Institut Curie, CNRS-UMR3347, INSERM-U1021, 91405 Orsay, France, DNA Therapeutics, Génopole, 91000 Evry, France, Institut Curie, CNRS-UMR3348, Plateforme PICT-IBiSA, 91405 Orsay, France and Museum National d'Histoire Naturelle, USM503, 75231 Paris, France.
Nucleic Acids Res. 2013 Aug;41(15):7344-55. doi: 10.1093/nar/gkt522. Epub 2013 Jun 12.
One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.
修复的早期主要步骤之一是在损伤部位募集修复蛋白，这是由磷脂酰肌醇 3-激酶相关激酶和/或多聚（ADP-核糖）聚合酶（PARP）控制的级联修饰协调的。我们使用模拟双链断裂（称为 Dbait）或单链断裂（称为 Pbait）的短干扰 DNA 分子来促进 DNA 依赖性蛋白激酶（DNA-PK）和 PARP 的激活。Dbait 结合并诱导 PARP 和 DNA-PK 活性，而 Pbait 仅作用于 PARP。因此，这两种分子的比较研究允许分析两条信号通路的各自作用：它们都募集参与单链断裂修复的蛋白质（PARP、XRCC1 和 PCNA），并防止它们在染色体损伤处募集。Dbait 但不是 Pbait，还抑制参与双链断裂修复的蛋白质（53BP1、NBS1、RAD51 和 DNA-PK）的募集。通过这些方式，Pbait 和 Dbait 扰乱 DNA 修复，从而使细胞对各种处理敏感。单链断裂修复的抑制取决于两种分子上主要蛋白质的直接捕获。双链断裂修复的抑制可能是间接的，是由激活的 DNA-PK 对双链断裂修复蛋白和染色质靶标的磷酸化引起的。这两种分子的 DNA 修复抑制作用通过它们与 BRCA 突变的合成致死性得到证实。
