title: 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.
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Erica
authors.initials: E
authors.lastname: Friedrich-Heineken
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Henneke
authors.forename: Elena
authors.initials: E
authors.lastname: Ferrari
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hübscher
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004706:Endodeoxyribonucleases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D045585:Flap Endonucleases; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D046911:Macromolecular Substances; D009711:Nucleotides; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D043211:Exodeoxyribonuclease V; D008274:Magnesium; D008239:Lysine