Asynchronous DNA Replication Research Articles

Same, same but different: Exploring Plasmodium cell division during liver stage development.

Plasmodium parasites have a complex life cycle alternating between a mosquito and a vertebrate host. Following the bite of an Anopheles female mosquito, Plasmodium sporozoites are transmitted from the skin to the liver; their first place of replication within the host. Successfully invaded sporozoites undergo a massive replication and growth involving asynchronous DNA replication and division that results in the generation of tens of thousands or even hundreds of thousands of merozoites depending on the Plasmodium species. The generation of a high number of daughter parasites requires biogenesis and segregation of organelles to finally reach a relatively synchronous cytokinesis event. At the end of liver stage (LS) development, merozoites are packed into merosomes and released into the bloodstream. They are then liberated and infect red blood cells to again produce merozoites by schizogony for the erythrocytic stage of the life cycle. Although parasite LS and asexual blood stage (ABS) differ in many respects, important similarities exist between the two. This review focuses on the cell division of Plasmodium parasite LS in comparison with other life cycle stages especially the parasite blood stage.

Differential condensation of sister chromatids acts with Cdc6 to ensure asynchronous S-phase entry in Drosophila male germline stem cell lineage.

During Drosophila melanogaster male germline stem cell (GSC) asymmetric division, preexisting old versus newly synthesized histones H3 and H4 are asymmetrically inherited. However, the biological outcomes of this phenomenon have remained unclear. Here, we tracked old and new histones throughout the GSC cell cycle through the use of high spatial and temporal resolution microscopy. We found unique features that differ between old and new histone-enriched sister chromatids, including differences in nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, a pre-replication complex component, and subsequent asynchronous DNA replication initiation in the resulting daughter cells. Disruption of asymmetric histone inheritance abolishes differential Cdc6 association and asynchronous S-phase entry, demonstrating that histone asymmetry acts upstream of these critical cell-cycle progression events. Furthermore, disruption of these GSC-specific chromatin features leads to GSC defects, indicating a connection between histone inheritance, cell-cycle progression, and cell fate determination.

Asynchronous nuclear cycles in multinucleated Plasmodium falciparum facilitate rapid proliferation.

Malaria-causing parasites proliferate within erythrocytes through schizogony, forming multinucleated stages before cellularization. Nuclear multiplication does not follow a strict geometric 2n progression, and each proliferative cycle produces a variable number of progeny. Here, by tracking nuclei and DNA replication, we show that individual nuclei replicate their DNA at different times, despite residing in a shared cytoplasm. Extrapolating from experimental data using mathematical modeling, we provide strong indication that a limiting factor exists, which slows down the nuclear multiplication rate. Consistent with this prediction, our data show that temporally overlapping DNA replication events were significantly slower than partially overlapping or nonoverlapping events. Our findings suggest the existence of evolutionary pressure that selects for asynchronous DNA replication, balancing available resources with rapid pathogen proliferation.

Asynchronous DNA Replication of Biallelically Expressed Genes in Human Peripheral Blood Lymphocytes as a Prognostic Sign of Cancer.

The aim of the study was to identify and quantify lymphocytes with asynchronous replication of the AURKA and TP53 genes in cancer patients versus controls and to assess the diagnostic capabilities of this approach.Materials and MethodsThe study was carried out with peripheral blood lymphocytes probed for the AURKA and TP53 genes using the interphase fluorescence in situ hybridization (FISH) method (Vysis, USA and Kreatech, The Netherlands). The control group included 70 people: clinically healthy donors and patients with non-oncological diseases such as gastritis, pancreatitis, chronic calculous cholecystitis, bronchial asthma, peptic ulcer disease, inguinal hernia, arthrosis, myoma, hepatitis, epilepsy, chronic prostatitis, chronic tonsillitis, and rectal adenoma. The group of cancer patients included 219 people with various oncological diseases: gastric cancer (n=68), colorectal cancer (n=30), chronic lymphocytic leukemia (n=52), Hodgkin lymphoma (n=33), and polyneoplasia (n=41).ResultsIn the control group, the mean frequency of lymphocytes with asynchronous gene replication (AGR) was 22.0±3.4% for AURKA and 18.0±3.2% for TP53; in the group of cancer patients, that was 36.8±4.8 and 28.4±5.1%, respectively. The excessive presence of lymphocytes with the AGR in cancer patients was consistent and statistically significant (p<0.0001). For the AURKA gene, the AGR-based cancer detection showed a sensitivity of 98.6±0.7%, a specificity of 100%, and an accuracy of 98.3±0.8%, and for the TP53 gene — 78.6±3.1, 98.5±0.9, and 85.9±2.6%, respectively.ConclusionThis pilot study on lymphocytes with AGR of AURKA and TP53 genes in cancer patients can serve a basis for creating a new molecular cytogenetic technology for detecting malignant neoplasms in humans.

