McNulty Walsh, the Lab's principal science writer.