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。这些途径受到双链断裂（DSB）和DNA损伤（如链间交叉汇（ICL））的刺激。我们利用三螺旋形成的寡核苷酸连接到补骨脂素（pso-TFO），在活哺乳动物细胞基因组的特定位点引入DNA ICL。与靶区同源性的双工DNA的共同引入导致供体的精确敲入频率比单独使用供体高2-3个数量级。敲入在缺乏ERCC1/XPF的细胞中被消除，ERCC1/XPF参与重组途径和交联修复。另外，单链寡核苷酸供体（SSO）与pso-TFO共同引入。它们的活性是双敲入供体的10倍。与双链供体相比，ERCC1/XPF缺失细胞的SSO疗效进一步提高。被切除的单链末端被认为是SSO序列调制的关键中间体，以及双链供体敲入。我们问，如果两个供体物种都与pso-TFO一起存在，是否会在这些目的之间存在竞争。双供体敲入的频率不受100倍摩尔过量SSO的影响。当使用归巢内切酶I-SceI在靶位点启动HDR时，获得了相同的结果。我们得出结论，DSB进入不同的HDR途径是由这些途径中的核酸伙伴以外的因素控制的。基于对酵母的研究，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，在哺乳动物细胞中，单链寡核苷酸供体更有可能进入双链断裂修复的非同源末端连接（NHEJ）途径。NHEJ被认为是一种非模板途径，我们的结论代表了对这种观点的一种新的修正。

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 in mammalian cells 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.