Flap endonuclease 1 (FEN1) is an essential protein involved in eukaryotic DNA replication. It has been demonstrated that FEN1 is involved in the maturation of Okazaki fragments. FEN1 forms a complex with proliferating cell nuclear antigen (PCNA), which stimulates the activity of this endonuclease. The lethal effects of FEN1 deficiency in mice and Arabidopsis confirm the fundamental role of this protein in both mammals and higher plants. In this study, we focused on the biochemical, biophysical, and structural analysis of AtFEN1 and its complexes formed with AtPCNA1 and DNA substrate. We found that the affinity of AtFEN1 for the double flap DNA substrate is two orders of magnitude stronger than for ssDNA or dsDNA molecules. The studies of AtFEN1 affinity to AtPCNA1 indicated a key role of the PCNA-interacting protein (PIP) box sequence of AtFEN1 for the formation of complexes with AtPCNA1. Analysis of kinetic parameters, including Km, of AtFEN1 in the presence of different concentrations of AtPCNA1 revealed that AtPCNA1 decreases the Km for this enzyme. Moreover, the enzymatic activity of AtFEN1 was stimulated by AtPCNA1 regardless of the presence or absence of the PIP-box sequence in AtFEN1. Using cryo-EM technique, we determined the three-dimensional structure of the AtFEN1/AtPCNA1/DNA substrate complex. Finally with the help of the bimolecular fluorescence complementation technique, we showed the formation of AtFEN1/AtPCNA1 complexes in plant cell nuclei.
Funding:
This project was founded by National Science Centre grant no. 2019/33/B/NZ3/01568 to WS.