Walsh Lindsey, Schmuckli-Maurer Jacqueline, Billinton Nicholas, Barker M Gordon, Heyer Wolf-Dietrich, Walmsley Richard M
Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester, M60 1QD, UK.
Curr Genet. 2002 Jul;41(4):232-40. doi: 10.1007/s00294-002-0302-2. Epub 2002 Jun 27.
DNA damage checkpoints regulate a number of physiological responses after DNA damage. The transcriptional level of many genes is specifically induced in response to genotoxic stress in a checkpoint-dependent manner. The regulation of DNA damage-induced transcription of RAD54 and RNR2 by RAD9, DDC1, DUN1, CRT1 and MBP1 was investigated in Saccharomyces cerevisiae, using green fluorescent protein reporter assays and Northern blots. RAD54 and RNR2 reporter activity in response to the DNA damaging agent, methyl methanesulphonate, was measured in ddc1-Delta, rad9-Delta, ddc1-Delta/rad9-Delta, dun1-Delta, crt1-Delta and mbp1-Delta mutants and was compared with that of the wild type. RAD9 and DDC1 were shown to be required for a full RNR2 transcriptional response, although with the double mutant, ddc1-Delta/rad9-Delta, no additive effect on RNR2 induction was observed. RAD54 promoter activity was not significantly reduced in either rad9-Delta or ddc1-Delta mutants and was only partially reduced in the rad9-Delta/ddc1-Delta strain, suggesting that DNA damage induction of RAD54 must depend on other genes, in addition to RAD9 and DDC1. In the dun1-Delta mutant, RNR2 promoter activity was lowered, whilst that of RAD54 was increased, confirming that DUN1 is required for transcriptional induction of RNR2, but is not required for damage-induced transcription of RAD54. Analysis of the crt1-Delta strain confirmed that RNR2 is regulated via the CRT1 repressor pathway, downstream of DUN1, but RAD54 is not. MBP1 was shown to be required for transcription of RNR2, but was not needed for transcription of RAD54. These results indicate that RNR2 and RAD54 are regulated in different ways.
DNA损伤检查点在DNA损伤后调节多种生理反应。许多基因的转录水平会以检查点依赖的方式，对基因毒性应激作出特异性诱导。利用绿色荧光蛋白报告基因检测法和Northern印迹法，在酿酒酵母中研究了RAD9、DDC1、DUN1、CRT1和MBP1对DNA损伤诱导的RAD54和RNR2转录的调控。在ddc1-Δ、rad9-Δ、ddc1-Δ/rad9-Δ、dun1-Δ、crt1-Δ和mbp1-Δ突变体中，检测了对DNA损伤剂甲磺酸甲酯作出反应的RAD54和RNR2报告基因活性，并与野生型进行了比较。结果显示，完整的RNR2转录反应需要RAD9和DDC1，不过在双突变体ddc1-Δ/rad9-Δ中，未观察到对RNR2诱导的累加效应。在rad9-Δ或ddc1-Δ突变体中，RAD54启动子活性并未显著降低，而在rad9-Δ/ddc1-Δ菌株中只是部分降低，这表明RAD54的DNA损伤诱导除了依赖RAD9和DDC1外，还必定依赖其他基因。在dun1-Δ突变体中，RNR2启动子活性降低，而RAD54的启动子活性增加，证实DUN1是RNR2转录诱导所必需的，但不是RAD54损伤诱导转录所必需的。对crt1-Δ菌株的分析证实，RNR2是通过DUN1下游的CRT1阻遏途径调控的，但RAD54并非如此。结果表明，MBP1是RNR2转录所必需的，但不是RAD54转录所必需的。这些结果表明，RNR2和RAD54的调控方式不同。
