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Two alternative cell cycle checkpoint pathways differentially control DNA damage-dependent induction of MAG1 and DDI1
1Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada.
Abstract:
Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression. In order to understand how damage checkpoints control the expression of DNA damage-inducible genes, the transcript level of two closely clustered genes, MAG1 and DDI1, was examined in a number of checkpoint mutants. We previously reported that MAG1 induction was abolished in pol2 and rad53 mutants, but not in the mec1-1 mutant. In this study, we found that mec1Delta and dun1Delta null mutants were defective in MAG1 induction, suggesting that MAG1 shares a common regulatory pathway with the RNR1,2,3,4 genes, which are also regulated by the POL2-MEC1-RAD53-DUN1 checkpoint pathway, and that the mec1-1 mutation probably represents a separation-of-function mutation. However, MAG1 is not activated in precisely the same way as the RNR genes, since mutations in CRT1, TUP1 and SSN6, which encode repressors of RNR genes, did not affect basal or induced expression of MAG1. In contrast, the DDI1 transcript level was not affected by any of the above checkpoint mutations. Interestingly, simultaneous inactivation of RAD53 or DUN1 with PDS1, a newly identified checkpoint gene, resulted in severe down-regulation of DDI1 expression, suggesting that DDI1 is controlled by two damage checkpoint pathways, one mediated by POL2-MEC1-RAD53-DUN1 and the other by CHK1-PDS1. On the other hand, deletion of TEL1, a structural homologue of MEC1, did not affect expression of MAG1, DDI1 or RNR3, suggesting that TEL1 plays no role in induction by DNA damage. Based on these and previous studies, we present a model for the role of checkpoint genes in transcriptional regulation in response to DNA damage.
Insights
DNA damage checkpoints regulate gene expression. MAG1 induction relies on the POL2-MEC1-RAD53-DUN1 pathway, while DDI1 is controlled by both this pathway and the CHK1-PDS1 pathway. TEL1 is not involved.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Eukaryotic cells activate DNA damage checkpoint pathways to halt cell cycle progression and induce gene expression.
- Understanding how these checkpoints regulate DNA damage-inducible genes is crucial for comprehending cellular responses to genotoxic stress.
Purpose of the Study:
- To investigate the transcriptional regulation of two clustered DNA damage-inducible genes, MAG1 and DDI1, within various DNA damage checkpoint mutants.
- To elucidate the specific roles of key checkpoint genes, including MEC1, RAD53, DUN1, and PDS1, in controlling MAG1 and DDI1 expression.
Main Methods:
- Analysis of MAG1 and DDI1 transcript levels in a series of yeast checkpoint mutants, including null mutants (mec1Δ, dun1Δ) and a separation-of-function mutant (mec1-1).
- Examination of gene expression in mutants affecting RNR gene regulation (crt1, tup1, ssn6).
- Assessment of DDI1 expression in combined mutants (e.g., rad53/pds1, dun1/pds1) and in the absence of TEL1.
Main Results:
- MAG1 induction was abolished in mec1Δ and dun1Δ mutants, indicating its dependence on the POL2-MEC1-RAD53-DUN1 pathway, distinct from RNR genes.
- DDI1 expression was unaffected by mutations in the POL2-MEC1-RAD53-DUN1 pathway or RNR repressor genes alone.
- Simultaneous inactivation of RAD53 or DUN1 with PDS1 led to severe DDI1 down-regulation, suggesting a dual-pathway control (POL2-MEC1-RAD53-DUN1 and CHK1-PDS1).
- Deletion of TEL1 did not impact MAG1, DDI1, or RNR3 expression, suggesting TEL1 is not involved in DNA damage-induced transcriptional regulation.
Conclusions:
- MAG1 and DDI1 are regulated by distinct, yet interconnected, DNA damage checkpoint pathways.
- The POL2-MEC1-RAD53-DUN1 pathway is essential for MAG1 induction, while DDI1 is under the control of both this pathway and the CHK1-PDS1 pathway.
- TEL1 plays no significant role in the transcriptional response to DNA damage investigated in this study.