Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.

During mitosis, the budding yeast, kinetochores remain attached to microtubules, except for a brief period during S phase. Sister-kinetochores separate into two clusters (bi-lobed’ organization) upon stable end-on attachment to microtubules emanating from opposite spindle poles. However, in meiosis, the outer kinetochore protein Ndc80 reassembles at the centromeres much later after prophase I, establishing new kinetochore-microtubule attachments. Perhaps due to this, despite homolog bi-orientation, we observed that the kinetochores (Ndc80) are linearly dispersed between spindle-poles during metaphase I of meiosis. The presence of end-on attachment marker Dam1 as a cluster near each pole suggests one of the other possibilities that the pole-proximal and pole-distal kinetochores are attached end-on and laterally to the microtubules, respectively. Colocalization studies of kinetochores and kinesin motors suggest that budding yeast kinesin 5, Cin8 and Kip1 perhaps localize to the end-on attached kinetochores while kinesin 8, Kip3 resides at all the kinetochores. Our findings, including kinesin 5 and Ndc80 co-appearance after prophase I and reduced Ndc80 levels in cin8 null mutant, suggest that kinesin motors are crucial for kinetochore reassembly and stability during early meiosis. Thus, this work reports yet another meiosis specific function of kinesin motors.

Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.

The characteristic bi-lobed organization of the kinetochores observed during mitotic metaphase is a result of separation of the sister kinetochores into two clusters upon their stable end-on attachment to the microtubules emanating from opposite spindle poles. In contrast, during metaphase I of meiosis despite bi-orientation of the homologs, we observe that the kinetochores are linearly dispersed between the two spindle poles indicating that pole-distal and pole-proximal kinetochores are attached laterally and end-on, respectively to the microtubules. Colocalization studies of kinetochores and kinesin motors suggest that budding yeast kinesin 5, Cin8 and Kip1 perhaps localize to the end-on attached kinetochores while kinesin 8, Kip3 resides at all the kinetochores. Unlike mitosis in budding yeast, in meiosis, the outer kinetochores assemble much later after prophase I. From the findings including co-appearance of kinesin 5 and the outer kinetochore protein Ndc80 at the centromeres after prophase I and a reduction in Ndc80 level in Cin8 null mutant, we propose that kinesin motors are required for reassembly and stability of the kinetochores during early meiosis. Thus, this work reports yet another meiosis specific function of kinesin motor.

14 15 Tel. (+ 91) 22 2576 7766; 16 Fax (+ 91) 222572 3480. 17 18 19 Running head: The association of 2 micron plasmid with chromosome 20 Abbreviations: CEN: Centromere; TEL: Telomere; 3-AT: 3-Amino-1,2,4-triazole 21 Keywords: 2-micron plasmid, extra-chromosomal element, budding yeast, centromere, 22 telomere, condensin complex, chromosome condensation. 23 24 25 26 27 28 2 Abstract 29Equipartitioning by chromosome hitchhiking and copy number correction by DNA amplification 30 are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present 31 analysis reveals plasmid presence near centromeres and telomeres in mitotic cells, with a 32 preference towards the latter. The observed correlation of plasmid missegregation with non-33 disjunction of rDNA and telomeres under Cdc14 inactivation, higher plasmid missegregation 34 upon induced missegregation of chromosome XII but not chromosome III, requirement of 35 condensin for plasmid stability and the interaction of the condensin subunit Brn1 with the 36 plasmid partitioning system lend functional credence to condensed chromatin being favored for 37 plasmid tethering. By homing to condensed/quiescent chromosome locales, and not over-38