Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.
AUTOR(ES)
Taghian, D G
RESUMO
Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=232490Documentos Relacionados
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