RESUMO
The nearly one million ALU: repeats in human chromosomes are a potential threat to genome integrity. ALU:s form dense clusters where they frequently appear as inverted repeats, a sequence motif known to cause DNA rearrangements in model organisms. Using a yeast recombination system, we found that inverted ALU: pairs can be strong initiators of genetic instability. The highly recombinagenic potential of inverted ALU: pairs was dependent on the distance between the repeats and the level of sequence divergence. Even inverted ALU:s that were 86% homologous could efficiently stimulate recombination when separated by <20 bp. This stimulation was independent of mismatch repair. Mutations in the DNA metabolic genes RAD27 (FEN1), POL3 (polymerase delta) and MMS19 destabilized widely separated and diverged inverted ALU:s. Having defined factors affecting inverted ALU: repeat stability in yeast, we analyzed the distribution of ALU: pairs in the human genome. Closely spaced, highly homologous inverted ALU:s are rare, suggesting that they are unstable in humans. ALU: pairs were identified that are potential sites of genetic change.
Assuntos
Elementos Alu/genética , DNA Fúngico/genética , Evolução Molecular , Genoma Humano , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae , Leveduras/genética , Biologia Computacional , DNA Polimerase III , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases Flap , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Marcadores Genéticos/genética , Humanos , Mutação/genética , Fatores de Transcrição , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Fen1/Rad27 nuclease activity, which is important in DNA metabolism, is stimulated by proliferating cell nuclear antigen (PCNA) in vitro. The in vivo role of the PCNA interaction was investigated in the yeast Rad27. A nuclease-defective rad27 mutation had a dominant-negative effect that was suppressed by a mutation in the PCNA binding site, thereby demonstrating the importance of the Rad27-PCNA interaction. The PCNA-binding defect alone had little effect on mutation, recombination, and the methyl methanesulfonate (MMS) response in repair-competent cells, but it greatly amplified the MMS sensitivity of a rad51 mutant. Furthermore, the PCNA binding mutation resulted in lethality when combined with a homozygous or even a heterozygous pol3-01 mutation in the 3'-->5' exonuclease domain of DNA polymerase delta. These results suggest that phenotypically mild polymorphisms in DNA metabolic proteins can have dramatic consequences when combined.
