The Fen1 extrahelical 3'-flap pocket is conserved from archaea to human and regulates DNA substrate specificity.
Fen1 is a key enzyme for the maintenance of genetic stability in archaea and eukaryotes and is classified as a tumor suppressor. Very recent structural data obtained from Archaeoglobus fulgidus Fen1 suggest that an extrahelical 3'-flap pocket is responsible for substrate specificity, by binding to the unpaired 3'-flap and by opening and kinking the DNA. Since the extrahelical 3'-flap pocket in archaeal Fen1 contains seven amino acids that are conserved to a great extent in human Fen1, we have mutated the four conserved or all seven amino acids in the human Fen1 extrahelical 3'-flap pocket to alanine. Our data suggest that the human extrahelical 3'-flap pocket mutants have lost substrate specificity to the double-flap DNA. Moreover, loss of high affinity for the unpaired 3'-flap suggests that the extrahelical 3'-flap pocket is essential for recognition and processing of the 'physiological' template. Human PCNA could stimulate the human Fen1 extrahelical 3'-flap pocket mutants but not restore their specificity. Thus the substrate specificity of Fen1 has been functionally conserved over a billion years from archaea to human.
Friedrich-Heineken
Erica
E
Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH 8057 Zürich, Switzerland.
Hübscher
Ulrich
U
D000595:Amino Acid Sequence; D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001483:Base Sequence; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity
D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D045585:Flap Endonucleases; C468317:FEN1 protein, human