Biological activity of Arabidopsis flap endonuclease 1 (FEN1) is modulated by nuclear factors that inhibit its aggregation.

Abstract

Flap endonuclease 1 (FEN1) is part of a group of nuclear enzymes involved in eukaryotic DNA replication and repair. In our studies, using both biochemical and biophysical approaches, we demonstrated that Arabidopsis thaliana FEN1 (AtFEN1) is unstable and prone to aggregation. To understand the reasons for AtFEN1 aggregation, we first analyzed the effects of heparin sodium and sodium chloride on its aggregation. We found that both heparin sodium and sodium chloride modulated the aggregation of this enzyme; however, achieving the same level of aggregation inhibition required using a sodium chloride concentration five orders of magnitude higher than that of heparin. Subsequently, to identify potential nuclear factors that may modulate the biological activity of AtFEN1 in vivo, we used DNA. Our experiments showed that negatively charged double-stranded DNA (dsDNA), similarly to the double-flap DNA (dfDNA) substrate of AtFEN1, inhibited AtFEN1 aggregation. This inhibitory effect was much less pronounced when single-stranded DNA (ssDNA) was used. Moreover, dfDNA prevented the loss of biological activity of AtFEN1. Finally, we revealed that AtFEN1 aggregation was also blocked by Arabidopsis proliferating cell nuclear antigen 1 (PCNA1), a natural interacting protein of AtFEN1. However, this effect was observed only when the putative PCNA-interacting protein (PIP)-box sequence was present in AtFEN1.