title: Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments.
abstract: There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG)n and (GAA)n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG)n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for single-stranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.
authors.affiliation: Department of Radiation Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010, USA.
authors.forename: Purnima
authors.initials: P
authors.lastname: Singh
authors.forename: Li
authors.initials: L
authors.lastname: Zheng
authors.forename: Valerie
authors.initials: V
authors.lastname: Chavez
authors.forename: Junzhuan
authors.initials: J
authors.lastname: Qiu
authors.forename: Binghui
authors.initials: B
authors.lastname: Shen
mesh_terms: D000409:Alanine / Q000235:genetics; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004271:DNA, Fungal / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D045585:Flap Endonucleases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D020690:GTPase-Activating Proteins / Q000502:physiology*; D018698:Glutamic Acid / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D018911:Trinucleotide Repeats / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D020690:GTPase-Activating Proteins; D009692:Nucleic Acid Heteroduplexes; C022262:Okazaki fragments; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D004247:DNA; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C028450:exodeoxyribonuclease I; C468323:RAD27 protein, S cerevisiae; D000409:Alanine