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 replication,’” 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 bacterial genome, comprised of several million base pairs—the
“letters” of the genetic code—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 DNA replication 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
protein machinery 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 eukaryotic organisms, 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.
Jingchuan (Jim) Sun and Huilin Li |
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 cell division 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 DNA double helix 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.
Additional
collaborators on this research include: Hironori Kawakami and Bruce
Stillman of Cold Spring Harbor Laboratory, and Juergen Zech and
Christian Speck of MRC Clinical Sciences Centre, Imperial College,
London.
The
research was funded by the U.S. National Institutes of Health, the UK
Medical Research Council, the Japan Society for the Promotion of
Science, and the Uehara Memorial Foundation. Cryo-EM was performed using
facilities at Brookhaven Lab supported by the DOE Office of Science.
Cdc6-Induced Conformational Changes in ORC Bound to Origin DNA Revealed by Cryo-Electron Microscopy
