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For Greene, the next natural question was what happens when proteins moving along the DNA run into nucleosomes. To study proteins that move randomly along DNA, Greene adjusted his DNA curtains to avoid having a constant flow. Instead, he attached not one but both ends of the DNA to the glass slide. He added nucleosomes first, and then two types of DNA repair factors—which constantly peruse DNA strands for mistakes. He tagged each type of protein complex with a different fluorescent color.
By following the proteins along thousands of DNA strands, Greene and his colleagues found that the two behave differently when they encounter nucleosomes. One hops off the DNA and reattaches on the other side of the nucleosome; the other protein stops, unable to continue. Inside a living cell, Greene's team hypothesizes, another protein couples with the stalled protein to help it cross the barrier. The work, published in the August 2010 issue of Nature Structural & Molecular Biology, would have been exceedingly difficult if the experiments had to be done by looking at one molecule at a time, says Greene. You would never know if what you were seeing on any given strand was the norm.
Greene got back to collisions in his most recent work, published online November 24, 2010, in Nature. He sent a fast-moving molecular motor protein toward other stationary proteins bound to the DNA. "This paper embodies all of the excitement I felt when I was a graduate student first thinking about these problems," says Greene. "In essence, this boils down to a very simple question: what happens when one protein rams into another? These types of collisions happen all of the time in our cells, but we have a very limited understanding of how they are resolved."
His lab chose a protein called RecBCD, which processes the ends of broken DNA strands in bacteria and can travel at 1,500 base pairs per second. The lab then picked four different proteins as potential roadblocks. When RecBCD encountered the first of the four—one of the tightest known DNA binding proteins—RecBCD rammed into the stationary protein and pushed it thousands of base pairs before finally kicking it off the DNA altogether.
"It's like running a car into a flower," says Greene. "The car feels nothing and keeps going, while the flower takes the brunt of the punishment." After this remarkable show of strength, it wasn't surprising that RecBCD did similar damage to the other three proteins that Greene's lab put in its way.
Power in Numbers
Greene is not the only one who sees the power of his new technique. HHMI investigator Michael O'Donnell at the Rockefeller University studies DNA replication—the process by which proteins copy DNA strands. He wanted to know how fast proteins produce new copies. Biochemical experiments in his lab had hinted that replisomes, the proteins involved in replication, move at different rates on different DNA strands. The lab was stumped as to how to quantify this variation until O'Donnell heard Greene talk about DNA curtains.
"The one disadvantage of single molecule work is that you're looking at a single molecule," says O'Donnell. "Greene's method got around that. I didn't want to look at one molecule and find out later that I was observing something that is not general."
O'Donnell's DNA curtains showed that individual replisomes' movement rates vary as much as 5-fold, his team reported in Proceedings of the National Academy of Sciences on August 11, 2009. Now they have a working average and a way to test what factors can shift that average.
As for Greene, he's ready to add complexity to his curtains. "We'd like to make arrays where the DNA is crisscrossed or supercoiled," he says. True to his love of molecular crashes, he also plans to see how RecBCD and other motor proteins behave when they encounter not just one protein in their path but a whole stretch of roadblocks. "If you think about what DNA looks like in the nucleus, it's jam-packed with proteins, so we would really like to mimic that environment by putting lots and lots of proteins on the DNA." On thousands of DNA strands, this will mean many thousands of collisions. It will be a fascinating show to watch.