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(UPPER RIGHT) "OPEN" DNA repair helicase in a green, blue, cyan, and gray protein. (MIDDLE) "CLOSED" DNA formation. (BACKGROUND) A combination of Optical Traps (RED CONES) and a (GREEN) single-molecule fluorescence microscope to measure relationship of OPEN to CLOSED DNA states. Image Credit: Matt Comstock, University of Illinois at Urbana-Champaign

Found — proteins critical for DNA repair

For the first time science has observed the structure and function of specific proteins critical in the repair of DNA. While providing much needed data on protein states, it also opens up exciting possibilities for bio-engineering a cell.

Scientists at the University of Illinois at Urbana-Champaign are specifically interested in how proteins perform within a cell. Their work has revealed clues to the shape of proteins, but only if the protein was either "closed" or "open." This restriction to one particular state (or form) means that a protein's function in another state is hidden.

Chemla's team looked at the structure-function relationship in a protein called UvrD which is found in E. coli bacteria. This protein separates strands of DNA in need of repair by unwinding and unzipping them. An equivalent protein performs the same function in humans.

Chemla wanted to know how many proteins are needed to unzip a gene - one or two?  Chemla: "We put a fluorescence dye molecule on each protein — so we could count them. Then we watched the unwinding with an optical trap. We found that a single UvrD helicase unwinds the DNA, but not very far. It just goes back and forth a small distance, so we call it 'frustrated'. When we have two UvrD molecules, it seems to unwind much further and doesn't go back and forth as much."

Chemla's team observed there are two distinct states associated with UvrD — it is organized in either an "open" or a "closed" position. The function of each state had been debated by experts for years.

"We used smFRET (single-molecule fluorescence resonance energy transfer) and put two dyes on the molecule. Based on the distance between these molecules, we could see one or another color of light, thus indicating whether the molecule was in the open or closed position. Then we used an optical trap to observe whether the molecule was unwinding the double-stranded DNA.

"We found that the molecules actually swiveled from open to closed and back again. As it turns out, the closed state unwinds the strands, using a torque wrench action. The open state allows the strands to zip back together."

Yann R. Chemla PhD, Professor, Department of Physics, University of Illinois at Urbana Champaign

In Ha's laboratory, the team engineered a protein called Rep locking it in a closed or open state by using another molecule as the "lock." The team found that when Rep was locked into a closed position, it became a "superhelicase" (an enzyme-protein that unpacks genes) that unwound double-stranded DNA over a long distance. But when locked in an open state, Rep couldn't do anything.

Bioengineering molecules is a needed step for gene manipulation, including rapid DNA sequencing. Adds Ha: "The superhelicase we engineered ... can be used as a powerful biotechnological tool for sensitive detection of pathogenic DNA..."

"Proteins are flexible and each may be able to serve multiple functions."

Taekjip Ha PhD, Professor, Department of Physics, University of Illinois at Urbana Champaign

Abstract (1)
Direct observation of structure-function relationship in a nucleic acid–processing enzyme
The relationship between protein three-dimensional structure and function is essential for mechanism determination. Unfortunately, most techniques do not provide a direct measurement of this relationship. Structural data are typically limited to static pictures, and function must be inferred. Conversely, functional assays usually provide little information on structural conformation. We developed a single-molecule technique combining optical tweezers and fluorescence microscopy that allows for both measurements simultaneously. Here we present measurements of UvrD, a DNA repair helicase, that directly and unambiguously reveal the connection between its structure and function. Our data reveal that UvrD exhibits two distinct types of unwinding activity regulated by its stoichiometry. Furthermore, two UvrD conformational states, termed “closed” and “open,” correlate with movement toward or away from the DNA fork.

Abstract (2)
Engineering of a superhelicase through conformational control
Conformational control of biomolecular activities can reveal functional insights and enable the engineering of novel activities. Here we show that conformational control through intramolecular cross-linking of a helicase monomer with undetectable unwinding activity converts it into a superhelicase that can unwind thousands of base pairs processively, even against a large opposing force. A natural partner that enhances the helicase activity is shown to achieve its stimulating role also by selectively stabilizing the active conformation. Our work provides insight into the regulation of nucleic acid unwinding activity and introduces a monomeric superhelicase without nuclease activities, which may be useful for biotechnological applications.

This work is published in the article "Engineering of a superhelicase through conformational control" in Science (April 17, 2015, v. 348, no. 6232, pp. 344-347; DOI: 10.1126/science.aaa0445).

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