Howard Hughes Medical Institute 2010 Annual Report

This image shows the locations of minor grooves in DNA in dark grey. Scientists are beginning to learn how DNA’s shape instructs proteins where to bind.

This image shows the locations of minor grooves in DNA in dark grey. Scientists are beginning to learn how DNA’s shape instructs proteins where to bind.

The Biophysics of the Cell

Scientists need to see and, in some cases, touch, poke or prod the molecules inside cells to learn how they work. In many HHMI laboratories, researchers are developing new ways to observe, manipulate, and measure molecules on the move. Their inventions are helping researchers understand how physical forces govern interactions between molecules.

DNA is often depicted as a long, elegant—but relatively static—double helix. But for HHMI investigator Barry Honig, DNA’s dynamic side is irresistible. Using tools he developed to help structural biologists predict how proteins work, Honig and his colleagues are now testing ideas about how proteins interact with DNA.

Scientists have long known that transcription factors—the proteins that activate and repress genes—look for their docking sites on DNA by scanning the genome for a specific nucleotide sequence that essentially says, “bind here.”

Honig’s group at Columbia University has shown that this isn’t the proteins’ only strategy for recognizing important DNA landmarks. The coiled, complementary strands of DNA form 'major' and 'minor' grooves, to which proteins can bind. Honig's team found that proteins can read nuances DNA’s shape by tracking the width of the minor grooves, which can be affected by single-letter changes in the DNA sequence.

“It is quite surprising, actually,” Honig says. “If you realize that those nucleotides determine which proteins bind the DNA, and they do it in part through their effect on shape, you begin to understand how sensitive and subtle the DNA structure really is.”

At the University of Illinois, Urbana-Champaign, physicist Taekjip Ha is working to understand how DNA is replicated in real time, and to clarify how DNA interacts with the proteins that shadow and groom it. Since no microscopy techniques can produce moving images at such fine resolution, he has been building a new toolkit for such studies.

Ha has been examining DNA-unwinding enzymes that crawl along the helix and separate the double strands as they go. He and colleague Timothy Lohman at Washington University School of Medicine in St. Louis literally pulled apart DNA from the aptly named single-strand DNA binding protein so they could see how the molecules interact. The researchers found that single-strand DNA binding protein, once thought to be stiff and immobile, actually slips and slides as it stabilizes DNA. The work is considered a landmark study because it helps clarify the interplay between DNA and its entourage of molecular attendants.

Visualizing this movement is important, Ha says, because it may lead to greater understanding of the machines that repair and replicate DNA, which are intimately linked to cancer and aging.

DNA repair is also of great interest to HHMI early career scientist Maria Spies at the University of Illinois, whose group uses classical and single-molecule biochemistry to study the proteins involved. Her goal is to find an Achilles' heel in the DNA repair process that can be exploited for the design of new drugs.

This year Spies teamed up with Ha to study how a specific kind of DNA helicase—a motor enzyme in the cell that converts chemical energy into mechanical work—helps repair DNA. Helicases, such as the XPD helicase studied by Spies and Ha, "unzip" DNA so it can be repaired or plow away obstacles that obstruct various DNA maintenance machineries. Spies’s work showed that XPD helicase has developed clever strategies for bypassing molecular traffic jams on the crowded DNA molecule. The studies are helping scientists see how the critical proteins involved in repair avoid "running off the rails," which can make cells weaker and more vulnerable to cancer, premature aging, and ultra-sensitivity to sunlight.

Seeing the movement of specific proteins inside cancer cells is an important challenge that motivates HHMI investigator Jay T. Groves, at the University of California, Berkeley. Groves would like to know if the aggressiveness of a tumor might be related to how much force a cancer cell uses to pull receptors on its surface–which are key to cancer’s spread–closer.

To answer that question, Groves and his colleagues built a half-biological, half-synthetic device to measure the mechanical pulling power of 26 well-studied breast cancer cell lines. Groves' team used nanofabrication techniques to engineer a synthetic cell surface to which they attached a type of receptor that helps cancer cells target and invade healthy cells. This synthetic cell surface served as a surrogate for a natural cell and formed an interface with live cancer cells bearing another protein that helped them lock on to the synthetic cell surface.

The synthetic cell surface allowed Groves' team to "reach in" and prod the living cancer cell at the molecular level. In this way, the team made detailed measurements that provide them with more information about the molecular dynamics and organization of the different cellular receptors and associated proteins.

When the researchers studied the pulling power of 26 other breast cancer epithelial cell lines, they used statistical analyses to compare the cells’ pulling strength, and discovered they could neatly order the tumor lines by invasiveness. Groves believes the discovery that this cytoskeletal pulling power correlates with cancer invasiveness could help researchers learn why some cancers spread rapidly.

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Photo Credit: Courtesy of Honig Lab