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Secrets of a precision protein machine
Date:4/15/2011

between two helical coils of amino-acid residues in the protein, which form an arch. But whether the end of the 5' flap threads through this arch or whether the arch closes around the flap was unknown. The Sakurai structure couldn't answer the question because this area of FEN1 was disordered in the model, with its associated structures free to move.

Considering FEN1's remarkable specificity and accuracy, Tainer saw that neither its structure nor mechanism could be solved unless DNA with a 5′ flap was also present. Determining the structure of the two together was essential.

Solving the compound structure

Using the SIBYLS beamline at Berkeley Lab's Advanced Light Source (ALS), designed specifically to tackle the kind of problem the FEN1 structure presented, Tsutakawa worked with Scott Classen at the ALS and Andy Arvai at the Scripps Institute. (SIBYLS stands for Structurally Integrated Biology for Life Sciences.) They rapidly obtained some 20 structures of FEN1 bound to DNA and used PHENIX software developed by Paul Adams of Berkeley Lab's Physical Biosciences Division to derive models of three conformations of DNA in the grip of FEN1. With the DNA in place, it was possible for the researchers to see what wasn't visible in the Sakurai model of FEN1.

"We saw that FEN1 binds the double-strand DNA on either side of the 5' flap and opens it by severely bending the template strand all the way back to 100 degrees, almost at right angles," says Tsutakawa. "Only because there's a break on one side of the double-stranded DNA at that point can the DNA bend that sharply."

"FEN1 is shaped roughly like a left-handed boxing glove," Tainer says, noting that FEN1 holds DNA at four binding sites, beginning with two widely separated regions where it grips the double-strand DNA by its template strand. One of these binding sites is near the tip of the glove, grasping the 3' overhang at the break so as to expose a sing
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Contact: Paul Preuss
paul_preuss@lbl.gov
510-486-6249
DOE/Lawrence Berkeley National Laboratory
Source:Eurekalert  

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