FEN Biology
Branched DNA and FEN Biology: Not Just a Plot from The X Files
Branched DNA has appeared in "The X Files", supposedly implanted into Agent Dana Scully by aliens so they could track her. Far from being science fiction, branched DNA plays important roles in biology. We usually think about our DNA as being double-helical in structure, but in fact, DNA adopts a number of different shapes as part of normal bilogical processes.
Flap endonucleases, 5' nucleases or 5'-3' exonucleases are some of the names given to a group of ubiquitous structure-specific nucleases that can cleave branched DNA, thus restoring the classical double helix. They occur in all living organisms from bacteria to Homo sapiens (recent FEN papers from Sheffield University).
Some viruses even carry genes encoding their own flap endonuclease enzymes. In the last few years most scientific papers describing these enzymes call them flap endonucleases (FENs), so that is what we will call them here. Apart from being essential for all cells (they participate in DNA replication and repair processes, e.g. see review by Lewis et al 2016), they are also widely used in biotechnology in genotyping, quantitative PCR, polymorphism screening and molecular biology.
DNA polymerase I possesses a flap endonuclease domain in addition to the well known Klenow (or large) fragment carrying the polymerase and proofreading polymerase domains. The DNA Pol1 FEN domain was known as the small fragment and was originally described as having 5'-3' exonuclease activity. However, this is indeed a FEN as can be appreciated from the crystal structures FENs and the Thermus aquaticus DNA Pol1.FENs are metalloenzymes, with binding sites for 2 or 3 divalent metal ions (Syson et al*). They bind but do not cut DNA in the absence of a suitable divalent metal ion. These enzymes can use a range of divalent metal cofactors ranging incuding Mg, Mn, Co, Ni, Fe, Ni, Zn and even Cu (Garforth & Sayers, Feng et al). The core structure consists of a central beta sheet with a number of helices adorning it. The active site contains several conserved carboxylates (mostly aspartic acid residues), a conserved tyrosine and important lysine and arginine residues.
There are a number of good reviews on biological roles of the FENs (e.g. Bob Bambara's Annual Review of Biochemistry or Peter Burgers' JBC review*). Basically, at least one FEN is required for cell viability as has been demonstrated in mammalian and bacterial cells. For example FEN knockout mice fail to develop through embryogenesis (Kucherlapati et al) and the FEN domain of PolI is required for cell viability in Streptococcus pneumoniae (Diaz et al).
The situation was a little confused regarding bacterial FENs until 2007. For example, Cathy Joyce at Yale showed that the gene encoding DNA PolI (the polA gene) can be deleted in E. coli resulting in bacteria that can grow, albeit slowly on minimal media yet Pol1 was essential for Streptococcus pneumoniae. Joyce also showed that adding back a gene encoding just the FEN-domain of PolI was enough to restore full viability (Joyce & Grindley, 1984).
However, at the time she did not know that many bacteria contain a second FEN-encoding gene (see Allen et al) which I hypothesized might be a backup for the polA-encoded FEN function in 1994 (Sayers, 1994*). Indeed, this seems to be the case and late in 2007, Fukushima et al showed that bacteria require at least one functional FEN activity for viability (Fukushima et al 2007).
So what do FENs do in the cell? They appear to play major roles in processing the remnants of the RNA primers that are used to initiate Okazaki fragment synthesis (so-called lagging strand synthesis), in maintaining genome stability and in DNA repair (e.g. see Greene et al and Lindahl & Wood* and the reviews above).