Characterization of chromatin at structurally abnormal inactive X chromosomes reveals potential evidence of a rare hybrid active and inactive isodicentric X chromosome.

X chromosome structural abnormalities are relatively common in Turner syndrome patients, in particular X isochromosomes. Reports over the last five decades examining asynchronous DNA replication between the normal X and isochromosome have clearly established that the structurally abnormal chromosome is the inactive X chromosome (Xi). Here the organization of chromatin at a deleted X chromosome, an Xq isochromosome, and two isodicentric chromosomes were examined. Consistent with previous differential staining methods, at interphase, the X isochromosome and isodicentric X chromosomes frequently formed bipartite Barr bodies, observed by fluorescence microscopy using numerous independent bona fide markers of Xi heterochromatin. At metaphase, with the exception of the pseudoautosomal region and the duplicated locus of the macrosatellite DXZ4 (if present on the abnormal X chromosome based on break points), euchromatin markers were absent from the Xi, whereas histone variant macroH2A formed reproducible banded mirror-image chromosomes. Unexpectedly, the isodicentric chromosome in 46,X,idic(X)(q28) cells, which carry a near full-length q-arm-to-q-arm fused chromosome, showed at interphase very rare instances of Xi chromatin bodies that were separated by large distances in the nucleus. Further examination using immunofluorescence and FISH support the possibility that these rare cells may represent ones in which one half of the isodicentric chromosome is active and the other half is inactive.

Chromatin Structure Dynamics Preserve Genome Stability in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a genetically complex disease with extensive clonal heterogeneity. Substantial genomic instability in MM is illustrated by extensive copy number variations (CNVs) that can be detected in almost every MM patient. The molecular basis of this genomic instability in MM is not clear. Linker histones are dynamic components of chromatin and mutations in these molecules are present in ~6% of MM patients. Two of the linker histone super-family, HIST1H1Eand HIST1H1Care the most frequently mutated members of the family in MM and their mutations mostly occur in a clonal fashion. Interestingly, it has been reported in human cell lines and in other species that linker histone loss affects DNA damage/repair pathways and leads to transcription-replication conflicts. Based on these data we hypothesized that mutation or genomic loss of linker histones affects the genome stability of MM cells. To test this hypothesis, we developed an experimental system using CRISPR/Cas9 genome editing to generate MM linker histone-deficient cells. Low-pass whole genome sequencing (LPWGS), immunoblotting and immunofluorescent experiments were performed for genomic, molecular and functional characterization. We found that HIST1H1E,HIST1H1Cand H1FXwere the most abundantly expressed members of the linker histone family in primary myeloma cells and that myeloma cells have the highest dependency on HIST1H1Eand HIST1H1Cwhen compared to all other cancer cell lines derived from other tissues. We used OPM2 and U266 myeloma cell lines and generated knock-out variants of HIST1H1E, HIST1H1Cand H1FXlinker histones by inserting a biallelic stop codon, followed by generation of individual single-cell clones that were used as replicates. We first asked if linker histone deficient cells preserve genome stability. To address this question, we performed low pass whole genome sequencing and found more copy number abnormalities in linker histone deficient myeloma cells, when compared to wild-type cells. Moreover, linker histone deficient cells showed increased DNA damage as indicated by higher frequency of nuclear foci that were positive for damage dependent phosphorylation of the histone variant H2AX ( γH2AX). This was associated with an increased frequency of micronuclei in linker histones deficient cells, suggesting defects in mitotic fidelity and in genome stability. These micronuclei were positive for γH2AX by microscopic staining, indicative of DNA damage. We then asked if the DNA damage in micronuclei is due to defective and asynchronous DNA replication when the myeloma cells are exposed to etoposide, a topoisomerase inhibitor that induces DNA replication stress and double-strand DNA breaks (DSBs). Etoposide treatment of myeloma cells caused DNA replication stress, as measured by immunofluorescent staining of micronuclei for Replication Protein A (RPA). Conclusions: Our results demonstrate that loss of linker histones is associated with increased copy number abnormalities, extensive DNA damage and increased frequency of micronuclei, most likely as a consequence of replication stress. These data provide a potential mechanism of how chromatin structure dynamics preserve genome stability in myeloma cells. Disclosures Lohr: Celgene: Research Funding; T2 Biosystems: Honoraria.

