Double strand break repair

来自外源供体DNA的信息可以通过同源定向修复（HDR）1途径引入基因组中。这些途径受到双链断裂（DSB）和DNA损伤（如链间交联（ICL））的刺激。我们已经采用了三螺旋形成寡核苷酸连接到peptide（pso-TFO），在一个特定的网站在活的哺乳动物细胞的基因组中的DNA ICL。具有靶区域同源性的双链体DNA的共引入导致供体的精确敲入，其频率比单独供体高2-3个数量级。 敲入在ERCC 1/XPF缺陷的细胞中被消除，ERCC 1/XPF参与重组途径以及交联修复。单独地，将单链寡核苷酸供体（SSO）与pso-TFO共引入。这些比双链体敲入供体的活性高10倍。与双链体供体相反，SSO功效在ERCC 1/XPF缺陷的细胞中进一步升高。切除的单链末端已被牵连作为关键中间体的序列调节SSO，以及双链体供体敲入。我们询问如果两者都与pso-TFO一起存在，供体物种之间是否会竞争这些末端。双链体供体敲入的频率不受100倍摩尔过量的SSO的影响。当归巢核酸内切酶I-SceI用于在靶位点启动HDR时，获得了相同的结果。我们的结论是，进入不同的HDR途径的DSB控制的因素以外的核酸合作伙伴在这些途径。基于在酵母中的工作，认为单链寡核苷酸供体进入单链退火途径。然而，我们的实验表明，单链寡核苷酸供体更可能进入双链断裂修复的非同源末端连接（NHEJ）途径。NHEJ被认为是一个非模板化的途径，我们的结论代表了这一观点的一个新的修改。

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.