The acetylatable lysines of human Fen1 are important for endo- and exonuclease activities.

Abstract

Human Fen1 can be acetylated in vivo and in vitro resulting in reduced endonuclease and exonuclease activities in vitro. Acetylation occurs at four lysines located at the C terminus of Fen1, which is important for DNA binding. In this paper we show that Fen1 mutant proteins lacking the lysines at the C terminus have both reduced PCNA independent exonucleolytic and endonucleolytic activities. However, lysines at the C terminus are not required for PCNA stimulation of human Fen1. A double flap substrate was optimal for human Fen1 endonuclease and did not require the C-terminal lysines. Similarly, a one nucleotide 3'-overhang nick substrate was optimal for human Fen1 exonuclease and also did not require the C-terminal lysines. Finally, we found by an electromobility shift assay that human Fen1 had a different mode of binding with a double flap substrate containing a one nucleotide 3'-tail when compared to various other flap substrates. Taken together, our results confirm the double flap substrate as the likely in vivo intermediate for human Fen1 and that the C-terminal lysines are important for the endonuclease and exonuclease activities likely through DNA binding.