HOW DNA MACHINERY IS A ‘TWO-WAY STREET’
07/01/04 -- Biochemists at Duke University Medical Center have discovered key components that enable
the cell's DNA repair machinery to adeptly launch its action in either direction along a DNA strand to strip out faulty DNA.
Such flexibility exemplifies the power of the repair machinery, which guards cells against mutations by editing out errors
that occur during the process of chromosome replication. Malfunction of the "mismatch repair" machinery is the cause of several
types of cancer, including relatively common forms of colon cancer.
The researchers, led by Howard Hughes Medical Institute investigator Paul Modrich, Ph.D., at Duke, reported
their findings in the July 2, 2004, issue of the journal Molecular Cell. Joint first authors on the paper were Leonid Dzantiev,
Ph.D., and Nicoleta Constantin, and the other co-authors were Jochen Genschel, Ph.D., Ravi Iyer, Ph.D., and Peter Burgers,
Ph.D. The research was supported by the National Institutes of Health.
Modrich and his colleagues have long studied the mismatch repair machinery of the cell. This machinery
detects and corrects errors in DNA replication in which the wrong DNA unit is stitched into place in a newly forming DNA strand.
Normally such units -- called nucleotides -- on one strand of the double-stranded DNA molecule bond with complementary nucleotides
on the other strand, like complementary pieces of a puzzle. Thus, an adenine on one strand is normally paired with a thymine
on the other, and a guanine on one strand with a cytosine on another.
The process of mismatch repair involves first recognizing the mismatch -- for example of an adenine with
a cytosine. The machinery then recognizes a break in the newly synthesized DNA strand, which triggers the machinery to excise
the section including the mismatch, starting at the strand break and working toward the mismatch and slightly beyond. The
system then replaces the mismatched strand with one containing the correct complementary nucleotide.
A central mystery is how the mismatch repair system is flexible enough to recognize such a triggering
strand break on either side of the mismatch along the DNA strand, said Modrich. In the Molecular Cell paper, he and his colleagues
have defined the protein components of the machinery that allows such bidirectionality and figured out how those components
assemble at the strand break to direct the excision.
Importantly, the researchers' biochemical experiments and analyses of mutations in the repair proteins
revealed how the machinery for excising the faulty DNA strand "knows" which way to go from the strand break to the mismatch.
Basically, they found that a protein called PCNA is clamped onto the DNA at the strand break. PCNA, together
with the protein that clamps PCNA onto the DNA double helix, regulate the enzyme whose job it is to snip out the segment containing
the mismatch, by "aiming" the enzyme -- called exonuclease I -- in the right direction to work itself along the strand, stripping
out the segment containing the mismatch.
"A surprising feature of the repair system that it can evaluate the placement of the strand signal to
one side or the other of the mismatch and work from there," said Modrich. According to Modrich placement of the strand break
that directs repair to one side or the other of the mismatch is likely a consequence of the mechanism by which DNA is copied
by the replication machinery.
Modrich and his colleagues are continuing their studies by seeking to identify other components of the
repair system, which could have implications for understanding how cancers become resistant to chemotherapy.
"This system does more than just repair DNA biosynthetic errors," he said. "Many cancer chemotherapeutic
drugs work by damaging DNA, which selectively kills cancer cells because they are proliferating more than resting cells. The
mismatch repair machinery senses certain types of DNA damage, which leads to activation of the cell's suicide machinery, called
apoptosis, resulting in cell death. Inactivation of the mismatch repair system not only predisposes cells to tumor development,
but also renders them resistant to certain anti-tumor drugs.
"Such findings as the ones we are reporting build on the basic understanding of mismatch repair and may
allow us to explore such possibilities," said Modrich. Source: Duke University Medical Center