RESUMO
During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Polimerase I/metabolismo , DNA/metabolismo , Endonucleases Flap/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Animais , Células Cultivadas , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , Embrião de Mamíferos , Feminino , Endonucleases Flap/classificação , Endonucleases Flap/genética , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Saccharomyces cerevisiaeRESUMO
Flap endonuclease-1 (FEN1) belongs to the Rad2 family of structure-specific nucleases. It is required for several DNA metabolic pathways, including DNA replication and DNA damage repair. Here, we have identified a shade avoidance mutant, sav6, which reduces the mRNA splicing efficiency of SAV6. We have demonstrated that SAV6 is an FEN1 homologue that shows double-flap endonuclease and gap-dependent endonuclease activity, but lacks exonuclease activity. sav6 mutants are hypersensitive to DNA damage induced by ultraviolet (UV)-C radiation and reagents that induce double-stranded DNA breaks, but exhibit normal responses to chemicals that block DNA replication. Signalling components that respond to DNA damage are constitutively activated in sav6 mutants. These data indicate that SAV6 is required for DNA damage repair and the maintenance of genome integrity. Mutant sav6 plants also show reduced root apical meristem (RAM) size and defective quiescent centre (QC) development. The expression of SMR7, a cell cycle regulatory gene, and ERF115 and PSK5, regulators of QC division, is increased in sav6 mutants. Their constitutive induction is likely due to the elevated DNA damage responses in sav6 and may lead to defects in the development of the RAM and QC. Therefore, SAV6 assures proper root development through maintenance of genome integrity.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Endonucleases Flap/genética , Genoma de Planta/genética , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Endonucleases Flap/classificação , Endonucleases Flap/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Histocitoquímica , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Dados de Sequência Molecular , Mutação , Hormônios Peptídicos/genética , Hormônios Peptídicos/metabolismo , Filogenia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
FENs (flap endonucleases) play essential roles in DNA replication, pivotally in the resolution of Okazaki fragments. In eubacteria, DNA PolI (polymerase I) contains a flap processing domain, the N-terminal 5'-->3' exonuclease. We present evidence of paralogous FEN-encoding genes present in many eubacteria. Two distinct classes of these independent FEN-encoding genes exist with four groups of eubacteria, being identified based on the number and type of FEN gene encoded. The respective proteins possess distinct motifs hallmarking their differentiation. Crucially, based on primary sequence and predicted secondary structural motifs, we reveal key differences at their active sites. These results are supported by biochemical characterization of two family members--ExoIX (exonuclease IX) from Escherichia coli and SaFEN (Staphylococcus aureus FEN). These proteins displayed marked differences in their ability to process a range of branched and linear DNA structures. On bifurcated substrates, SaFEN exhibited similar substrate specificity to previously characterized FENs. In quantitative exonuclease assays, SaFEN maintained a comparable activity with that reported for PolI. However, ExoIX showed no observable enzymatic activity. A threaded model is presented for SaFEN, demonstrating the probable interaction of this newly identified class of FEN with divalent metal ions and a branched DNA substrate. The results from the present study provide an intriguing model for the cellular role of these FEN sub-classes and illustrate the evolutionary importance of processing aberrant DNA, which has led to their maintenance alongside DNA PolI in many eubacteria.