|Structure of DNA-Bound FEN1 Reveals Mechanism of Action|
DNA replication is a critical step in the life of all organisms, insuring that each new cell gets an accurate copy of the genome. Among the legions of proteins required to do this work, the DNA-slicing “flap endonuclease” FEN1 plays a key role. Much of FEN1’s structure was solved previously, but the DNA-free structure failed to expose information about the mechanics of how it works. An international team of scientists led by researchers from Berkeley Lab and the Scripps Research Institute has solved the structure of human FEN1 bound to DNA using ALS Beamline 12.3.1, revealing the surprising mechanism behind FEN1’s speed, accuracy, and versatility.
DNA replication is the first and most critical step in cell reproduction. (For an in-depth overview of DNA replication, see this highlight’s sidebar, “A Recipe for Rigorous Replication.”) During the replication of the lagging strand, numerous short, discrete nucleotide fragments must be joined to create the nascent strand of DNA. This is where FEN1 comes in, preparing adjacent fragments by slicing the 5’ flap of one so it can be connected to the 3’ overhang of the other.
During each replication, FEN1 performs this operation approximately 50 million times without introducing any errors. But FEN1 is also important in DNA repair, targeting specific repair pathways, which presents different challenges than does replication. Researchers were looking for clues as to how FEN1 can do different jobs.
Using the SIBYLS (Structurally Integrated Biology for Life Sciences) beamline at the ALS, researchers rapidly obtained some 20 structures of FEN1 bound to DNA and used PHENIX software to derive models of three conformations of DNA bound to FEN1. What was not visible in previous models of FEN1 is this: the protein binds the double-stranded DNA on either side of the 5’ flap and opens it by severely bending the template strand to a 100-degree angle. The DNA is only able to bend this sharply because one side of the double-stranded DNA is broken at that point.
The secret of how FEN1 interacts with DNA lies in its structure. FEN1 binds DNA at four sites. At two of these sites, FEN1 holds the template DNA strand opposite the open junction. At the third site, FEN1 grasps the 3’ overhang, exposing a single unpaired nucleotide. Lastly, FEN1 grips the 5’ flap.
The 5’ flap threads through an opening between two helical coils in FEN1, an archway too narrow for double-stranded DNA. The helicity of the double strand positions the flap to thread through the arch. The flap is then cut to match the exposed overhang at a place called the “scissile phosphate.” Phosphates are the backbone of DNA to which bases are anchored. The nucleotide with the scissile phosphate and its neighboring nucleotide are initially base-paired to the template strand, holding the flap firmly in place for cutting. The bend in the template strand opens these two base pairs and lines up the target nucleotides near two metal ions that catalyze the hydrolysis (cutting) of the flap.
FEN1 was long thought to move into position by sliding down the 5’ flap to the incision site. But this research shows that the arch does not close around the flap as was originally hypothesized; instead, the end of the 5’ flap threads through the arch. Solving FEN1’s structure and mechanism required the presence of DNA with a 5′ flap.
Just as FEN1 moves the template into position so the 5’ flap will be cut precisely, FEN1 also contributes to recombination and repair by cutting where the strand has already been accurately paired to the template, leaving just one unpaired base to pair with the 3′ overhang. Its ability to target specific repair pathways makes it of interest for cancer research. FEN1 plays a dual role in cancer—it is essential to health, but it’s also overproduced in many cancers and associated with aggressive tumors. Designing drugs that induce changes in specific structures is the first step in creating more effective, more targeted, and less damaging treatment.
Research conducted by: S. Classen, P.K. Cooper, L.D. Finger, A.H. Sarker, S.E. Tsutakawa, and J.A. Tainer (Berkeley Lab); A.S. Arvai, B.R. Chapados, and G. Guenther (The Scripps Research Institute and Skaggs Institute for Chemical Biology); J.A. Grasby, C.G. Tomlinson, and P. Thompson (University of Sheffield, UK); and B. Shen (Zhejiang University, China).
Research funding: National Institutes of Health. U.S. Department of Energy (DOE), Office of Basic Energy Sciences (BES). Operation of the ALS and SSRL is supported by DOE BES.
S.E. Tsutakawa, S. Classen, B.R. Chapados, A. Arvai, L.D. Finger, G. Guenther, C.G. Tomlinson, P. Thompson, A.H. Sarker, B. Shen, P.K. Cooper, J.A. Grasby, and J.A. Tainer, “Human flap endonuclease structures, DNA double base flipping and a unified understanding of the FEN1 superfamily,” Cell 145, 198 (2011).
ALS Science Highlight #240