Mitotic DNA damage promotes chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

DNA damage response (DDR) during interphase involves active signaling and repair to ensure genomic stability. However, how mitotic cells respond to DNA damage remains poorly understood. Supported by correlative live-/fixed-cell microscopy, it was found that mitotic cells exposed to several cancer chemotherapy compounds acquire and signal DNA damage, regardless of how they interact with DNA. In-depth analysis upon DNA damage during mitosis revealed a spindle assembly checkpoint (SAC)-dependent, but ataxia telangiectasia mutated-independent, mitotic delay. This delay was due to the presence of misaligned chromosomes that ultimately satisfy the SAC and missegregate, leading to micronuclei formation. Mechanistically, it is shown that mitotic DNA damage causes missegregation of polar chromosomes due to the action of arm-ejection forces by chromokinesins. Importantly, with the exception of DNA damage induced by etoposide-a topoisomerase II inhibitor-this outcome was independent of a general effect on kinetochore microtubule stability. Colony formation assays in pan-cancer cell line models revealed that mitotic DNA damage causes distinct cytotoxic effects, depending on the nature and extent of the damage. Overall, these findings unveil and raise awareness that therapeutic DNA damage regimens may contribute to genomic instability through a surprising link with chromokinesin-mediated missegregation of polar chromosomes in cancer cells.

Micronuclei from misaligned chromosomes that satisfy the spindle assembly checkpoint in cancer cells.

Chromosome alignment to the spindle equator is a hallmark of mitosis thought to promote chromosome segregation fidelity in metazoans. Yet chromosome alignment is only indirectly supervised by the spindle assembly checkpoint (SAC) as a byproduct of chromosome bi-orientation, and the consequences of defective chromosome alignment remain unclear. Here, we investigated how human cells respond to chromosome alignment defects of distinct molecular nature by following the fate of live HeLa cells after RNAi-mediated depletion of 125 proteins previously implicated in chromosome alignment. We confirmed chromosome alignment defects upon depletion of 108/125 proteins. Surprisingly, in all confirmed cases, depleted cells frequently entered anaphase after a delay with misaligned chromosomes. Using depletion of prototype proteins resulting in defective chromosome alignment, we show that misaligned chromosomes often satisfy the SAC and directly missegregate without lagging behind in anaphase. In-depth analysis of specific molecular perturbations that prevent proper kinetochore-microtubule attachments revealed that misaligned chromosomes that missegregate frequently result in micronuclei. Higher-resolution live-cell imaging indicated that, contrary to most anaphase lagging chromosomes that correct and reintegrate the main nuclei, misaligned chromosomes are a strong predictor of micronuclei formation in a cancer cell model of chromosomal instability, but not in non-transformed near-diploid cells. We provide evidence supporting that intrinsic differences in kinetochore-microtubule attachment stability on misaligned chromosomes account for this distinct outcome. Thus, misaligned chromosomes that satisfy the SAC may represent a previously overlooked mechanism driving chromosomal/genomic instability during cancer cell division, and we unveil genetic conditions predisposing for these events.

Mps1-mediated release of Mad1 from nuclear pores ensures the fidelity of chromosome segregation.

The spindle assembly checkpoint (SAC) relies on the recruitment of Mad1-C-Mad2 to unattached kinetochores but also on its binding to Megator/Tpr at nuclear pore complexes (NPCs) during interphase. However, the molecular underpinnings controlling the spatiotemporal redistribution of Mad1-C-Mad2 as cells progress into mitosis remain elusive. Here, we show that activation of Mps1 during prophase triggers Mad1 release from NPCs and that this is required for kinetochore localization of Mad1-C-Mad2 and robust SAC signaling. We find that Mps1 phosphorylates Megator/Tpr to reduce its interaction with Mad1 in vitro and in Drosophila cells. Importantly, preventing Mad1 from binding to Megator/Tpr restores Mad1 accumulation at kinetochores, the fidelity of chromosome segregation, and genome stability in larval neuroblasts of mps1-null mutants. Our findings demonstrate that the subcellular localization of Mad1 is tightly coordinated with cell cycle progression by kinetochore-extrinsic activity of Mps1. This ensures that both NPCs in interphase and kinetochores in mitosis can generate anaphase inhibitors to efficiently preserve genomic stability.

Coherent-hybrid STED: high contrast sub-diffraction imaging using a bi-vortex depletion beam.