Asynchronous replication in oncological patients

Epigenetics is a science studying mechanisms of heritable changes in gene function that occur without a change in the DNA sequence. One of the most important marks of epigenetic misbalance of cell genome is an replication asynchrony of genes with biallelic expression. Interphase fluorescence in situ hybridization (I-FISH) in phytohaemagglutinin-stimulated lymphocytes of peripheral blood is a proper method of estimation of aberrant DNA replication time e. g. DNA replication asynchrony. In this review we analyzed reports referring to asynchronous DNA replication of biallelically expressed genes in lymphocytes of peripheral blood of cancer patients. Analysis shows the DNA replication asynchrony is a nonspecific tumor marker observing both in tumor cells and lymphocytes of peripheral blood in oncohematological patients and patients with solid tumors. It’s stated the frequency of lymphocytes with asynchronous DNA replication of studied genes in cancer patients is increased significantly compares with healthy donors and is enhanced during malignance process. It gives the opportunity of potential using asynchronous replication as molecular genetic marker for cancer patients early revealing.

Papillomavirus genomes associate with BRD4 to replicate at fragile sites in the host genome.

It has long been recognized that oncogenic viruses often integrate close to common fragile sites. The papillomavirus E2 protein, in complex with BRD4, tethers the viral genome to host chromatin to ensure persistent replication. Here, we map these targets to a number of large regions of the human genome and name them Persistent E2 and BRD4-Broad Localized Enrichments of Chromatin or PEB-BLOCs. PEB-BLOCs frequently contain deletions, have increased rates of asynchronous DNA replication, and are associated with many known common fragile sites. Cell specific fragile sites were mapped in human C-33 cervical cells by FANCD2 ChIP-chip, confirming the association with PEB-BLOCs. HPV-infected cells amplify viral DNA in nuclear replication foci and we show that these form adjacent to PEB-BLOCs. We propose that HPV replication, which hijacks host DNA damage responses, occurs adjacent to highly susceptible fragile sites, greatly increasing the chances of integration here, as is found in HPV-associated cancers.

Asynchronous DNA replication and aneuploidy in lymphocytes of hepatocellular carcinoma patients

Using fluorescence in situ hybridization (FISH) analysis, we examined the replication mode of the centromere region (homologous counterpart) and the aneuploidy level of chromosome 17 in the interphase nuclei of phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes from (1) patients with hepatocellular carcinoma (HCC); (2) patients with liver cirrhosis (LC) due to hepatitis C viral infection who are individuals at a higher increased risk for HCC; and (3) healthy control participants. We also compared the allelic-replication asynchrony and aneuploidy frequencies with serum alpha-fetoprotein (AFP) levels. We found a significant increase in centromeric replication asynchrony accompanied by a high frequency of aneuploidy in lymphocytes of HCC patients compared with those of LC patients and healthy control participants. These changes are similar to those previously observed in other types of malignancy (hematological, ovarian, prostate, and breast cancer). The cytogenetic alterations of aneuploidy and strong asynchronous replication displayed in the lymphocytes of HCC patients arose from malignancy, as they were associated neither with an increased risk for cancer nor with an infection. The cytogenetic cancer-associated markers observed in patients' lymphocytes appeared to be superior to serum AFP, the marker currently used for HCC. Thus, the cytogenetic cancer-associated markers may be potentially useful in noninvasive cancer detection.

Micronuclear Chromosome Pulverization May Underlie Chromothripsis

Abstract Major finding: Mitotic errors produce micronuclear DNA that can be incorporated into the genome.Mechanism: Defective, asynchronous DNA replication damages chromosomes in micronuclei.Impact: Whole-chromosome aneuploidy can cause DNA damage and potentially produce mutations.

DNA breaks and chromosome pulverization from errors in mitosis

Whether whole-chromosome aneuploidy promotes tumorigenesis has been controversial, in large part because of the paucity of insight into underlying mechanisms. Here we identify a mechanism by which mitotic chromosome segregation errors generate DNA breaks via the formation of structures called micronuclei. Whole chromosome-containing micronuclei form when mitotic errors produce lagging chromosomes. We tracked the fate of newly generated micronuclei and found that they undergo defective and asynchronous DNA replication, resulting in DNA damage and frequently pulverization of the chromosome in the micronucleus. Micronuclei can persist in cells over several generations but the chromosome in the micronucleus can also be distributed to daughter nuclei. Thus, chromosome segregation errors potentially lead to mutations and chromosome rearrangements that can integrate into the genome. Pulverization of chromosomes in micronuclei may also be one explanation for “chromothripsis” in cancer and developmental disorders, where isolated chromosomes or chromosome arms undergo massive local DNA breakage and rearrangement.

