An official website of the United States government
Accurate chromosome segregation is vital for cell and organismal viability. The mitotic spindle, a bipolar macromolecular machine composed largely of dynamic microtubules, is responsible for chromosome segregation during each cell replication cycle. Prior to anaphase, a bipolar metaphase spindle must be formed in which each pair of chromatids is attached to microtubules from opposite spindle poles. In this bipolar configuration pulling forces from the dynamic microtubules can generate tension across the sister kinetochores. The tension status acts as a signal that can destabilize aberrant kinetochore-microtubule attachments and reinforces correct, bipolar connections. Historically it has been challenging to isolate the specific role of tension in mitotic processes due to the interdependency of attachment and tension status at kinetochores. Recent technical and experimental advances have revealed new insights into how tension functions during mitosis. Here we summarize the evidence that tension serves as a biophysical signal that unifies multiple aspects of kinetochore and centromere function to ensure accurate chromosome segregation.
more »
« less
Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer
Abstract Oncogenic extrachromosomal DNA elements (ecDNA) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency varies greatly. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. ecTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. After mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis. Significance: ecDNAs are vehicles for oncogene amplification. The circular nature of ecDNA affords unique properties, such as mobility and ecDNA-specific replication and segregation behavior. We uncovered fundamental ecDNA properties by tracking ecDNAs in live cells, highlighting uneven and random segregation and ecDNA hubs that drive cargo gene transcription. See related commentary by Henssen, p. 293. This article is highlighted in the In This Issue feature, p. 275
more »
« less
- PAR ID:
- 10349580
- Date Published:
- Journal Name:
- Cancer Discovery
- Volume:
- 12
- Issue:
- 2
- ISSN:
- 2159-8274
- Page Range / eLocation ID:
- 468 to 483
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Gene amplification has been observed in many bacteria and eukaryotes as a response to various selective pressures, such as antibiotics, cytotoxic drugs, pesticides, herbicides, and other stressful environmental conditions. An increase in gene copy number is often found as extrachromosomal elements that usually contain autonomously replicating extrachromosomal circular DNA molecules (eccDNAs). Amaranthus palmeri , a crop weed, can develop herbicide resistance to glyphosate [ N -(phosphonomethyl) glycine] by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase ( EPSPS ) gene, the molecular target of glyphosate. However, biological questions regarding the source of the amplified EPSPS , the nature of the amplified DNA structures, and mechanisms responsible for maintaining this gene amplification in cells and their inheritance remain unknown. Here, we report that amplified EPSPS copies in glyphosate-resistant (GR) A. palmeri are present in the form of eccDNAs with various conformations. The eccDNAs are transmitted during cell division in mitosis and meiosis to the soma and germ cells and the progeny by an as yet unknown mechanism of tethering to mitotic and meiotic chromosomes. We propose that eccDNAs are one of the components of McClintock’s postulated innate systems [McClintock B (1978) Stadler Genetics Symposium ] that can rapidly produce soma variation, amplify EPSPS genes in the sporophyte that are transmitted to germ cells, and modulate rapid glyphosate resistance through genome plasticity and adaptive evolution.more » « less
-
All plants, animals and fungi share a common ancestor, and though they have evolved to become very distinct over billions of years, they all share the essential machinery needed for cells to grow and divide. At the heart of this is the complex interaction of proteins involved in DNA replication, the process of duplicating the genetic material every time a cell divides. DNA replication needs to be done with great care, with error rates as small as one mistake in a billion. Otherwise, mutations can accumulate in the genome, causing problems for long-term survival. Despite the overall principles of DNA replication remaining the same, the underlying mechanisms vary across different organisms. Given the precision and complexity of replicating DNA, it was not clear how the process had evolved mechanistic differences. Fumasoni and Murray set out to answer this by forcing a strain of budding yeast to evolve by removing the gene for an important, but not essential, component of DNA replication. The cells were still able to reproduce, but they were hampered by this mutation. Fumasoni and Murray studied the yeast after it had reproduced for a thousand generations, giving it enough time to acquire new mutations that would allow it to compensate for the initial defects. In eight separate samples, the yeast had made many of the same changes in order to overcome the original mutation. These mutations altered conserved features of DNA replication and the segregation of genetic material and inactivated a third feature that would normally protect the cell against the accumulation of damaged DNA. These findings show how reproducible the evolutionary pathways can be in a controlled, laboratory environment and that cells can evolve quickly after conserved processes in the cell are damaged. The behavior of the mutated yeast mimicked that of cancer cells, which are often struggling to adapt to mutations in their replication machinery. Studying the rapid evolution that follows genetic perturbations could help researchers to better deal with challenges in cancer treatment and the development of antibiotic resistance in bacteria, as well as leading to a deeper understanding of both evolution and cell biology.more » « less
-
SUMMARY Proper function in a bacterial cell relies on intrinsic cell size regulation. The molecular mechanisms underlying how bacteria maintain their cell size remain unclear. The conserved regulator DnaA, the initiator of chromosome replication, is associated to size regulation by controlling the number of origins of replication (oriC) per cell. In this study, we identify and characterize a new mechanism in which DnaA modulates cell size independently oforiC-copy number. By altering the levels of DnaA without impacting chromosome replication, we demonstrate that DnaA’s activity as a transcription factor can slow down cell elongation rate resulting in cells that are ∼20% smaller. We identify the peptidoglycan biosynthetic enzyme MurD as a key player of cell size regulation inCaulobacter crescentusand in the evolutionarily distant bacteriumEscherichia coli. Collectively, our findings provide mechanistic insights to the complex regulation of cell size in bacteria.more » « less
-
Abstract Lung cancer sequencing efforts have uncovered mutational signatures that are attributed to exposure to the cigarette smoke carcinogen benzo[a]pyrene. Benzo[a]pyrene metabolizes in cells to benzo[a]pyrene diol epoxide (BPDE) and reacts with guanine nucleotides to form bulky BPDE adducts. These DNA adducts block transcription and replication, compromising cell function and survival, and are repaired in human cells by the nucleotide excision repair pathway. Here, we applied high-resolution genomic assays to measure BPDE-induced damage formation and mutagenesis in human cells. We integrated the new damage and mutagenesis data with previous repair, DNA methylation, RNA expression, DNA replication, and chromatin component measurements in the same cell lines, along with lung cancer mutagenesis data. BPDE damage formation is significantly enhanced by DNA methylation and in accessible chromatin regions, including transcribed and early-replicating regions. Binding of transcription factors is associated primarily with reduced, but also enhanced damage formation, depending on the factor. While DNA methylation does not appear to influence repair efficiency, this repair was significantly elevated in accessible chromatin regions, which accumulated fewer mutations. Thus, when damage and repair drive mutagenesis in opposing directions, the final mutational patterns appear to be dictated by the efficiency of repair rather than the frequency of underlying damages.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1158/2159-8290.CD-21-1376
