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"All cancers are marked by some form of DNA
replication gone awry, so a basic understanding of DNA replication is
of paramount importance to those designing cancer-fighting drugs,"
said lead author Yousif Shamoo, assistant professor of biochemistry
and cell biology. "In addition, almost every form of life – including
bacteria – use a variant of the protein that we studied, and we
believe the work may also aid drug makers who are developing new forms
of antibiotics."
In the study, Shamoo and graduate student John
Bruning used x-ray crystallography and isothermal titration
calorimetry to determine the structures of two variants of a protein
called Human Proliferating Cellular Nuclear Antigen, or PCNA.
PCNA is a member of the "sliding clamp" family of
proteins, which are so-named because of their unique shape and
function. Sliding clamps are ring-shaped proteins that slide along
strands of DNA. DNA is fed through the hole in the center, and the
PCNA acts as a docking mechanism for other proteins that need to
interact with the DNA to make repairs or copies or to take part in
other genetically regulated tasks. Genes that code for sliding clamp
proteins are present in all forms of life except for some viruses.
In humans, at least a dozen proteins are known to
dock with PCNA. Each of them docks with PCNA by inserting a kind of
key known as a PCNA-interacting protein, or "PIP-box," which binds
chemically to the PCNA and holds the docked protein on the DNA strand.
"Each protein that binds with PCNA has its own
version of the key, but all the keys fit into the same lock," said
Shamoo. "There is a hierarchy among the PIP-box proteins, with some
winning out and trumping others before they get a chance to bind. By
deciphering the structure of two of these keys, while they were in the
lock, we were able to determine their binding energies and find out
how the strongest key -- the trump card -- blocks the others and shuts
down DNA replication."
The structure of PCNA containing the trump key, the
PIP-box from a cell regulatory protein called p21, was solved by
researchers at the Rockefeller University. P21 is important because it
is produced by cells with damaged DNA. In healthy cells, p21 binds
strongly with PCNA to prevent the cells from making copies of DNA
until the genetic damage is repaired.
Shamoo and Bruning solved the structure of PCNA
containing two other forms of PIP-box keys, both of which are involved
in DNA replication. By comparing the chemical structure of the weaker
keys against the stronger p21 key, they were able to determine how p21
optimizes its connection to PCNA.
If drug makers can replicate p21's strategy in
targeted cancer-fighting compounds, they could attack cancer cells'
ability to reproduce at the most basic level.
"The sliding clamp protein that's used by bacteria
has the same function as PCNA in humans, but the keys for bacteria are
very different from those in humans," said Bruning. "If bacteria use a
similar hierarchy to access to their PCNA, it might be possible to
design an antibiotic that plays the bacterial trump card without
affecting human cells at all." |