Asynchronous DNA replication and origin licensing in the mouse one‐cell embryo

To prevent duplicate DNA synthesis, metazoan replication origins are licensed during G1. Only licensed origins can initiate replication, and the cytoplasm interacts with the nucleus to inhibit new licensing during S phase. DNA replication in the mammalian one-cell embryo is unique because it occurs in two separate pronuclei within the same cytoplasm. Here, we first tested how long after activation the oocyte can continue to support licensing. Because sperm chromatin is licensed de novo after fertilization, the timing of sperm injection can be used to assay licensing initiation. To experimentally skip some of the steps of sperm decondensation, we injected mouse sperm halos into parthenogenetically activated oocytes. We found that de novo licensing was possible for up to 3 h after oocyte activation, and as early as 4 h before DNA replication began. We also found that the oocyte cytoplasm could support asynchronous initiation of DNA synthesis in the two pronuclei with a difference of at least 2 h. We next tested how tightly the oocyte cytoplasm regulates DNA synthesis by transferring paternal pronuclei from zygotes generated by intracytoplasmic sperm injection (ICSI) into parthenogenetically activated oocytes. The pronuclei from G1 phase zygotes transferred into S phase ooplasm were not induced to prematurely replicate and paternal pronuclei from S phase zygotes transferred into G phase ooplasm continued replication. These data suggest that the one-cell embryo can be an important model for understanding the regulation of DNA synthesis.

Asynchronous DNA replication detected by fluorescence in situ hybridisation as a possible indicator of genetic damage in human lymphocytes

The present study aimed to correlate the DNA replication timing of different genes with genetic damage and frequency of cancer. Using a fluorescence in situ hybridisation (FISH) approach, the replication timing of three loci, two human genes possessing transcriptional capability and involved in both the cellular response to genetic damage and cancer development (TP53 and RB1) and the non-coding locus D22S163, was evaluated. The data obtained show that normal human lymphocytes exposed in vitro to known DNA-damaging agents, e.g. H2O2, ionizing radiation and mitomycin C, exhibit an asynchronous replication of the genes TP53 and RB1. In vivo studies were performed in three different populations from Kazakhstan. In two of these populations that are living in polluted areas and have higher cancer mortalities than people living in a control area, a DNA replication behaviour similar to that observed in human lymphocytes exposed in vitro to known genotoxic agents was detected. The results obtained further indicate that DNA damage hampers replication and FISH represents a fast and accurate method of assessing asynchronous replication by providing an important tool to evaluate DNA damage at a populational level.

Gene cluster lock after pheromone receptor gene choice

In mammals, perception of pheromones is based on the expression in each vomeronasal sensory neuron of a limited set of receptor genes, chosen among a large repertoire. Here, we report an extremely tight control of the monogenic and monoallelic transcription of the V1rb2 receptor gene. Combining genetic and electrophysiological approaches, we show that the transcription of a non-functional V1r allele leads to the coexpression of another, functional V1r gene. The choice of this coexpressed gene surprisingly includes genes located on the cluster homologous to the one from which the mutant allele is transcribed. However, V1r genes located in cis relative to the transcribed mutant allele are excluded from the coexpression choice. Our observations strongly suggest a monogenic regulatory mechanism acting (a) at a general level, via the expression of the V1r receptor itself, and (b) at a more local level, defined by the V1r gene cluster.

An epigenetic state associated with areas of gene duplication.

Asynchronous DNA replication is an epigenetically determined feature found in all cases of monoallelic expression, including genomic imprinting, X-inactivation, and random monoallelic expression of autosomal genes such as immunoglobulins and olfactory receptor genes. Most genes of the latter class were identified in experiments focused on genes functioning in the chemosensory and immune systems. We performed an unbiased survey of asynchronous replication in the mouse genome, excluding known asynchronously replicated genes. Fully 10% (eight of 80) of the genes tested exhibited asynchronous replication. A common feature of the newly identified asynchronously replicated areas is their proximity to areas of tandem gene duplication. Testing of other clustered areas supported the idea that such regions are enriched with asynchronously replicated genes.

