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

le unpaired nucleotide. The other double-strand binding site is at the wrist of the boxing glove, near the 5' flap.

Two other sites hold the template strand opposite the open junction and, crucially, at the crease between thumb and forefinger, where the 5' flap will be cut.

In human FEN1, the arch between the thumb and the rest of the glove is indeed capped, although this is not true for other members of FEN1's family. In FEN1 the 5' flap is forced to thread through the arch, which is too narrow for double-strand DNA to enter and thus selects for the single-stranded flap.

The flap is cut at a place called the "scissile phosphate." Phosphates (and sugars) are like the vertebrae of a single strand's backbone, to which the bases are anchored (a phosphate plus a base makes one nucleotide). The flap is held firmly in the right place for cutting because the nucleotide with the scissile phosphate, plus its neighboring nucleotide, are initially base-paired to the template strand. The bend in the template strand opens these pairs and lines up the target nucleotides near two metal ions, which catalyze the hydrolysis the clipping of the flap at the precise scissile-phosphate location.

"The structure was unexpected, but it made clear how FEN1 works," says Tsutakawa. "At the same time it explains how the superfamily of similar endonucleases can operate in such different situations."

Motion and precision

"How does FEN1 contribute to recombination and repair accurately, without mistakes?" Tainer asks. "We see how moving the template will position the 5' flap for precise cutting. Incision is equally precise. FEN1 cuts it where it knows the strand has already been accurately paired to the template, leaving just one unpaired base, which can then pair with the unpaired 3′ overhang."

Tsutakawa summarizes the findings: "FEN1 grasps the template of the double-strand DNA where the junction is be
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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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