DNA-damage induction of RAD54 can be regulated independently of the RAD9- and DDC1-dependent checkpoints that regulate RNR2

Abstract.

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- Δ, rad9- Δ, ddc1- Δ /rad9- Δ, dun1- Δ, crt1- Δ and mbp1- Δ 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- Δ /rad9- Δ, no additive effect on RNR2 induction was observed. RAD54 promoter activity was not significantly reduced in either rad9- Δ or ddc1- Δ mutants and was only partially reduced in the rad9- Δ /ddc1- Δ strain, suggesting that DNA damage induction of RAD54 must depend on other genes, in addition to RAD9 and DDC1. In the dun1- Δ 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- Δ 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.