SummaryThe minichromosome maintenance genes, MCM21 and MCM22, have been cloned and are shown to code for the ORFs YDR318W and YJR135C respectively. Mutations in these genes caused a decrease in the stability of the minichromosome. This decrease in stability was associated with an increase in the copy number of the minichromosome in cells carrying it. Small circular dicentric plasmids were maintained relatively stably and structurally intact in the mutants compared with the wild-type strain. In the latter, such plasmids were mitotically unstable and, upon recovery, showed frequent rearrangements of their DNA. A centromere offered less obstruction to transcription in mutant cells than in the wild type, showing that both these mutants had a more relaxed kinetochore assembly. The mutant strains showed elevated rates of chromosome loss but not those of recombination. Both the mutations caused the cells to display a higher sensitivity towards the anti-mitotic drug benomyl. All these observations suggest that MCM21 and MCM22 are important for chromosome segregation with a potential role in kinetochore function. These genes are nonessential, as their deletions from chromosomes did not cause loss of cell viability. However, exponentially growing mutant cells carrying the deletion of the MCM21 gene had a significant population of largebudded cells with a single nucleus at the neck. Furthermore, the DNA content of these cells showed a shift towards 2N, suggesting a temporary pause of cells in G 2 or in an early phase of mitosis. The mcm21 and mcm22 mutations do not show synthetic lethality or any further enhancement of growth defects, implying that they could be carrying out non-overlapping functions in chromosome segregation.
For many papillomaviruses, the viral protein E2 tethers the viral genome to the host mitotic chromosomes to ensure persistent, long-term maintenance of the genome during cell division. Our previous studies of E2 proteins from different genera of papillomaviruses have shown that they bind to different regions of the host chromosomes during mitosis. For example, bovine papillomavirus type 1 (BPV-1) E2 binds to all chromosomes as small speckles in complex with the cellular protein Brd4. In contrast, the human papillomavirus type 8 (HPV-8) E2 protein binds as large speckles at the pericentromeric regions of chromosomes. Here we show that these speckles do not contain Brd4, and unlike that of BPV-1, the N-terminal Brd4-interacting domain of HPV-8 E2 is not required for chromosome binding. In contrast to BPV-1 E2, the HPV-8 E2 protein targets the short arms of acrocentric mitotic chromosomes. Furthermore, the E2 protein interacts with the repeated ribosomal DNA genes found in this location and colocalizes with UBF, the RNA polymerase I transcription factor. Therefore, HPV-8 E2 genome tethering occurs by a Brd4-independent mechanism through a novel interaction with specific regions of mitotic chromosomes. Thus, a wide range of viruses have adopted the strategy of linking their genomes to host chromosomes, but individual viruses use different chromosomal targets. Characterization of these targets will enable the development of antiviral therapies to eliminate the viral genomes from infected cells.Papillomaviruses infect the basal layer of stratified epithelia. The viral genomes are stably maintained in the nuclei of the dividing cells as low copy number, extrachromosomal elements for many years. Error-free replication and equal distribution of the replicated copies of the viral DNA to the dividing cells are key to the persistence of papillomavirus infection. The mechanism of viral genome partitioning is different from that of host chromosomes, as the viral DNA does not possess any sequence equivalent to the centromere to utilize the mitotic segregation machinery of the host. Instead, E2, a multifunctional viral protein, attaches the viral DNA to the host chromosomes (15,21,34). As the host cell chromosomes are divided equally into daughter cells during mitotic division, the viral DNA is passively segregated by being tethered to the host chromosomes. Chromosomal tethering mediated by a virus-encoded protein is a common tactic for maintaining the genomes of other persistent viruses. Gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus use this strategy, which is mediated by the EBNA-1 and LANA proteins, respectively (reviewed in reference 23).Among the papillomaviruses, bovine papillomavirus type 1 (BPV-1) is best studied for chromosomal segregation. The multifunctional viral protein E2 has been shown to play an important role in this process. Both the BPV-1 E2 protein and viral genomes are localized on mitotic chromosomes as small speckles on the arms of all chromosomes, in complex wi...
Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes. Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs. In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes. When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants. One mutant, mcm17, belonging to the size-dependent class was further characterized. In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation. This defect was largely suppressed in the 60-kb mini-chromosome. A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance. This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination. Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability. This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 degrees C or 37 degrees C. This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length.
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