Posttranscriptional regulation of the RAD5 DNA repair gene by the Dun1 kinase and the Pan2-Pan3 poly(A)-nuclease complex contributes to survival of replication blocks.
The yeast Dun1 kinase has complex checkpoint functions including DNA damage-dependent cell cycle arrest in G(2)/M, transcriptional induction of repair genes, and regulation of postreplicative DNA repair pathways. Here we report that the Dun1 forkhead-associated domain interacts with the Pan3 subunit of the poly(A)-nuclease complex and that dun1pan2 and dun1pan3 double mutants are dramatically hypersensitive to replicational stress. This phenotype was independent of the function of Dun1 in regulating deoxyribonucleotide levels as it was also observed in strains lacking the ribonucleotide reductase inhibitor Sml1. dun1pan2 mutants initially arrested normally in response to replication blocks but died in the presence of persistent replication blocks with considerably delayed kinetics compared with mutants lacking the Rad53 kinase, indicating that the double mutation does not compromise the intra-S phase checkpoint. Interestingly, the RAD5 gene involved in error-free postreplication repair pathways was specifically up-regulated in dun1pan2 double mutants. Moreover, inducible overexpression of RAD5 mimicked the double mutant phenotype by hypersensitizing dun1 mutants to replication blocks. The data indicate that Dun1 and Pan2-Pan3 cooperate to regulate the stoichiometry and thereby the activity of postreplication repair complexes, suggesting that posttranscriptional mechanisms complement the transcriptional response in the regulation of gene expression by checkpoint signaling pathways in Saccharomyces cerevisiae.
Hammet
Andrew
A
St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, The University of Melbourne, 9 Princes Street, Fitzroy, Victoria 3065, Australia.
Pike
Brietta L
BL
Heierhorst
Jörg
J
D000251:Adenosine Triphosphatases; D015152:Blotting, Northern; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D064447:Checkpoint Kinase 2; D003593:Cytoplasm / Q000378:metabolism; D004265:DNA Helicases; D004305:Dose-Response Relationship, Drug; D005095:Exoribonucleases / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008957:Models, Genetic; D010641:Phenotype; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation
D000251:Adenosine Triphosphatases; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004265:DNA Helicases; D005095:Exoribonucleases; D005656:Fungal Proteins; D006918:Hydroxyurea; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D012313:RNA; D029701:Saccharomyces cerevisiae Proteins; C104169:DUN1 protein, S cerevisiae; C067022:RAD53 protein, S cerevisiae; C101747:PAN3 protein, S cerevisiae; C076225:RAD5 protein, S cerevisiae