DNA-damage induction of RAD54 can be regulated independently of the RAD9- and DDC1-dependent checkpoints that regulate RNR2.
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.
Walsh
Lindsey
L
Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester, M60 1QD, UK.
Schmuckli-Maurer
Jacqueline
J
Billinton
Nicholas
N
Barker
M Gordon
MG
Heyer
Wolf-Dietrich
WD
Walmsley
Richard M
RM
D018797:Cell Cycle Proteins / Q000235:genetics; D004249:DNA Damage; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; D008164:Luminescent Proteins; D009153:Mutagens; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D008741:Methyl Methanesulfonate; D012264:Ribonucleotide Reductases; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes