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RAD9 and RAD24 define two additive, interacting branches of the DNA damage checkpoint pathway in budding yeast
M A de la Torre-Ruiz1, C M Green, N F Lowndes
1Imperial Cancer Research Fund, Clare Hall Laboratories, CDC Laboratory, South Mimms, Herts EN6 3LD, UK.
Abstract:
In budding yeast, RAD9 and RAD24/RAD17/MEC3 are believed to function upstream of MEC1 and RAD53 in signalling the presence of DNA damage. Deletion of any one of these genes reduces the normal G1/S and G2/M checkpoint delays after UV irradiation, whereas in rad9Delta-rad24Delta cells the G1/S checkpoint is undetectable, although there is a residual G2/M checkpoint. We have shown previously that RAD9 also controls the transcriptional induction of a DNA damage regulon (DDR). We now report that efficient DDR induction requires all the above-mentioned checkpoint genes. Residual induction of the DDR after UV irradiation observed in all single mutants is not detectable in rad9Delta-rad24Delta. We have examined the G2/M checkpoint and UV sensitivity of single mutants after overexpression of the checkpoint proteins. This analysis indicates that RAD9 and the RAD24 epistasis group can be placed onto two separate, additive branches that converge on MEC1 and RAD53. Furthermore, MEC3 appears to function downstream of RAD24/RAD17. The transcriptional response to DNA damage revealed unexpected and specific antagonism between RAD9 and RAD24. Further support for genetic interaction between RAD9 and RAD24 comes from study of the modification and activation of Rad53 after damage. Evidence for bypass of RAD53 function under some conditions is also presented.
Insights
DNA damage response in yeast requires RAD9 and RAD24/RAD17/MEC3 checkpoint genes. These genes function in parallel pathways converging on MEC1 and RAD53, with antagonism between RAD9 and RAD24 impacting DNA repair.
Area of Science:
- Cellular and Molecular Biology
- Genetics and Genomics
- Biochemistry
Background:
- RAD9 and RAD24/RAD17/MEC3 are key components of the DNA damage response (DDR) pathway in budding yeast.
- These genes are known to function upstream of MEC1 and RAD53, crucial for cell cycle checkpoint control after DNA damage.
- Previous work established RAD9's role in the transcriptional induction of the DDR.
Purpose of the Study:
- To elucidate the precise roles and genetic interactions of RAD9 and the RAD24 epistasis group in DNA damage signaling and checkpoint control.
- To investigate the requirement of these checkpoint genes for the transcriptional induction of the DDR.
- To determine the relationship between RAD9, RAD24/RAD17/MEC3, MEC1, and RAD53 in DNA damage response pathways.
Main Methods:
- Analysis of G1/S and G2/M checkpoint delays in yeast mutants after UV irradiation.
- Assessment of transcriptional induction of the DNA damage regulon (DDR) in various mutant strains.
- Overexpression of checkpoint proteins to examine G2/M checkpoint and UV sensitivity.
- Study of Rad53 modification and activation following DNA damage.
Main Results:
- Efficient DDR induction requires RAD9, RAD24/RAD17, and MEC3; residual induction in single mutants is absent in rad9Δ-rad24Δ double mutants.
- RAD9 and the RAD24 epistasis group operate on separate, additive branches converging on MEC1 and RAD53.
- MEC3 functions downstream of RAD24/RAD17, and specific antagonism exists between RAD9 and RAD24 in the transcriptional response.
- Genetic interactions were further supported by Rad53 modification studies, with evidence for Rad53 bypass under certain conditions.
Conclusions:
- RAD9 and the RAD24/RAD17/MEC3 complex are essential for robust DNA damage checkpoint activation and DDR induction in yeast.
- These pathways converge additively on MEC1 and RAD53, highlighting a complex signaling network.
- Antagonistic interactions between RAD9 and RAD24 reveal intricate regulatory mechanisms within the DDR, influencing checkpoint control and DNA repair efficiency.