In bulk studies, rather than single-molecule kinetic observations, the ATP doesn't produce a signal from unwound DNA because the slippage masks the signal.
They then surmised that different mixtures of nucleotides might allow them to investigate helicase subunit coordination. They found that very small amounts of dTTP mixed with large amounts of ATP were enough to decrease the "slippage" events they saw with the ATP alone.
Further inspection revealed that while two subunits of the T7 helicase are binding and releasing nucleotides, the other four can remain bound to nucleotides to anchor the DNA and prevent it from slipping. It only takes one subunit bound to dTTP to decrease slippage almost entirely a little goes a long way.
Such studies can help scientists gain a deeper understanding of helicase mechanics and, in the case of medicine, what happens when helicases go awry or don't bind correctly.
Smita Patel, Rutgers University biochemistry professor and paper co-author, says helicase defects are associated with cancer predisposition, premature aging and many other genetics-related conditions.
"This study provides fundamental new knowledge about a cellular process that is essential to all forms of life," said Catherine Lewis, who oversees single-molecule biophysics grants at the National Institute of General Medical Sciences of the National Institutes of Health. "By using single-molecule methods to study how helicases work, Dr. Wang has resolved several longstanding questions about how the enzyme is coordinated, and possibly regulated, during replication."
|Contact: Blaine Friedlander|