Loader of the Rings in DNA
DNA clamp loader positions the DNA polymerase machinery
onto the DNA for replication. Chemistry professor John Kuriyan
and his colleagues recently solved the structure of the
yeast's complex. Image courtesy
on the image to see a larger version.
do DNA replication and a new bottle of wine have in common? According
to a new model proposed by chemistry professor John Kuriyan
and his colleagues, they both employ a corkscrew motif. Based
on the crystal structure of a donut-shaped ring complex that encircles
DNA during replication, the new model helps to explain how DNA
is snugly wrapped by the replication complex as DNA is duplicated.
details of the structure and the model are reported in the June
17 edition of Nature by Kuriyan, postdoctoral fellow Gregory
Bowman and Mike O'Donnell, who is at Rockefeller University.
first glance, DNA replication seems simple: as the double helix
is unwound into two single strands, the principle of complementarity
dictates what base is added at each position of the two newly
synthesized chains. However, the two template strands run in different
directions, but DNA polymerase, the protein complex that copies
DNA, works in only one direction. One strand, the leading strand,
is copied continuously while the other, the lagging strand, is
replicated in discontinuous loops, with each strand being handled
simultaneously by a polymerase machine containing a dozen or more
polymerases move remarkably fast, giving rise to many topological
issues, said Kuriyan. "If you scale this process up to our
level, it is comparable to a car moving at 350 mph while rotating
quickly around an axis. In bacteria, the polymerase spins around
the DNA at an amazing rate of 100 times per second."
way the cell deals with all of this twisting and turning is to
attach ring-shaped proteins, known as sliding clamps, to DNA.
The DNA polymerase is connected to the sliding clamp, and the
DNA is threaded through the ring. The polymerase can then detach
from the DNA but remain tethered through the clamps.
found that subunits in this complex interact to give a symmetric
spiral conformation with the same curvature as DNA," said
Bowman. "The protein is poised to encircle DNA's corkscrew
shape and load the clamp protein, which can slide down DNA and
position the polymerase machinery."
new crystal structure, the first from a eukaryotic source, included
both the DNA clamp and ATP, the common energy currency of the
cell. The clamp loader binds to the clamp and places it onto double-stranded
DNA. "The two general functions of the clamp loader are to
open, load and unload the clamp, and to target the clamp to the
DNA as it becomes unwound for replication," said Kuriyan.
With the bacterial clamp loader structure in hand, scientists
knew what it looked like, but for loading to occur, ATP must be
new structure also provided insight into how the ATPase activity
of the clamp loader is triggered, leading to the loading of the
clamp and the dissociation of the loader. "It was known that
DNA binding triggers the ATPase of the clamp loader, but it was
not clear how," said Bowman. "From the model, we see
that the DNA itself, fitting cleanly inside the clamp loader,
may very well trigger the activity directly."
polymerase in charge of replicating the lagging strand must hop
from one completed fragment to the next start site, called a primer-template
junction. By specifically placing clamps onto these junctions,
the clamp-loader effectively provides a landing pad that helps
to target the polymerase for the next round of synthesis. How
the clamp-loader recognized these junctions has been a mystery,
but may very well be answered by the new structure. The primer-template
junctions are short stretches of double-stranded DNA, somewhat
like a stiff rod, that are flanked by the much more flexible single-stranded
DNA. "The spiraling of the subunits forms a pocket on the
underside of the clamp-loader which appears well suited to fit
both the more rigid double helix, and a single-stranded extension,"
said Bowman. "In short, this pocket looks like an ideal binding
site for a primer-template junction."
group was the first to solve the structure of the ring-shaped
clamp protein more than a decade ago, and in 2002 published the
structure of the bacterial clamp-loader assembly, the five-subunit
machine that loads the clamps onto DNA. This clamp-loader structure
provided a good blueprint for the complex's architecture, but
many details still needed to be filled in. "X-ray crystallography
takes a snapshot of the protein complex and its components, a
static picture of a dynamic process," said Bowman, "but
with enough pictures, the movement and interactions in the complex
can be seen." One analogy is a child's drawings of a dinosaur
battle on corner of the pages in a book that, when flipped through
quickly, becomes a rudimentary movie.
"The first structure was a picture of the complex in the
'off' state, and this is a snapshot of the activated state,"
structure is also the first from the AAA+ family to be solved
in complex with a binding partner. The AAA+ superfamily proteins
(ATPase Associated with various cellular Activities) have diverse
cellular functions, including membrane fusion and protein degradation,
but they share the ability to bind to their target and somehow
change the structure. So far, the particular spiral seen in this
structure is quite different than other AAA+ proteins, which have
a more flattened disk-like shape. Thus, while other AAA+ proteins
perform very different tasks in the cell, this X-ray snapshot
of the clamp-loader may also indicate something about how this
family of machines jostle about in their daily routines.