Mutations of theDrosophilaZinc Finger-encoding GenevielfältigImpair Mitotic Cell Divisions and Cause Improper Chromosome Segregation

We describe the molecular characterization and function of vielfältig (vfl), a X-chromosomal gene that encodes a nuclear protein with six Krüppel-like C2H2 zinc finger motifs. vfl transcripts are maternally contributed and ubiquitously distributed in eggs and preblastoderm embryos, excluding the germline precursor cells. Zygotically, vfl is expressed strongly in the developing nervous system, the brain, and in other mitotically active tissues. Vfl protein shows dynamic subcellular patterns during the cell cycle. In interphase nuclei, Vfl is associated with chromatin, whereas during mitosis, Vfl separates from chromatin and becomes distributed in a granular pattern in the nucleoplasm. Functional gain-of-function and lack-of-function studies show that vfl activity is necessary for normal mitotic cell divisions. Loss of vfl activity disrupts the pattern of mitotic waves in preblastoderm embryos, elicits asynchronous DNA replication, and causes improper chromosome segregation during mitosis.

Chicken orthologues of mammalian imprinted genes are clustered on macrochromosomes and replicate asynchronously

In the chicken genome, most orthologues of mouse imprinted genes are clustered on macrochromosomes. Only a few orthologues are located in the microchromosome complement. Macrochromosomal and, to a lesser extent, microchromosomal regions containing imprinted gene orthologues exhibit asynchronous DNA replication. We conclude that highly conserved arrays of imprinted gene orthologues were selected during vertebrate evolution, long before these genes were recruited for parent-specific gene expression by genomic imprinting mechanisms. Evidently, the macrochromosome complement provides a better chromatin environment for the establishment of asynchronous DNA replication and imprinted gene expression later in evolution than microchromosomes.

Crystal structure of a SeqA–N filament: implications for DNA replication and chromosome organization

Escherichia coli SeqA binds clusters of transiently hemimethylated GATC sequences and sequesters the origin of replication, oriC, from methylation and premature reinitiation. Besides oriC, SeqA binds and organizes newly synthesized DNA at replication forks. Binding to multiple GATC sites is crucial for the formation of stable SeqA-DNA complexes. Here we report the crystal structure of the oligomerization domain of SeqA (SeqA-N). The structural unit of SeqA-N is a dimer, which oligomerizes to form a filament. Mutations that disrupt filament formation lead to asynchronous DNA replication, but the resulting SeqA dimer can still bind two GATC sites separated from 5 to 34 base pairs. Truncation of the linker between the oligomerization and DNA-binding domains restricts SeqA to bind two GATC sites separated by one or two full turns. We propose a model of a SeqA filament interacting with multiple GATC sites that accounts for both origin sequestration and chromosome organization.

Monoallelic Expression and Asynchronous Replication of p120 Catenin in Mouse and Human Cells

The number of autosomal mammalian genes subject to random monoallelic expression has been limited to genes highly specific to the function of chemosensory neurons or lymphocytes, making this phenomenon difficult to address systematically. Here we demonstrate that asynchronous DNA replication can be used as a marker for the identification of novel genes with monoallelic expression and identify p120 catenin, a gene involved in cell adhesion, as belonging to this class. p120 is widely expressed; its presence in available cell lines allowed us to address quantitative aspects of monoallelic expression. We show that the epigenetic choice of active allele is clonally stable and that biallelic clones express p120 at twice the level of monoallelic clones. Unlike previous reports about genes of this type, we found that expression of p120 can be monoallelic in one cell type and strictly biallelic in another. We show that in human lymphoblasts, the silencing of one allele is incomplete. These unexpected properties are likely to be wide-spread, as we show that the Tlr4 gene shares them. Identification of monoallelic expression of a nearly ubiquitous gene indicates that this type of gene regulation is more common than previously thought. This has important implications for carcinogenesis and definition of cell identity.

Monoallelic gene expression: a repertoire of recurrent themes.

The development of mature B and T cells in the lymphoid system involves a series of molecular decisions that culminate in the expression of a single antigen receptor on the cell surface, a phenomenon termed allelic exclusion. While feedback inhibition of the recombinase-activation gene proteins evidently plays an important role in the maintenance of allelic exclusion, the initial restriction of rearrangement to only one allele in each cell seems to be achieved through monoallelic epigenetic changes. Epigenetic mechanisms involved in the establishment of allelic exclusion also play a central role in other types of monoallelic expression, including X-chromosome inactivation in female cells, and parental imprinting. In all three systems, the inequality of the two alleles seems to be achieved mainly by differential DNA methylation, asynchronous DNA replication, differential chromatin modifications, unequal nuclear localization, and non-coding RNA. In this review, we discuss the unifying features among these monoallelically expressed systems and the unique characteristics displayed by each of them.