Stimulated emission depletion (STED) fluorescence microscopy squeezes an excited spot well below the wavelength scale using a doughnut-shaped depletion beam. To generate a doughnut, a scale-free vortex phase modulation (2D-STED) is often used because it provides maximal transverse confinement and radial-aberration immunity (RAI) to the central dip. However, RAI also means blindness to a defocus term, making the axial origin of fluorescence photons uncertain within the wavelength scale provided by the confocal detection pinhole. Here, to reduce the uncertainty, we perturb the 2D-STED phase mask so as to change the sign of the axial concavity near focus, creating a dilated dip. By providing laser depletion power, the dip can be compressed back in three dimensions to retrieve lateral resolution, now at a significantly higher contrast. We test this coherent-hybrid STED (CH-STED) mode in x-y imaging of complex biological structures, such as the dividing cell. The proposed strategy creates an orthogonal direction in the STED parametric space that uniquely allows independent tuning of resolution and contrast using a single depletion beam in a conventional (circular polarization-based) STED setup.

Mitotic progression, arrest, exit or death relies on centromere structural integrity, rather than de novo transcription.

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain a robust spindle assembly checkpoint (SAC) response. Here, we used live-cell imaging of human cells, RNA-seq and qPCR to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC signaling and cell fate. However, we were unable to detect significant changes in mitotic transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on Aurora B centromeric localization, SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, mitotic progression, arrest, exit or death is determined by centromere structural integrity, rather than de novo transcription.

Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions.

In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa-Atr-Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects.

Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis.

Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1­c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1­c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.

The bracovirus genome of the parasitoid wasp Cotesia congregata is amplified within 13 replication units, including sequences not packaged in the particles.

The relationship between parasitoid wasps and polydnaviruses constitutes one of the few known mutualisms between viruses and eukaryotes. Viral particles are injected with the wasp eggs into parasitized larvae, and the viral genes thus introduced are used to manipulate lepidopteran host physiology. The genome packaged in the particles is composed of 35 double-stranded DNA (dsDNA) circles produced in wasp ovaries by amplification of viral sequences from proviral segments integrated in tandem arrays in the wasp genome. These segments and their flanking regions within the genome of the wasp Cotesia congregata were recently isolated, allowing extensive mapping of amplified sequences. The bracovirus DNAs packaged in the particles were found to be amplified within more than 12 replication units. Strikingly, the nudiviral cluster, the genes of which encode particle structural components, was also amplified, although not encapsidated. Amplification of bracoviral sequences was shown to involve successive head-to-head and tail-to-tail concatemers, which was not expected given the nudiviral origin of bracoviruses.

Expression analysis of MLH3, MLH1, and MSH4 in maturation arrest.

The expression of DNA mismatch repair (DMMR) genes in patients with maturation arrest (MA) was analyzed. Samples were subjected to mutL homolog 3 (MLH3) mutation analysis by denaturing high-performance liquid chromatography/sequencing and quantification of MMR expression in testicular tissue by real-time polymerase chain reaction (PCR). Microsatellite instability assays were negative. Two missense and 1 intronic mutations were found. The missense mutation 2531C/T (P844 L), predicted to affect MLH3 function, was found in 3 MA cases in association with the intronic variant IVS9 + 66G/A. Relative messenger RNA (mRNA) quantification identified 2 patients who overexpressed MLH3, 1 of them also overexpressing mutL homolog 1 (MLH1). The latter also presented the 2531C/T-IVS9 + 66G/A mutation. In conclusion, we suggest that a predominance of MLH3 expression might favor the MLH1/MLH3 complex which then would compete with the MLH1/PMS2 complexes. This could convey disruption of the relative stoichiometry between MLH1/MLH3 and MLH1/PMS2 complexes, thus causing meiosis failure, as MLH1/PMS2 complexes are supposed to replace MLH1/MLH3 during diplonema.

Abrogation of microsatellite-instable tumors using a highly selective suicide gene/prodrug combination.

A substantial fraction of sporadic and inherited colorectal and endometrial cancers in humans is deficient in DNA mismatch repair (MMR). These cancers are characterized by length alterations in ubiquitous simple sequence repeats, a phenotype called microsatellite instability. Here we have exploited this phenotype by developing a novel approach for the highly selective gene therapy of MMR-deficient tumors. To achieve this selectivity, we mutated the VP22FCU1 suicide gene by inserting an out-of-frame microsatellite within its coding region. We show that in a significant fraction of microsatellite-instable (MSI) cells carrying the mutated suicide gene, full-length protein becomes expressed within a few cell doublings, presumably resulting from a reverting frameshift within the inserted microsatellite. Treatment of these cells with the innocuous prodrug 5-fluorocytosine (5-FC) induces strong cytotoxicity and we demonstrate that this owes to multiple bystander effects conferred by the suicide gene/prodrug combination. In a mouse model, MMR-deficient tumors that contained the out-of-frame VP22FCU1 gene displayed strong remission after treatment with 5-FC, without any obvious adverse systemic effects to the mouse. By virtue of its high selectivity and potency, this conditional enzyme/prodrug combination may hold promise for the treatment or prevention of MMR-deficient cancer in humans.

