The transcriptional regulator p53 has an essential role in tumor suppression. Almost 50% of human cancers are associated with the loss of p53 functions, where p53 often accumulates in the nucleus as well as in cytoplasm. Although it has been previously suggested that amyloid formation could be a cause of p53 loss-of-function in subset of tumors, the characterization of these amyloids and its structure-function relationship is not yet established. In the current study, we provide several evidences for the presence of p53 amyloid formation (in human and animal cancer tissues); along with its isolation from human cancer tissues and the biophysical characterization of these tissue-derived fibrils. Using amyloid seed of p53 fragment (P8, p53(250-257)), we show that p53 amyloid formation in cells not only leads to its functional inactivation but also transforms it into an oncoprotein. The in vitro studies further show that cancer-associated mutation destabilizes the fold of p53 core domain and also accelerates the aggregation and amyloid formation by this protein. Furthermore, we also show evidence of prion-like cell-to-cell transmission of different p53 amyloid species including full-length p53, which is induced by internalized P8 fibrils. The present study suggests that p53 amyloid formation could be one of the possible cause of p53 loss of function and therefore, inhibiting p53 amyloidogenesis could restore p53 tumor suppressor functions.

The 2-m yeast plasmid, a benign high-copy nuclear parasite, propagates itself with nearly the same fidelity as the chromosomes of its host. Equal plasmid segregation is absolutely dependent on the cohesin complex assembled at the plasmid partitioning locus STB. However, the mechanism of cohesin action in the context of multiple plasmid copies, resident within two separate clusters after DNA replication, is unknown. By using ''single-copy'' derivatives of the 2-m plasmid, we demonstrate that recruitment of cohesin at STB during S phase indeed translates into cohesion between plasmid molecules. Through binary fluorescence tagging, we reveal that segregation of replicated plasmids occurs in a sister-tosister fashion. Thus, cohesin serves the same fundamental purpose in plasmid and chromosome segregation.2-m circle ͉ cohesin complex ͉ plasmid cohesion ͉ sister-to-sister segregation T he 2-m plasmid, a high-copy extrachromosomal selfish DNA element resident in the yeast nucleus, propagates itself stably with the assistance of a partitioning system (1). The plasmid-coded proteins Rep1p and Rep2p and the cis-acting locus STB comprise the partitioning system, which is designed to channel central components of the chromosome segregation machinery toward plasmid segregation (2). The histone H3 variant Cse4p, which has so far been thought to be unique to centromeres (3), is present also at STB and is essential for equal plasmid segregation (4). The maturation of Cse4p-containing STB chromatin into its functional state appears to be mediated through the RSC2 chromatin remodeling complex (4-6). The yeast cohesin complex, required for one-to-one segregation of sister chromatids, is assembled at STB in a Rep1p-and Rep2p-assisted manner during early S phase, and this association lasts until anaphase (7,8). Like chromosome-cohesin association, plasmid-cohesin association also requires the loading factors Scc2 and Scc4 (ref. 9; S. Mehta and M.J., unpublished work). However, in contrast to chromosomes, the plasmid fails to acquire cohesin when the mitotic spindle is disassembled (10). Consistent with a potential role for the RSC2 complex in remodeling STB chromatin, inactivation of the complex blocks cohesin assembly on the plasmid (5, 11). The timely recruitment of cohesin at STB and its timely disassembly are critical events in 2-m-plasmid segregation (7).An amplification system consisting of the Flp recombinase and its target sites (FRTs) augments the partitioning system in the high-copy persistence of the 2-m circle. Under steady state conditions, the amplification system appears to be negatively regulated by a bipartite Rep1p-Rep2p repressor complex (12). Amplification is triggered only when a rare missegregation event leads to a copy-number drop in the plasmid. The generally accepted amplification mechanism is a carefully timed Flp recombination event that converts a pair of bidirectional replication forks into unidirectional ones by DNA inversion (13). The dual rolling-circle replication generates a plasmid concatamer that ...

The centromere protein A homologue Cse4p is required for kinetochore assembly and faithful chromosome segregation in Saccharomyces cerevisiae. It has been regarded as the exquisite hallmark of centromeric chromatin. We demonstrate that Cse4 resides at the partitioning locus STB of the 2-μm plasmid. Cse4p-STB association is absolutely dependent on the plasmid partitioning proteins Rep1p and Rep2p and the integrity of the mitotic spindle. The kinetochore mutation ndc10-1 excludes Cse4p from centromeres without dislodging it from STB. Cse4p-STB association lasts from G1/S through late telophase during the cell cycle. The release of Cse4p from STB chromatin is likely mediated through spindle disassembly. A lack of functional Cse4p disrupts the remodeling of STB chromatin by the RSC2 complex, negates Rep2p binding and cohesin assembly at STB, and causes plasmid missegregation. Poaching of a specific histone variant by the plasmid to mark its partitioning locus with a centromere tag reveals yet another one of the molecular trickeries it performs for achieving chromosome- like fidelity in segregation.

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata C holera still continues to be an important cause of human infection, especially in developing countries that lack access to safe drinking water and proper sanitation. The recent devastating cholera outbreak in Haiti (13), for the first time in almost a century, placed this ancient disease at the forefront of the global public health agenda. In May 2011, the World Health Assembly recognized the reemergence of cholera as a significant global public health problem and called for the implementation of an integrated and comprehensive global approach to cholera control (17). This dreadful diarrheal disease is caused by the Gram-negative toxigenic bacterium Vibrio cholerae (7). To date, more than 200 serogroups of V. cholerae are known, but only serogroups O1 and O139 cause epidemic and pandemic cholera (7, 16). To date, the world has experienced seven pandemics of cholera. Among these, the first six were caused by the classical biotype strains, whereas the ongoing seventh pandemic has been caused by the El Tor biotype (16). In recent years, the emergence and dissemination of novel pathogenic variants of V. cholerae O1 throughout many Asian and African countries (1,2,3,5,9,10,11,14,15) indicated a cryptic change in cholera epidemiology. Our recent study showed that the El Tor variant strains of V. cholerae O1 have replaced the prototype El Tor biotype strains in Kolkata, India, since 1995 (15). This report, together with the recent massive cholera outbreak in Haiti, caused by V. cholerae organisms with a mutation in the 58th nucleotide of ctxB (3), motivated us to investigate the emergence and dissemination of this new variant of V. cholerae O1 biotype El Tor strains, if any, in Kolkata.In this study, we have developed a double-mismatch-amplification mutation assay (DMAMA) to accurately discriminate the classical, El Tor, and Haitian type ctxB alleles through a rapid and simple PCR-based assay. A total of 142 V. cholerae O1 strains were included in this study. These strains were selected from the repository of the National Institute of Cholera and Enteric Diseases, Kolkata, India, covering different months of each year from 2004 to 2011. V. cholerae O1 strains O395 (serotype Ogawa), N16961 (serotype Inaba), and EL-1786 (Ogawa, El Tor) were used as standard strains for the classical, El Tor, and Haitian type, respectively.Development of the DMAMA-PCR. All 142 tested strains, along with the control strains, were grown in Luria-Bertani broth (Becton Dickinson, Sparks, MD) for 18 h and then streaked on Luria agar (LA) plates. In this study, we focused on ctxB in V. cholerae O1 strains to confirm the strains carrying Haitian, classical, and El Tor alleles in a simple PCR-based assay. Current methods for differentiating the biotype-specific cholera toxin B (CTB) subunit of V. cholerae O1 necessitate MAMA-PCR with ...

a b s t r a c tFaithful segregation of chromosomes during mitosis and meiosis is the cornerstone process of life. Cohesin, a multi-protein complex conserved from yeast to human, plays a crucial role in this process by keeping the sister chromatids together from S-phase to anaphase onset during mitosis and meiosis. Technological advancements have discovered myriad functions of cohesin beyond its role in sister chromatid cohesion (SCC), such as transcription regulation, DNA repair, chromosome condensation, homolog pairing, monoorientation of sister kinetochore, etc. Here, we have focused on such functions of cohesin that are either independent of or dependent on its canonical role of sister chromatid cohesion. At the end, human diseases associated with malfunctioning of cohesin, albeit with mostly unperturbed sister chromatid cohesion, have been discussed.