COLLEGE OF ARTS AND SCIENCE GRADUATE SCHOOL OF ARTS AND SCIENCE Tuesday Seminar Series - Eric Greene, 3/31/15 at 2:00 p.m. Eric Greene, Columbia and HHMI James W. Canary, NYU Chemistry Eric GreeneColumbia UniversityDepartment of Biochemistry and Molecular BiophysicsHoward Hughes Medical InstituteGreene HHMI Scientist ProfileGreene Lab Research Page Our group uses single-molecule optical microscopy to study fundamental interactions between proteins and nucleic acids. Our overall goal is to reveal the molecular mechanisms that cells use to repair, maintain, and decode their genetic information. This research combines aspects of biochemistry, physics, and nanoscale technology to answer questions about complex biological problems that simply can not be addressed through traditional biochemical approaches. The advantages of our approaches are that we can actually see what proteins are bound to DNA, where they are bound, how they move, and how they influence other components of the system - all in real-time, at the level of a single reaction.

We are particularly interested in determining the physical basis for the mechanisms that proteins use to survey DNA molecules for damage and initiate repair processes, and how these initial steps are coordinated with downstream events that lead to completion of repair. As part of our work, we are also actively pursuing the development of novel experimental tools that can be used to facilitate the study of single biochemical reactions. In particular, we are applying techniques derived from nanotechnology to our biological research, and using nano- and micro-scale engineering to facilitate the development of new, robust experimental platforms that enable "high throughput" single molecule imaging.Eric Greene smashes molecules together. He precisely arranges proteins on strands of DNA and sends one zipping toward another, headed for an impact. Sometimes, a weaker molecule falls off the DNA immediately. Other times, in a feat of acrobatics, one protein hurdles another. Recently, Greene saw a protein push another one backward when the two collided.

How proteins behave in such a crash test gives scientists data about their structural integrity, how they attach to DNA, and how they behave in a cell—where molecular fender-benders are likely a commonplace occurrence in the jam-packed nucleus. Greene, an HHMI early career scientist at Columbia University, is doing these kinds of experiments on an entirely new scale. Rather than looking at the behavior of just one molecule at a time, he lines up hundreds of DNA strands and watches hundreds or thousands of proteins as they interact with the DNA. The aligned molecules of DNA with bright dots of fluorescent protein look like glittering, beaded curtains—leading Greene to dub them "DNA curtains." But these flashing curtains provide more than stunning images. They give researchers powerful insights into how DNA-bound proteins interact with the DNA and with each other. As a postdoc at the National Institutes of Health in the late 1990s, Greene learned how to watch the dynamics of DNA-bound proteins under the microscope.

The ability to see the physical wrangling at such a small scale fascinated him. But limitations of the technique—which involved essentially dumping proteins into a vat of DNA and watching one at a time—became frustrating. "I'd spend hours doing tedious manipulations," says Greene, "and at the end of the day have only one or two data points. This really limited what we could do. blackout curtains galwayIt often took weeks or months just to figure out if an experiment was even working." sears rod pocket curtainsMoreover, in many instances the proteins would stick to the microscope slide, and this nonspecific sticking was a huge problem in the field. fairy light curtain hire brisbaneGreene struggled to find a surface that worked for all his experiments.buy elvis '77 the final curtain

By the time he started his own lab at Columbia, Greene had a plan. He realized that one surface was natural to all proteins: the lipid bilayer, which surrounds every living cell. Greene figured that a bilayer would provide an inert environment to prevent proteins from sticking to a glass slide and also a way to tether DNA in place. Page 2 of 3 The crux of his method lies in the ability to corral lipids into separate areas of a glass slide. He scratches miniscule lines into the glass, which keep lipids in separate areas—they can't move across the blemishes. He then encloses special lipids, tagged with a molecule called biotin, in one linear area. Unmarked lipids cover the rest of the glass slide. Specially engineered strings of DNA attach only to the biotin-marked lipids, so the ends of the DNA are arranged in a line. Finally, Greene turns on a flow of liquid across the surface, sweeping all the strands in the same direction, forming a curtain of aligned DNA. This movie shows a DNA curtain (green) bound by fluorescently tagged nucleosomes (magenta).

When a constant flow of liquid runs through the setup, the DNA extends in parallel lines, letting researchers see the exact placement of the nucleosomes. When the flow lets up, as seen at 7.4 seconds on the timer, the DNA snaps back up into a more relaxed conformation—until the flow resumes. The first time Greene tried this technique, he used a razor blade to scratch the glass—a rudimentary trial, but a successful one. Now, his lab uses electron-beam lithography, a technology developed for manufacturing electronic circuits, to make precisely controlled barriers on the glass that can be used to define the organization of thousands of DNA molecules. In principle, the technique behind DNA curtains can be used to study any kind of linear molecule. A scientist could tether RNA, or long filaments that act as cellular highways, to the lipids. But Greene's passion is understanding how proteins interact with DNA. "These relatively simple interactions can drive what an organism looks like, what an organism does, or how it evolves," says Greene.

"It's always fascinated me." Some of the most important protein complexes on DNA are the nucleosomes, the packaging units that consist of DNA wrapped tightly around a protein core. Once Greene established that his curtains worked, he wanted to know whether they would still work for DNA wrapped around nucleosomes. Greene and his team added nucleosomes to DNA before lining up the strands. When the strands were aligned, the scientists saw that nucleosomes are static once they've attached to DNA. Moreover, by recording the positions of thousands of nucleosomes at one time, Greene's lab had a powerful case for understanding where on DNA nucleosomes attach. The results appeared in the October 2009 issue of Nature Structural & Molecular Biology. The experiment was the first proof of concept that the technique worked on DNA associated with nucleosomes, says Greene. Page 3 of 3 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. 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."