Structural basis of 5′ flap recognition and protein–protein interactions of human flap endonuclease 1

Abstract

Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5′ flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein–protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5′ flap prior to its threading into the active site for cleavage. We also found that the β-pin region is rigidified into a short helix in R192F hFEN1–DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the β-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.

Functional regulation of the structure-specific endonuclease FEN1 by the human cytomegalovirus protein IE1 suggests a role for the re-initiation of stalled viral replication forks

“…In contrast, phosphorylation at serine 187 was shown to inhibit flap endonuclease activity by preventing PCNA binding [20]. All in all, Xu and colleagues proposed a model whereby methylation and phosphorylation antagonistically act on FEN1 by either promoting flap endonuclease or gap endonuclease activity, respectively [24]. Interestingly, phosphorylation of FEN1 stimulates its own SUMOylation [21,24].…”

“…All in all, Xu and colleagues proposed a model whereby methylation and phosphorylation antagonistically act on FEN1 by either promoting flap endonuclease or gap endonuclease activity, respectively [24]. Interestingly, phosphorylation of FEN1 stimulates its own SUMOylation [21,24]. While modification with SUMO3 was shown to stimulate FEN1 ubiquitination and subsequent proteasomal degradation in a cell cycle dependent manner, modification with SUMO1 prevents SUMO3-mediated degradation and promotes interaction of FEN1 with the Rad9-Rad1-Hus1 complex [21,25].…”

“…As phosphorylation of FEN1 is described to stimulate its double strand break (DSB)-generating gap endonuclease activity [24], we propose that FEN1 might be involved in the induction of γH2AX, a sensitive marker for DSBs, during the late phase of HCMV infection [6][7][8] This finding strongly indicates that FEN1 might be required for the IE1-driven activation of either mock infected or infected with AD169 at an MOI of 3 (E) or with AD169 (wt) at an MOI of 7 and equivalent genome copy numbers of a recombinant strain lacking IE1 (ΔIE1) (F). At the indicated times post infection, cells were harvested and analyzed by Western blotting for FEN1, pFEN1, IE1, IE2 (for F), UL44, MCP, and β-actin.…”

“…Methylation of FEN1 at arginine 192 was shown to promote binding to the DNA sliding clamp PCNA, which in turn stimulates FEN1’s flap endonuclease activity, while FEN1 phosphorylation at serine 187 is simultaneously inhibited [ 22 , 23 ]. Meanwhile, an additional structural analysis could provide evidence that methylation at arginine 192 prevents FEN1 phosphorylation by preventing access of protein kinases [ 24 ]. Furthermore, by utilizing the FEN1 methylation mimic R192F, the authors could not only show that FEN1 phosphorylation and kinase binding were impaired but also its gap endonuclease activity [ 24 ].…”

“…Meanwhile, an additional structural analysis could provide evidence that methylation at arginine 192 prevents FEN1 phosphorylation by preventing access of protein kinases [ 24 ]. Furthermore, by utilizing the FEN1 methylation mimic R192F, the authors could not only show that FEN1 phosphorylation and kinase binding were impaired but also its gap endonuclease activity [ 24 ]. In contrast, phosphorylation at serine 187 was shown to inhibit flap endonuclease activity by preventing PCNA binding [ 20 ].…”