G₁/S and G₂/M Cyclin-Dependent Kinase Activities Commit Cells to Death in the Absence of the S-Phase Checkpoint

Abstract

The Mec1 and Rad53 protein kinases are essential for budding yeast cell viability and are also required to activate the S-phase checkpoint, which supports DNA replication under stress conditions. Whether these two functions are related to each other remains to be determined, and the nature of the replication stress-dependent lethality of mec1 and rad53 mutants is still unclear. We show here that a decrease in cyclin-dependent kinase 1 (Cdk1) activity alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of a certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G 1 and mitosis. In fact, delaying either the G 1 /S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to hydroxyurea and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions. T he integrity of the genome is constantly challenged by DNA damage caused by environmental and intracellular factors. Aberrant DNA replication is a major source of mutations and chromosome rearrangements that can lead to cancer and other diseases in metazoans (reviewed in reference 23). Replication fork progression can be hampered by exogenous or endogenous DNA damage. Furthermore, faithful replication depends on a balanced supply of deoxyribonucleotides (deoxyribonucleoside triphosphates [dNTPs]), whose levels are maintained during S-phase through the action of the ribonucleotide reductase (RNR) activity that converts the ribonucleotides to dNTPs (reviewed in reference 37). Indeed, replication fork pausing can be experimentally induced by genotoxic drugs, such as hydroxyurea (HU), which reduces dNTP pools by inhibiting RNR activity, and the DNA alkylating agent methyl methanesulfonate (MMS) that causes intra-S damage.Eukaryotic cells respond to replication interference through a complex signal-transduction pathway, known as the S-phase checkpoint, whose key players in the budding yeast Saccharomyces cerevisiae are the Mec1 and Rad53 kinases (reviewed in references 5 and 63). Mec1, together with its interacting protein Ddc2, is recruited to stalled forks, where it activates the effector kinase Rad53. Both kinases act in various ways to respond to replication interference. They are needed to complete DNA replication after exposure to HU or MMS (16, 55) by maintaining the integrity and/or activity of the replication forks (11,15,26,34). Furthermore, they stimulate dNTP production (1, 25, 64, 65) and the transcription of several MCB binding factor (MBF)-regulated genes that are involved in DNA replication...

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress

“…Several suppressors of rad53-HU-sensitivity have been identified. Deletion of either the G1/S or G2/M factor delays cell cycle transition 86 ; THO, TREX-2 components alter nascent RNA exportation 17 ; and Rrm3 and Pif1 DNA helicases 34 Fig. 8 Model for the effect of H3K4 methylation in relieving transcription-replication conflicts.…”

A humanized yeast phenomic model of deoxycytidine kinase to predict genetic buffering of nucleoside analog cytotoxicity

“…Moreover, the observation that CLB2 deletion (suppressing effect) opposes that of CLB5 (deletion enhancing; see above Fig. 6D, 1-0-0) has been previously described in the context of loss of the S-phase checkpoint [263] ; (8) SKY1/SRPK1 (serine-arginine rich serine-threonine kinase), which is overexpressed in glioma, and prostate, breast, and lung cancer [207] ; (9) SNC2/VAMP8 , which functions in fusion of Golgi-derived vesicles with the plasma membrane and is overexpressed in glioma and breast cancer [208,209] ; (10) YPT6/RAB34 , which functions in fusion of endosome-derived vesicles with the late Golgi and is overexpressed in glioma, breast cancer, and hepatocellular carcinoma [211-213]; (11) TOP1/TOP1/TOP1MT , Topoisomerase I, which has increased copy number in pancreatic and bile duct cancer [210]; (12) ALD6 , which encodes cytosolic aldehyde dehydrogenase, and was a deletion suppressor for both gemcitabine and cytarabine, having multiple homologs that were OES ( ALDH1A1 , ALDH1A2 , ALDH1B1 , and ALDH7A1. ALDH1B1 ).…”

A Humanized Yeast Phenomic Model of Deoxycytidine Kinase to Predict Genetic Buffering of Nucleoside Analog Cytotoxicity

“…(7) CLB2/CCNA2/CCNB1 , a B-type cyclin involved in cell cycle progression, of which both CCNA2 and CCNB1 are overexpressed in breast and colorectal cancer [194,195,196,197]. Moreover, the observation that CLB2 deletion (suppressing effect) opposes that of CLB5 (deletion enhancing; see above Figure 6D, 1-0-0) has been previously described in the context of loss of the S-phase checkpoint [263]. (8) SKY1/SRPK1 (serine–arginine-rich serine–threonine kinase), which is overexpressed in glioma and prostate, breast, and lung cancer [207].…”