When PcrA reaches the end of its DNA rope, it releases it and starts the reeling in process over again, removing any additional problematic proteins that have bound to the damaged DNA as it reels.
By using FRET, a technique Ha developed, the team also was able to answer another question about PcrA: How consistent is its motor function? Researchers agree that on average, PcrA moves one DNA unit, called a base pair, for each unit of cellular energy it uses, called ATP. But because researchers traditionally study the enzyme in relatively large samples, broad distributions of data have led to conflicting views on whether the helicase moves in uniform steps or those of varying lengths even up to six base pairs per ATP.
Since FRET is a single-molecule technique, the researchers were able to document a single enzyme's function, step by step, and found that PcrA does, in fact, move in uniform steps of one base pair per ATP.
Next, the team plans to create a reaction environment more similar to that in vivo, using three and four colors of FRET dyes to measure activities of multiple proteins simultaneously. They are also working toward understanding why helicase moves only in one direction.
"This is an ideal marriage of a new technology and an interesting biological problem," Ha said.
|Contact: Liz Ahlberg|
University of Illinois at Urbana-Champaign