Web Date: September 13, 2012
The Curtain Rises On DNA–Protein Interactions
For the first time, scientists have watched proteins interact with long single-stranded DNA molecules in real time (Anal. Chem., DOI: 10.1021/ac302117z). The new method should yield insights into DNA replication and repair, the researchers say.
Until now, scientists could only watch proteins interacting with double-stranded DNA or very short single strands, says Eric Greene of Columbia University, who developed the new method. But, he says, “Basically anything to do with DNA metabolism or DNA repair involves single-stranded DNA intermediates.”
To find a way to monitor long single strands, Greene built upon a method his team devised six years ago, which visualizes proteins binding to double-stranded DNA (Langmuir, DOI: 10.1021/la051944a). They tethered fluorescent DNA molecules to lipids in a bilayer that coated the surface of a microfluidic sample chamber. When the researchers flowed buffer through the chamber, the DNA molecules diffused within the bilayer, eventually aligning along a nanofabricated barrier in the bilayer. The researchers added proteins to the chamber and observed their interactions with DNA in real time by noting changes in the DNA’s fluorescent signal. The team called the method “DNA curtains” because the hundreds of aligned DNA strands—visualized by total internal reflection fluorescence microscopy—reminded them of a window curtain.
But the technique didn’t work with single-stranded DNA, which is so flexible that it often folds upon itself into extensive secondary structures that preclude protein binding. Also, the intercalating fluorescent dyes that label double-stranded DNA can break single strands of DNA.
So Greene and his coworkers found a way to simultaneously unravel folded-up single-stranded DNA and fluorescently label it. The key was a protein called replication protein A (RPA), which binds single strands during DNA replication to prevent them from folding up. When the researchers added a fluorescently labeled version of the protein to a DNA-curtains assay, it coated single-stranded DNA molecules, helping extend the DNA and keep it accessible to proteins. Because RPA coats all single-stranded DNA in cells, the researchers say, the ubiquitous protein is unlikely to interfere with the binding of other proteins.
To test whether the assay could visualize interactions of single-stranded DNA with other proteins, the team injected fluorescently tagged Sgs1 protein into a flow cell containing RPA-bound DNA curtains. Sgs1 is a yeast helicase protein involved in DNA repair. Using fluorescence microscopy to visualize the labeled RPA and Sgs1 proteins, the researchers watched Sgs1 bind single-stranded DNA in real time.
Greene says that the new assay will allow researchers to probe when and where proteins bind single-stranded DNA, and how multiple proteins influence each other’s binding. “We can watch all of this stuff as it happens,” he says.
Maria Spies of the University of Iowa says, “The DNA curtains approach combines the benefits of single-molecule and bulk studies.” That’s because, she explains, researchers can use it to visualize hundreds of single-stranded DNA molecules at the same time. “I can’t wait to try this system in my lab,” she says.
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