LA JOLLA, CA—Researchers at the Scripps Research Institute and Lawrence Berkeley National Laboratory have uncovered the capabilities of an essential enzyme in DNA replication and repair, one that has potential as an anti-cancer therapy. Thanks to the production of clearly defined crystal structures of FEN1, a member of a group of enzymes known as flap endonucleases, researchers were able to discover that its function and effects are even more precise than was originally thought.
"This work represents a seminal advance in the understanding of FEN1," says John Tainer, professor and member of the Skaggs Institute for Chemical Biology at Scripps Research, senior scientist at Lawrence Berkeley National Lab and team leader of the study. "The research produced very accurate structures showing DNA before and after being cut by FEN1 activity, providing a basis for understanding a whole superfamily of enzymes that must cut specific DNA structures in order for DNA to be replicated and repaired."
DNA replication begins when the DNA double helix is unwound by a replication fork, separating the two strands, which then form two prongs of the replication fork and serve as templates for creating the new complementary strands. Creating the complementary strand on what is called the "leading" of the two strands is fairly simple, as the replication fork moves from the 3' (three prime) end of the strand to the 5' (five prime) end, and the enzyme DNA polymerase creates a 5' to 3' complementary strand.
However, the "lagging" strand is oriented in the opposite direction, so DNA polymerase, which works in the 5' to 3' direction, has a harder time replicating that second strand. The strand is instead replicated in pieces, known as Okazaki fragments, found near the replication fork. The fragments include something known as a "primer," a strand of RNA that provides a starting point for synthesizing DNA. FEN1, which is expressed in all cells, is responsible for removing the RNA primer.
"To replicate one DNA double helix in one cell you have to cut off a 5' flap so that you don't have one base pair too many or one base pair too few, and you have to do this accurately with 50 million Okazaki primers in each cell cycle," Tainer says. "It has always been a mystery as to how FEN1 can precisely cut this flap so efficiently and so rapidly. It's an amazing, efficient molecular machine for precisely cutting DNA."
It was previously thought that FEN1 grabs the flap of the 5' single stranded DNA, moves to the joint where DNA is replicated, and then cuts and patches the primer at that spot. The researchers first became interested in FEN1 because of its involvement in DNA repair, says Susan Tsutakawa, one of the first authors of the paper. The enzyme "is used in both DNA replication and in certain DNA repair pathways that are needed all the time."
For this study, Andy Arvai, co-author for the study and a scientific associate at Scripps Research, led the team of researchers in growing crystals of FEN1 when bound to DNA. The team used X-ray crystallography to identify the complex's atomic structure, and by using Lawrence Berkeley National Laboratory's Advanced Light Source beamline, known as SIBYLS, the researchers were able to solve three different crystal structures, resulting in a model of DNA before and after it is cut by FEN1. Thanks to the crystal structures, the researchers discovered that FEN1 first binds the DNA, then bends, frays and cuts it.
"It binds duplex DNA, bends it into a single-stranded DNA right at the flap, flips out two base pairs, and cuts between them," says Tainer.
FEN1 is part of a superfamily of enzymes that all play significant roles in the replication and maintenance of DNA. The 5' nuclease superfamily, Tsutakawa explains, includes three other enzymes: Exo1 and XPG, which are both involved in several DNA repair pathways, and GEN1, which is involved in the homologous recombination repair pathway. She notes that "all these proteins are potential targets, although none have been developed to drug trials."
Mutations in FEN1 can predispose cells to cancer because errors in DNA replication, specifically in flap removal, can produce unstable DNA. Studies have shown that mice with only one of the pair of inherited FEN1 genes are predisposed to developing cancer if damage occurs to any of their DNA. It is a catch-22, because while the absence of one of the genes for the enzyme can predispose cells to cancer, it is over-expressed in cancer cells, which makes them resistant to therapy. According to Tsutakawa, the researchers hope that by eliminating the enzyme within cancer cells, thereby taking away a pivotal enzyme in DNA replication and repair, they "will topple the already stressed cancer cell."
Tsutakawa notes that while the researchers don't fully understand what FEN1 does in cancer cells, "there is correlation with increased levels of this protein in certain cancers."
In a paper published in Nature Medicine in 2007, "Fen1 mutations result in autoimmunity, chronic inflammation and cancers," the study's authors examined the enzyme and its presence in various cancers. The abstract notes that "mutations…affecting the protein's activity for DNA repair and apoptotic DNA degradation could be more common." Their results showed FEN1 mutations in "five of 71 non-small cell lung carcinoma specimens."
A paper published in 2009 in Human Mutation, entitled "Functional FEN1 polymorphisms are associated with DNA damage levels and lung cancer risk," examined FEN1's influence in terms of lung cancer, one of the cancers the protein is most associated with. The researchers note in their abstract that they identified "two single nucleotide polymorphisms (SNPs)…that were associated with reduced FEN1 expression" in their sample of coke-oven workers. Their results, they state, "highlight FEN1 as an important gene in human carcinogenesis and genetic polymorphisms in FEN1 confer susceptibility to lung cancer."
Given the enzyme's potential and its connection to cancer, Arvai and Tsutakawa say the next step for this project is to identify compounds that will inhibit FEN1's activity. Arvai is convinced that "a better understanding of FEN1 structure and function may have long-term positive benefits to human health," an opinion that Tainer shares.
"My hope is that our finding of how FEN1 works mechanistically might provide a foundation for a next-generation cancer drug," says Tainer. "We need to cut as many lifelines as possible in cancer cells in order to provide an effective treatment."
The study received financial support from grants from the National Institutes of Health and the Biotechnology and Biological Sciences Research Council (BBSRC) in the United Kingdom.
The paper, titled "Human Flap Endonuclease Structures, DNA Double-Base Flipping, and a Unified Understanding of the FEN1 Superfamily," was published in the April 15, 2011 issue of Cell. The first authors of the paper are Tsutakawa and Scott Classen of Lawrence Berkeley National Laboratory and Brian R. Chapados and Arvai of Scripps Research. Additional authors of the paper, other than Tainer, Tsutakawa, Classen, Chapados and Arvai, are L. David Finger at the University of Sheffield; Grant Guenther of Scripps Research; Christopher G Tomlinson, Peter Thompson and Jane A. Grasby of the University of Sheffield; Altaf H. Sarker and Priscilla K. Cooper of Lawrence Berkeley National Laboratory; and Binghui Shen of City of Hope National Medical Center and Beckman Research Institute and Zhejiang University (China).
