Bracesco Nelson, Candreva Ema C, Keszenman Deborah, Sánchez Ana G, Soria Sandra, Dell Mercedes, Siede Wolfram, Nunes Elia
Lab. Radiobiología, Departamento Biofísica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Radiat Environ Biophys. 2007 Nov;46(4):401-7. doi: 10.1007/s00411-007-0119-y. Epub 2007 Jul 12.
Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Delta/chk1Delta and rad17Delta/rad17Delta, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Delta/rad17Delta cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Delta/rad17Delta cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Delta/chk1Delta cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Delta/chk1Delta mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.
关卡是参与基因组稳定性的信号通路的组成部分。我们使用酿酒酵母突变株分析了Rad17和Chk1作为关卡以及在DNA修复中的假定双重功能。将二倍体关卡缺陷突变体chk1Delta/chk1Delta和rad17Delta/rad17Delta以及同基因野生型菌株的对数期群体暴露于拟放射性药物博来霉素（BLM）。通过脉冲场电泳、存活分数和增殖动力学测定的DNA双链断裂（DSB）在处理后或在有或无环己酰亚胺（CHX）的营养培养基中孵育后立即测量。处理后，野生型菌株中由BLM诱导的DSB随着孵育时间的延长而减少，染色体修复受到CHX的抑制。暴露于低浓度BLM的rad17Delta/rad17Delta细胞未显示DSB修复、低存活率，且CHX无影响。相反，暴露于高浓度BLM的rad17Delta/rad17Delta细胞显示DSB修复受到CHX的抑制。chk1Delta/chk1Delta细胞显示DSB修复，且CHX无影响；这些细胞在高浓度BLM处理后存活率最低。目前的结果表明，Rad17对于低浓度BLM（低水平氧化损伤）诱导后的DSB修复至关重要。chk1Delta/chk1Delta突变株中的观察结果表明，组成型非同源末端连接参与了BLM诱导的DSB的修复。与野生型细胞相比，关卡突变体中DNA修复和存活的差异表达表明存在一个调节开关网络，该网络根据DNA损伤的程度和遗传背景控制DSB修复并将其引导至替代途径。