Identification of new breakpoints in AZFb and AZFc.

Microdeletions in AZFa, AZFb and AZFc regions lead to different patterns of male infertility, from severe oligozoospermia to non-obstructive azoospermia. Intrachromosomal homologous recombination mechanisms were already identified in patients with simultaneous microdeletions in the AZFb and AZFc regions. Ten patients with atypical AZFb and AZFc deletion patterns were studied. The definition of those microdeletions and the fine characterization of the respective breakpoints were performed using sequence tagged sites/single nucleotide variants-PCR and DNA sequencing. Y-chromosome haplogroups were determined to establish a putative association with the patterns obtained. Seven deletion patterns were identified, P5/terminal (30%; 3/10), P5/P1 distal (20%; 2/10), IR4/distal-P2, IR2/proximal-P1, IR4/distal-P1, P4/terminal and complete AZFb/c deletion (10%; 1/10). Breakpoint sequence analysis suggests that only in one patient the P5/P1 distal deletion pattern was due to a homologous recombination mechanism. Sequence alignment of the other deletion patterns suggest that they have resulted from non-homologous recombination mechanisms.

DNA mismatch repair gene hMLH3 variants in meiotic arrest.

Defects of the DNA mismatch repair gene hMLH3 were screened by denaturing high-performance liquid chromatography and sequencing in germinal tissue DNA from patients with spermatogenic arrest, with sequence variations being confirmed in genomic DNA by polymerase chain reaction (PCR) direct sequencing analysis. Four missense (2896T/C, 2531C/T) and eight intronic (IVS9+66G/A) variants were found, with the combination of 2531C/T and IVS9+66G/A being identified only in patients with primary meiotic arrest, thus suggesting that two simultaneous hMLH3 variants might predispose to spermatogenic arrest.

DAZ gene copies: evidence of Y chromosome evolution.

The DAZ gene, a contributing factor in infertility, lies on the human Y chromosome's AZFc region, whose deletion is a common cause of spermatogenic failure. Y chromosome binary polymorphisms on the non-recombining Y (NRY) region, believed to be a single occurrence on an evolutionary scale, were typed in a sample of fertile and infertile men with known DAZ backgrounds. The Y single-nucleotide polymorphisms (Y-SNPs) with low mutation rates are currently well characterized and permit the construction of a unique phylogeny of haplogroups. DAZ haplotypes were defined using single-nucleotide variant (SNV)/sequence tagged-site (STS) markers to distinguish between the four copies of the gene. The variation of 10 Y chromosome short tandem repeat (STRs) was used to determine the coalescence age of DAZ haplotypes in a comparable time frame similar to that of SNP haplogroups. An association between DAZ haplotypes and Y chromosome haplogroups was found, and our data show that the DAZ gene is not under selective constraints and its evolution depends only on the mutation rate. The same variants were common to fertile and infertile men, although partial DAZ deletions occurred only in infertile men, suggesting that those should only be used as a tool for infertility diagnosis when analysed in combination with haplogroup determinations.

Unique t(Y;1)(q12;q12) reciprocal translocation with loss of the heterochromatic region of chromosome 1 in a male with azoospermia due to meiotic arrest: a case report.

A de novo reciprocal translocation 46,X,t(Y;1)(q12;q12) was found in an azoospermic male with meiotic arrest. Cytogenetics and fluorescent in situ hybridization (FISH) were used to define the karyotype, translocation breakpoints and homologue pairing. SRY (Yp), Yq11.2-AZF regions, DAZ gene copies and the distal Yq12 heterochromatin were studied by PCR and restriction analysis using sequence-tagged sites and single nucleotide variants. High resolution GTL, CBL and DA-DAPI staining revealed a (Y;1) translocation in all metaphases and a normal karyotype in the patient's father. FISH showed the presence of the distal Yq12 heterochromatic region in der(1) and loss of the heterochromatic region of chromosome 1. PCR demonstrated the intactness of the Y chromosome, including the SRY locus, AZF regions, DAZ genes and distal heterochromatin. A significant decrease (P = 0.005) of Xp/Yp pairing (18.6%), as compared with controls (65.7%), was found in arrested primary spermatocytes, and cell culture and mRNA expression studies confirmed an irreversible arrest at meiosis I, with induction of apoptosis and removal of germ cells by Sertoli cells. We characterized a de novo t(Y;1)(q12;q12) balanced reciprocal translocation with loss of the heterochromatic region of chromosome 1, that caused unpairing of sex chromosomes followed by meiosis I arrest, apoptotic degeneration of germ cells and azoospermia.