The 2-micron plasmid of the budding yeast Saccharomyces cerevisiae encodes copy-number amplification and partitioning systems that enable the plasmid to persist despite conferring no advantage to its host. Plasmid partitioning requires interaction of the plasmid Rep1 and Rep2 proteins with each other and with the plasmid-partitioning locus STB. Here we demonstrate that Rep1 stability is reduced in the absence of Rep2, and that both Rep proteins are sumoylated. Lysine-to-arginine substitutions in Rep1 and Rep2 that inhibited their sumoylation perturbed plasmid inheritance without affecting Rep protein stability or two-hybrid interaction between Rep1 and Rep2. One-hybrid and chromatin immunoprecipitation assays revealed that Rep1 was required for efficient retention of Rep2 at STB and that sumoylation-deficient mutants of Rep1 and Rep2 were impaired for association with STB. The normal co-localization of both Rep proteins with the punctate nuclear plasmid foci was also lost when Rep1 was sumoylation-deficient. The correlation of Rep protein sumoylation status with plasmid-partitioning locus association suggests a theme common to eukaryotic chromosome segregation proteins, sumoylated forms of which are found enriched at centromeres, and between the yeast 2-micron plasmid and viral episomes that depend on sumoylation of their maintenance proteins for persistence in their hosts.
The yeast 2-μm plasmid is a remarkable genetic parasite, managing efficient maintenance at high-copy number with minimal impact on the host. Equal partitioning of the plasmid upon host cell division requires plasmid proteins Rep1 and Rep2 and the plasmid STB locus. The Rep proteins and the plasmid-encoded Raf protein also regulate plasmid gene transcription. In this study, protein interaction assays, sequence analyses and mutational approaches were used to identify domains and residues in Rep2 and Raf required for association with Rep1 and Rep2 and to delineate the Rep2 DNA-binding domain. Rep2 and Raf displayed similarities in interactions with Rep1 and Rep2, in having Rep1 promote their STB association in vivo, and in stabilizing Rep protein levels. Rep2 mutants impaired for self-association were competent for transcriptional repression while those deficient for Rep1 association were not. Surprisingly, Rep2 mutants impaired for either Rep1 interaction or self-association were able to maintain efficient plasmid inheritance provided Raf was present and competent for Rep protein interaction. Our findings provide insight into the Rep protein complexes required for partitioning and transcriptional repression, and suggest that in addition to its transcriptional function, Raf stabilization of Rep partitioning proteins contributes to the remarkable persistence of the 2-μm plasmid.
Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.
Equal partitioning of the multi-copy yeast 2-micron plasmid requires association of plasmid proteins Rep1 and Rep2 with tandem repeats at the plasmid STB locus. To identify sequence elements required for these associations we generated synthetic versions of a 63-bp section of STB, encompassing one repeat. A single copy of this sequence was sufficient for Rep protein association in vivo, while two directly arrayed copies provided partitioning function to a plasmid lacking all other 2-micron sequences. Partitioning efficiency increased with increasing repeat number, reaching that conferred by the native STB repeat array. By altering sequences in synthetic repeats, we identified the TGCA component of a TGCATTTTT motif as critical for Rep protein recognition, with a second TGCA sequence in each repeat also contributing to association. Mutation of TGCATTTTT to TGTATTTT, as found in variant 2-micron STB repeats, also allowed Rep protein association, while mutation to TGCATTAAT impaired inheritance without abolishing Rep protein recognition, suggesting an alternate role for the T-tract. Our identification of sequence motifs required for Rep protein recognition provides the basis for understanding higher-order Rep protein arrangements at STB that enable the yeast 2-micron plasmid to be efficiently partitioned during host cell division.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.