Institute for Cancer Genetics, Vagelos College of Physicians and Surgeons, Columbia University, New York City, NY 10032.
Department of Medicine, Columbia University Medical Center, New York, NY 10032.
Proc Natl Acad Sci U S A. 2023 Aug;120(31):e2301972120. doi: 10.1073/pnas.2301972120. Epub 2023 Jul 24.
PARP1 (poly-ADP ribose polymerase 1) is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes chromatin and recruits other DNA repair factors, including XRCC1 and DNA Ligase 3, to maintain genomic stability. Here we show that, in contrast to the normal development of Parp1-null mice, heterozygous expression of catalytically inactive Parp1 (E988A, ) acts in a dominant-negative manner to disrupt murine embryogenesis. As such, all the surviving F1 mice are chimeras with mixed (neoR retention) cells that act similarly to . Pure F2 embryos were found at Mendelian ratios at the E3.5 blastocyst stage but died before E9.5. Compared to cells, genotype and expression-validated pure cells retain significant ADP-ribosylation and PARylation activities but accumulate markedly higher levels of sister chromatid exchange and mitotic bridges. Despite proficiency for homologous recombination and nonhomologous end-joining measured by reporter assays and supported by normal lymphocyte and germ cell development, cells are hypersensitive to base damages, radiation, and Topoisomerase I and II inhibition. The sensitivity of cells to base damages and Topo inhibitors exceed controls. The findings show that the enzymatically inactive PARP1 dominant negatively blocks DNA repair in selective pathways beyond wild-type PARP1 and establishes a crucial physiological difference between PARP1 inactivation vs. deletion. As a result, the expression of enzymatically inactive PARP1 from one allele is sufficient to abrogate murine embryonic development, providing a mechanism for the on-target side effect of PARP inhibitors used for cancer therapy.
PARP1（多聚 ADP 核糖聚合酶 1）被 DNA 链断裂招募并激活，催化 NAD+ 生成多聚 ADP-核糖（PAR）链。PAR 松弛染色质并招募其他 DNA 修复因子，包括 XRCC1 和 DNA 连接酶 3，以维持基因组稳定性。在这里，我们表明，与 Parp1 缺失型小鼠的正常发育相反，催化失活的 Parp1（E988A， ）的杂合表达以显性负性方式作用，破坏小鼠胚胎发生。因此，所有存活的 F1 小鼠都是嵌合体，具有混合的 （neoR 保留）细胞，其作用类似于 。在 E3.5 胚泡阶段，以孟德尔比率发现纯 F2 胚胎，但在 E9.5 之前死亡。与 细胞相比，经基因型和表达验证的纯 细胞保留了显著的 ADP-核糖基化和 PAR 化活性，但积累了明显更高水平的姐妹染色单体交换和有丝分裂桥。尽管通过报告基因测定和正常淋巴细胞和生殖细胞发育支持同源重组和非同源末端连接的效率，但 细胞对碱基损伤、辐射和拓扑异构酶 I 和 II 抑制非常敏感。 细胞对碱基损伤和 Topo 抑制剂的敏感性超过了对照。这些发现表明，酶失活的 PARP1 显性负性地在超过野生型 PARP1 的选择性途径中阻断 DNA 修复，并在 PARP1 失活与缺失之间建立了关键的生理差异。因此，来自一个等位基因的酶失活 PARP1 的表达足以废除小鼠胚胎发育，为用于癌症治疗的 PARP 抑制剂的靶标副作用提供了一种机制。
