Analysis of human flap endonuclease 1 mutants reveals a mechanism to prevent triplet repeat expansion.
Flap endonuclease 1 (FEN1), involved in the joining of Okazaki fragments, has been proposed to restrain DNA repeat sequence expansion, a process associated with aging and disease. Here we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity. Introduction of these mutants into yeast led to sequence expansions. Reconstituting triplet repeat expansion in vitro, we previously found that DNA ligase I promotes expansion, but FEN1 prevents the ligation that forms expanded products. Here we show that among the intermediates that could generate sequence expansion, a bubble is necessary for ligation to produce the expansion product. Severe exonuclease defects in the mutant FEN1 suggested that the inability to degrade bubbles exonucleolytically leads to expansion. However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated. Instead, we propose that FEN1 suppresses sequence expansion by degrading flaps that equilibrate with bubbles, thereby reducing bubble concentration. In this way FEN1 employs endonuclease rather than exonuclease to prevent expansions. A model is presented describing the roles of DNA structure, DNA ligase I, and FEN1 in sequence expansion.
Liu
Yuan
Y
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, New York 14642, USA.
Bambara
Robert A
RA
D001483:Base Sequence; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D045585:Flap Endonucleases; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D019680:Trinucleotide Repeat Expansion
C000607892:LIG1 protein, human; D009841:Oligonucleotides; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D043211:Exodeoxyribonuclease V; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D005998:Glycine