Double strand break repair

点击翻译按钮获取中文摘要

Information from exogenous donor DNA can be introduced into the genome via homology directed repair (HDR)1 pathways. These pathways are stimulated by double strand breaks (DSB) and by DNA damage such as interstrand crossinks (ICL). We have employed triple helix forming oligonucleotides linked to psoralen (pso-TFO) to introduce a DNA ICL at a specific site in the genome of living mammalian cells. Co-introduction of duplex DNA with target region homology resulted in precise knock-in of the donor at frequencies 2-3 orders of magnitude greater than with donor alone. Knock-in was eliminated in cells deficient in ERCC1/XPF which is involved in recombinational pathways as well as crosslink repair. Separately, single strand oligonucleotide donors (SSO) were co-introduced with the pso-TFO. These were 10 fold more active than the duplex knock-in donor. SSO efficacy was further elevated in cells deficient in ERCC1/XPF, in contrast to the duplex donor. Resected single strand ends have been implicated as critical intermediates in sequence modulation by SSO, as well as duplex donor knock-in. We asked if there would be a competition between the donor species for these ends if both were present with the pso-TFO. The frequency of duplex donor knock-in was unaffected by a 100-fold molar excess of the SSO. The same result was obtained when the homing endonuclease I-SceI was used to initiate HDR at the target site. We conclude that the entry of DSB into distinct HDR pathways is controlled by factors other than the nucleic acid partners in those pathways. Based on work in yeast it was believed that the single strand oligonuceotide donors entered a single strand annealing pathway. However our experiments suggest that the single strand oligonucleotide donors are more likely to enter the Non Homologous End Joining (NHEJ) pathway of double strand break repair. NHEJ is regarded as a nontemplated pathway and our conclusion represents a novel modification of this view.