The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair.
The conserved, structure-specific flap endonuclease FEN1 cleaves 5' DNA flaps that arise during replication or repair. To address in vivo mechanisms of flap cleavage, we developed a screen for human FEN1 mutants that are toxic when expressed in yeast. Two targets were revealed: the flexible loop domain and the catalytic site. Toxic mutants caused G(2) arrest and cell death and were unable to repair methyl methanesulfonate lesions. All the mutant proteins retained flap binding. Unlike the catalytic site mutants, which lacked cleavage of any 5' flaps, the loop mutants exhibited partial ability to cut 5' flaps when an adjacent single nucleotide 3' flap was present. We suggest that the flexible loop is important for efficient cleavage through positioning the 5' flap and the catalytic site.
Storici
Francesca
F
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Henneke
Ghislaine
G
Ferrari
Elena
E
Gordenin
Dmitry A
DA
Hübscher
Ulrich
U
Resnick
Michael A
MA
D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D045585:Flap Endonucleases; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation
D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D043211:Exodeoxyribonuclease V