RESUMO
Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.
Assuntos
Dano ao DNA , Endonucleases Flap/fisiologia , Recombinação Genética , Animais , Sequência de Bases , Ciclo Celular , Galinhas , DNA/química , Reparo do DNA , DNA Complementar/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Endonucleases Flap/metabolismo , Citometria de Fluxo , Raios gama , Cinética , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência do Ácido Nucleico , Troca de Cromátide Irmã , Fatores de Tempo , TransfecçãoRESUMO
Flap endonuclease 1 (FEN-1) is a nuclear enzyme involved in DNA metabolism, such as replication, repair, and recombination. Here, we report the comparative genomic organization of the chicken, mouse, and human FEN1 genes as well as the comparative organization of a small gene (C11orf10) located immediately upstream of the FEN1 gene in reverse orientation. Immunostaining revealed that the C11orf10 protein, unlike FEN-1, is located in the cytoplasm, suggesting that these two proteins do not form a physical complex. Importantly, in the human genome, the two mRNAs are overlapping (14 bp) in their 5' ends. Thus, the FEN1/C11orf10 locus is a new example of two overlapping, divergent transcription units in the human genome.
Assuntos
Endodesoxirribonucleases/genética , Genoma Humano , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/enzimologia , Galinhas , Clonagem Molecular , DNA Complementar/metabolismo , Células Epiteliais/citologia , Endonucleases Flap , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The structure-specific FEN-1 endonuclease has been implicated in various cellular processes, including DNA replication, repair and recombination. In vertebrate cells, however, no in vivo evidence has been provided so far. Here, we knocked out the FEN-1 gene (FEN1) in the chicken DT40 cell line. Surprisingly, homozygous mutant (FEN1-/-) cells were viable, indicating that FEN-1 is not essential for cell proliferation and thus for Okazaki fragment processing during DNA replication. However, compared with wild-type cells, FEN1-/- cells exhibited a slow growth phenotype, probably due to a high rate of cell death. The mutant cells were hypersensitive to methylmethane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and H2O2, but not to UV light, X-rays and etoposide, suggesting that FEN-1 functions in base excision repair in vertebrate cells.
