Defects in anaphase-promoting complex (APC) activity, which regulates mitotic progression and chromatin assembly, results in genomic instability, a hallmark of premature aging and cancer. We investigated whether APC-dependent genomic stability affects aging and life span in yeast. Utilizing replicative and chronological aging assays, the APC was shown to promote longevity. Multicopy expression of genes encoding Snf1p (MIG1) and PKA (PDE2) aging-pathway components suppressed apc5 CA phenotypes, suggesting their involvement in APC-dependent longevity. While it is known that PKA inhibits APC activity and reduces life span, a link between the Snf1p-inhibited Mig1p transcriptional modulator and the APC is novel. Our mutant analysis supports a model in which Snf1p promotes extended life span by inhibiting the negative influence of Mig1p on the APC. Consistent with this, we found that increased MIG1 expression reduced replicative life span, whereas mig1⌬ mutations suppressed the apc5 CA chronological aging defect. Furthermore, Mig1p and Mig2p activate APC gene transcription, particularly on glycerol, and mig2⌬, but not mig1⌬, confers a prolonged replicative life span in both APC5 and acp5 CA cells. However, glucose repression of APC genes was Mig1p and Mig2p independent, indicating the presence of an uncharacterized factor. Therefore, we propose that APC-dependent genomic stability is linked to prolonged longevity by the antagonistic regulation of the PKA and Snf1p pathways.
The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5 CA mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5 CA phenotypes in a CAF-1-and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1⌬ cac2⌬ msi1⌬ (caf1⌬) cells expressing apc5 CA exhibit a phenotype more severe than that of apc5 CA or caf1⌬. The Ts ؊ phenotypes observed in apc5 CA and apc5 CA caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts ؊ defects. Synthetic genetic interactions were also observed in apc5 CA asf1⌬ cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5 CA Ts ؊ defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APCdependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5 CA phenotypes. We discuss the implications of our observations.
Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model system Saccharomyces cerevisiae. Modifications to an in vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s): RSP5, and a subunit of the Anaphase Promoting Complex (APC), APC5. Additional modifications are described that allow for a rapid analysis and an in vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that the in vitro and in vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks.
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