Abstract
Extrachromosomal telomeric circles are commonly invoked as important players in telomere maintenance, but their origin has remained elusive. Using electron microscopy analysis on purified ...telomeres we show that, apart from known structures, telomeric repeats accumulate internal loops (i-loops) that occur in the proximity of nicks and single-stranded DNA gaps. I-loops are induced by single-stranded damage at normal telomeres and represent the majority of telomeric structures detected in ALT (Alternative Lengthening of Telomeres) tumor cells. Our data indicate that i-loops form as a consequence of the exposure of single-stranded DNA at telomeric repeats. Finally, we show that these damage-induced i-loops can be excised to generate extrachromosomal telomeric circles resulting in loss of telomeric repeats. Our results identify damage-induced i-loops as a new intermediate in telomere metabolism and reveal a simple mechanism that links telomere damage to the accumulation of extrachromosomal telomeric circles and to telomere erosion.
Abstract
Homologous recombination factors play a crucial role in protecting nascent DNA during DNA replication, but the role of chromatin in this process is largely unknown. Here, we used the ...bacterial Tus/
Ter
barrier known to induce a site-specific replication fork stalling in
S. cerevisiae
. We report that the Set1C subunit Spp1 is recruited behind the stalled replication fork independently of its interaction with Set1. Spp1 chromatin recruitment depends on the interaction of its PHD domain with H3K4me3 parental histones deposited behind the stalled fork. Its recruitment prevents the accumulation of ssDNA at the stalled fork by restricting the access of Exo1. We further show that deleting SPP
1
increases the mutation rate upstream of the barrier favoring the accumulation of microdeletions. Finally, we report that Spp1 protects nascent DNA at the Tus/
Ter
stalled replication fork. We propose that Spp1 limits the remodeling of the fork, which ultimately limits nascent DNA availability to nucleases.
Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that ...aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.
Abstract
Cell polarization is of paramount importance for proliferation, differentiation, development, and it is altered during carcinogenesis. Polarization is a reversible process controlled by ...positive and negative feedback loops. How polarized factors are redistributed is not fully understood and is the focus of this work. In
Saccharomyces cerevisiae
, mutants defective in haspin kinase exhibit stably polarized landmarks and are sensitive to mitotic delays. Here, we report a new critical role for haspin in polarisome dispersion; failure to redistribute polarity factors, in turn, leads to nuclear segregation defects and cell lethality. We identified a mitotic role for GTP-Ras in regulating the local activation of the Cdc42 GTPase, resulting in its dispersal from the bud tip to a homogeneous distribution over the plasma membrane. GTP-Ras2 physically interacts with Cdc24 regulateing its mitotic distribution. Haspin is shown to promote a mitotic shift from a bud tip-favored to a homogenous PM fusion of Ras-containing vesicles. In absence of haspin, active Ras is not redistributed from the bud tip; Cdc24 remains hyperpolarized promoting the activity of Cdc42 at the bud tip, and the polarisome fails to disperse leading to erroneously positioned mitotic spindle, defective nuclear segregation, and cell death after mitotic delays. These findings describe new functions for key factors that modulate cell polarization and mitotic events, critical processes involved in development and tumorigenesis.
Symmetry breaking by cellular polarization is an exquisite requirement for the cell-cycle of
cells, as it allows bud emergence and growth. This process is based on the formation of polarity clusters ...at the incipient bud site, first, and the bud tip later in the cell-cycle, that overall promote bud emission and growth. Given the extreme relevance of this process, a surveillance mechanism, known as the morphogenesis checkpoint, has evolved to coordinate the formation of the bud and cell cycle progression, delaying mitosis in the presence of morphogenetic problems. The atypical protein kinase haspin is responsible for histone H3-T3 phosphorylation and, in yeast, for resolution of polarity clusters in mitosis. Here, we report a novel role for haspin in the regulation of the morphogenesis checkpoint in response to polarity insults. Particularly, we show that cells lacking the haspin ortholog Alk1 fail to achieve sustained checkpoint activation and enter mitosis even in the absence of a bud. In
Δ cells, we report a reduced phosphorylation of Cdc28-Y19, which stems from a premature activation of the Mih1 phosphatase. Overall, the data presented in this work define yeast haspin as a novel regulator of the morphogenesis checkpoint in
, where it monitors polarity establishment and it couples bud emergence to the G2/M cell cycle transition.
The telomerase reverse transcriptase elongates telomeres to prevent replicative senescence. This process requires exposure of the 3'-end, which is thought to occur when two sister telomeres are ...generated at replication completion. Using two-dimensional agarose gel electrophoresis (2D-gels) and electron microscopy, we found that telomeric repeats are hotspots for replication fork reversal. Fork reversal generates 3' telomeric ends before replication completion. To verify whether these ends are elongated by telomerase, we probed de novo telomeric synthesis in situ and at replication intermediates by reconstituting mutant telomerase that adds a variant telomere sequence. We found variant telomeric repeats overlapping with telomeric reversed forks in 2D-gels, but not with normal forks, nontelomeric reversed forks, or telomeric reversed forks with a C-rich 3'-end. Our results define reversed telomeric forks as a substrate of telomerase during replication.
Inertial technology has spread widely for its comfortable use and adaptability to various motor tasks. The main objective of this study was to assess the validity of inertial measurements of the ...cervical spine range of motion (CROM) when compared to that of the optoelectronic system in a group of healthy individuals. A further aim of this study was to determine the optimal placement of the inertial sensor in terms of reliability of the measure, comparing measurements obtained from the same device placed at the second cervical vertebra (C2), the forehead (F) and the external occipital protuberance (EOP). Twenty healthy subjects were recruited and asked to perform flexion-extension, lateral bending, and axial rotation movements of the head. Outcome measurements of interest were CROM and mean angular velocities for each cervical movement. Results showed that inertial measurements have good reliability (0.75 < ICC < 0.9). Excellent reliability (ICC > 0.9) was found in both flexion and right lateral bending angles. All parameters extracted with EOP placement showed ICC > 0.62, while ICC < 0.5 was found in lateral bending mean angular velocities both for F and C2 placements. Therefore, the optimal sensor's positioning emerged to be EOP. These results suggest that inertial technology could be useful and reliable for the evaluation of the CROM.
A promising but still scarcely explored strategy for the estimation of gait parameters based on inertial sensors involves the adoption of machine learning techniques. However, existing approaches are ...reliable only for specific conditions, inertial measurements unit (IMU) placement on the body, protocols, or when combined with additional devices. In this paper, we tested an alternative gait-events estimation approach which is fully data-driven and does not rely on a priori models or assumptions. High-frequency (512 Hz) data from a commercial inertial unit were recorded during 500 steps performed by 40 healthy participants. Sensors' readings were synchronized with a reference ground reaction force system to determine initial/terminal contacts. Then, we extracted a set of features from windowed data labeled according to the reference. Two gray-box approaches were evaluated: (1) classifiers (decision trees) returning the presence of a gait event in each time window and (2) a classifier discriminating between stance and swing phases. Both outputs were submitted to a deterministic algorithm correcting spurious clusters of predictions. The stance vs. swing approach estimated the stride time duration with an average error lower than 20 ms and confidence bounds between ±50 ms. These figures are suitable to detect clinically meaningful differences across different populations.
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