The regulatory network controlling DNA damage responses in Saccharomyces cerevisiae
DNA is subject to attack by DNA damaging agents from both environmental and endogenous sources. In response to DNA damage, living organisms enhance expression of many related genes to facilitate DNA repair and survival. The SOS response is a well-understood prokaryotic regulatory cascade that controls the expression of more than 30 genes in response to DNA damage. However, in eukaryotic organisms from simple budding yeast to human, such a regulatory network has not been reported.Previous research in our laboratory found that among DNA repair mutants of Saccharomyces cerevisiae, only rad6 and rad18 defective in the post-replication repair pathway significantly affected DNA damage induction of several genes examined. Rad6 and Rad18 form a ubiquitin conjugation-ligase complex and are required for the cellular tolerance to damaged DNA. Since the Rad6-Rad18 complex binds to single-stranded DNA, it may act as a DNA damage sensor required for the activation of DNA damage-induced transcription. We performed microarray analysis and found that the induction of up to 379 genes, including those involved in DNA repair, control of replication and transcription, regulation of the cell cycle and cell metabolism, are compromised in the rad6 and rad18 mutants. Although Rad6/Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA) following DNA damage to initiate a damage tolerance response, PCNA ubiquitination is not required for DNA damage induction. In budding yeast, cell-cycle checkpoints are involved in the control of DNA damage induction of gene expression through phosphorylation of a protein kinase Rad53 by two pathways represented by Rad24 and Sgs1. The Rad6-Rad18 complex appears to function in the Rad24 pathway and parallel to Sgs1. We further demonstrated that the Rad17 subunit of the 9-1-1 complex is subject to Rad6/Rad18- and DNA damage-dependent mono-ubiquitination and that the Rad17-Lys197 residue with flanking sequences homologous to Lys164 of PCNA is absolutely required for the DNA damage induction by Rad6-Rad18. Hence, by ubiquitinating two DNA clamps, PCNA and 9-1-1, the Rad6-Rad18 complex plays a central role in the cellular response to DNA damage by coordinating translesion synthesis, error-free bypass, homologous recombination, as well as transcriptional regulation, reminiscent of roles of RecA in E. coli cells.Several individual genes have also been examined in this study to elucidate the regulatory mechanisms acting on specific DNA damage-inducible genes. In the microarray analysis, DDI2 and DDI3, two identical genes located in duplicated chromosomal regions, were identified due to the highest induction ratio (122-fold) after MMS treatment. Interestingly, DDI2/DDI3 can only be highly induced by SN2-type alkylating agents. Promoter deletion analysis mapped the putative upstream acting sequence (UASDDI2) responsible for 40% of basal expression and 90% of induced expression by MMS.The CRT10 gene was identified through screening of the yeast deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistasis analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.
DegreeDoctor of Philosophy (Ph.D.)
DepartmentMicrobiology and Immunology
ProgramMicrobiology and Immunology
CommitteeLoh, Lambert; Krone, Patrick H.; Howard, S. Peter; Deneer, Harry