Protein ’key’ could aid search for cancer drugs

Scientists determine how ’trump card’ protein blocks DNA replication

New research at Rice University is allowing biochemists to understand a key hierarchy of protein interactions that occurs in DNA replication, showing for the first time how a key protein “trumps” its rivals and shuts down cell division while DNA repairs take place.
The work, sponsored by the American Cancer Society, appears in the Dec. 8 issue of the journal Structure. It could aid drug makers in designing targeted therapies that block cancer cells from multiplying.

“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.”