August 09, 2001
Repair Protein Cradles Broken DNA
A space-filling model showing Ku bound to DNA. Two components of Ku are colored red (Ku70) and yellow (Ku80). DNA is depicted with one dark gray and one light gray strand.
Howard Hughes Medical Institute researchers have produced the first
detailed images of a protein that performs the crucial task of
detecting and repairing broken strands of DNA. The images show that the
protein is constructed to cradle DNA while the DNA is repaired and
rejoined with great precision.
The images of the DNA-repair protein Ku were published in the August
9, 2001, issue of Nature by Howard Hughes Medical Institute
investigator Jonathan Goldberg and colleagues John R. Walker and
Richard A. Corpina at Memorial Sloan-Kettering Cancer Center.
Breaks in double-stranded DNA can occur randomly as a result of
exposure to ionizing radiation or as programmed events during the gene
shuffling that is necessary to create infection-fighting lymphocytes.
The Ku heterodimer, consisting of two subunits, Ku70 and Ku80, is a
member of a family of DNA repair proteins that fixes damaged DNA in
order to preserve the integrity of the genome. When Ku encounters
damaged DNA, it initiates a repair process, called non-homologous end
joining (NHEJ), which stitches double-stranded broken ends back
together even though the ends of each DNA strand may not be
complementary.
Ku’s role in maintaining the integrity of the genome was
established by earlier studies in which HHMI investigator Frederick W.
Alt and his colleagues knocked out Ku70 and other NHEJ components and
found that DNA repair was compromised and that aberrant rearrangements
of chromosomes occurred with high frequency. Although these studies
reinforced the role of Ku in DNA repair, the details of how Ku senses
and initiates repair were still sketchy. “The biochemistry is
very clear,” Goldberg said. “Ku is sitting in the nucleus
ready to sense DNA damage and to bind to DNA ends.”
What remained unclear, however, was how Ku was able to distinguish
between broken ends and intact DNA with such precision. “Also,
stitching DNA back together sounds dangerous because of the likelihood
of losing genetic information,” Goldberg said. “But
actually, the non-homologous end joining process is quite accurate, and
we wanted to find out why Ku appears to be so necessary for that
accuracy.”
Goldberg and his colleagues believed that seeing how Ku binds to DNA
might provide answers to some of the questions about the Ku-DNA
interaction. The researchers used a technique called x-ray
crystallography to visualize the interplay between the Ku heterodimer
and DNA. In x-ray crystallography, protein crystals are bombarded with
intense x-ray beams. As the x-rays bounce off atoms in the crystal,
they leave a diffraction pattern, which can then be analyzed to
determine the three-dimensional structure of the protein.
Before they could get a full picture of the Ku-DNA complex, Goldberg
and his colleagues decided to study the Ku heterodimer by itself. Their
attempts to prepare crystals of the Ku protein yielded only a few
usable crystals out of the hundreds they prepared. Fortunately, the
scientists were able to use a technique pioneered by HHMI investigator
Wayne Hendrickson to solve the complete structure of the Ku heterodimer
from a single crystal. The technique, called multiple wavelength
anomalous diffraction, was applied during crystallographic analyses
performed at the National Synchrotron Light Source at Brookhaven
National Laboratory. After the Ku structure was determined, the
scientists moved on to solving the structure of the Ku-DNA complex.
In their studies, Goldberg and his colleagues had to mimic DNA
breakage, ensuring that their test DNA fragment had only one accessible
end — in order to avoid Ku attaching at more than one site on the DNA.
They accomplished this by blocking the other end of the DNA with a
bulky DNA motif.
After solving the structure of Ku bound to DNA, Goldberg and his
colleagues could see how the Ku heterodimer manages to
“find” a broken DNA end regardless of its sequence.
“The problem is that Ku is not like a transcription factor that
binds to a specific DNA sequence,” said Goldberg. “Rather,
it wants to recognize any broken DNA. And, the structure showed us that
Ku is a ring-shaped molecule that can slide onto the end as soon as the
break is formed.”
The structure of the Ku-DNA complex reveals that the Ku heterodimer
forms a ring that encircles and “cradles” the end of the
strand of DNA. “We believe that the Ku proteins have to hold the
DNA ends together,” said Goldberg. “The question is how
they hold the end of a piece of DNA without obscuring the end. We found
that our protein has an extensive base that cradles the DNA, with a
very narrow bridge that lies over the top — holding one side of the
DNA extensively, but leaving the other side almost completely exposed.
We think this exposure might allow other repair factors to act on the
broken ends to repair them.”
The scientists speculate that the Ku proteins on two broken ends
link to one another to hold the two ends in position for joining the
DNA back together. Goldberg and his colleagues also found that the Ku
heterodimer makes no contact with the DNA bases, but rather grasps the
sugar backbone of the DNA strand — meaning that the protein does not
“care” about the sequence of the DNA that it binds. The
scientists also have evidence that Ku holds the DNA in precise
alignment to allow ready joining by repair enzymes. “It’s
logical that if the protein precisely aligns the DNA ends, that gives
an advantage to the repair proteins and the ligases that are going to
ultimately join the DNA ends together,” said Goldberg.
Next, Goldberg and his colleagues plan to explore the structure of
the Ku proteins attached to two broken strands of DNA in order to
understand the mechanism by which they precisely align the ends. This
precision is a key to the accuracy of the joining process in the
absence of natural homology of the separated strands that could aid
repair, Goldberg said.
Image: Jonathan Goldberg
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