PMC:4103454 / 14561-16770 JSONTXT

{"project":"2_test","denotations":[{"id":"24981866-10493868-20021742","span":{"begin":747,"end":751},"obj":"10493868"}],"text":"FAN1 Enzymes Are Monomeric in Solution\nThe isolated VRR-Nuc domains studied here form dimers, consistent with an important role in the recognition of four-way DNA junctions, whereas the FAN1 orthologs exhibited no activity on four-way DNA junctions, instead processing 5′ flaps very efficiently. This suggested that the FAN1 VRR-Nuc domains may act as monomeric enzymes, and this hypothesis was investigated further.\nUsing analytical gel filtration, column retention times of hFAN1 and mFAN1 were consistent with a molecular weight of approximately 200 kDa, raising the possibility of a dimeric interaction (Figure 4A). By contrast, the column retention time of pFAN1 was consistent with a monomer. PSIPRED secondary structure predictions (Jones, 1999) suggest that both hFAN1 and mFAN1 are essentially unstructured over the first ∼370 residues, potentially explaining the low retention times in gel filtration experiments. To test this, we removed the first 359 residues of hFAN1 leaving the region of homology to pFAN1. This protein (hFAN1_360) was fully functional in nuclease assays (not shown) and eluted from gel filtration with a retention time consistent with a monomer. Although it remained possible that the N-terminal regions within mouse and human FAN1 might be mediating self-association, sedimentation velocity analysis showed unambiguously that, like pFAN1, full-length mouse FAN1 is monomeric (Figure 4B). Finally, limited tryptic proteolysis of hFAN1_360 produced three protected fragments identified by mass spectrometry as amino acids 360–507, 515–790, and 795–1,012. Gel filtration performed on a preparative scale tryptic digest gave rise to a single eluted peak containing three species with a retention time almost identical to the undigested molecule (Figure 4C). Indeed, attempts to express constructs corresponding to the tryptic fragments either in isolation or as pairs were unsuccessful, suggesting an intimate association of all three domains. Together, these results support the hypothesis that eukaryotic FAN1 proteins comprise an unstructured N terminus and a compact C terminus comprised of three domains that broadly correspond to the SAP, TPR, and VRR-Nuc homology regions."}