Replication Fork Velocity Research Articles

Combing of picogram level DNA equivalent to genomic DNA present in single human cell by self propelled droplet motion over a stable gradient surface

DNA combing is a powerful technique for studying replication profile, fork-directionality and fork velocity. At present, there is requirement of a methodology to comb DNA present in a single human cell for studying replication dynamics at early embryonic stage. In our study, a surface having dual characteristics i.e., affinity towards negatively charged single DNA molecules and a hydrophobic gradient for self propelled droplet motion of combing solution was developed. The surface was made by coating of TCOS (trichloro-octylsilane) by vapor diffusion on APTES (Aminopropyl-triethoxysilane) coated glass slides. A gradient surface having high deposition efficiency (DE) was developed on which 5 picogram DNA equivalent to genomic DNA present in one single human cell can be combed. The gradient surface was thermostable in nature having the ability to sustain boiling temperature for two hours and sustain anisotropy in 70 % ethanol for 80 h. Applicability for multiple runs was enhanced such that the surface can be used for 13–14 times. Factors associated with gradient surface are unidirectional movement of combing solution droplet over the gradient surface for combing straight DNA molecules and a longer gradient surface of more than 1 cm such that long size DNA molecules can be combed. Ellipsometry and contact angle hysteresis confirmed the presence of hydrophobic gradient. XPS (X-ray photoelectron spectroscopy) and FTIR (Fourier Transform Infrared Spectroscopy) confirmed the presence of characteristic affinity towards negatively charged DNA molecules on the gradient surface. Combing solution was optimized for increasing deposition efficiency and for increasing the applicability of gradient surface for multiple runs. High temperature of combing solution was found to increase Deposition Efficiency. Combing solution was also optimized for combing single DNA molecules over the gradient surface. Single DNA molecules were combed by reducing pH and lowering concentration of triton-X in the combing solution. Dye: bp ratio was optimized for high fluorescent intensity and low surface background.

Excessive reactive oxygen species induce transcription-dependent replication stress

Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.

The in vivo measurement of replication fork velocity and pausing by lag-time analysis

An important step towards understanding the mechanistic basis of the central dogma is the quantitative characterization of the dynamics of nucleic-acid-bound molecular motors in the context of the living cell. To capture these dynamics, we develop lag-time analysis, a method for measuring in vivo dynamics. Using this approach, we provide quantitative locus-specific measurements of fork velocity, in units of kilobases per second, as well as replisome pause durations, some with the precision of seconds. The measured fork velocity is observed to be both locus and time dependent, even in wild-type cells. In this work, we quantitatively characterize known phenomena, detect brief, locus-specific pauses at ribosomal DNA loci in wild-type cells, and observe temporal fork velocity oscillations in three highly-divergent bacterial species.

Genome-wide mapping of individual replication fork velocities using nanopore sequencing

Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed, a nanopore sequencing-based method to map and extract the velocity of individual forks detected as tracks of the thymidine analogue bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae mean fork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficient bromodeoxyuridine incorporation and wild-type growth, and precisely quantifies speed changes in cells with altered replisome progression or exposed to hydroxyurea. The positioning of >125,000 fork velocities provides a genome-wide map of fork progression based on individual fork rates, showing a uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites.

Quantitative in vivo measurement of fork velocity by deep sequencing

DNA is the substrate for many competing biological processes in vivo. Recent experiments have revealed that transcription is subject to conflicts which stall the replication fork and predict that the replication fork velocity may vary significantly over the genome. Here, we demonstrate a novel approach to measuring the locus-dependent in vivo replication fork velocity by deep sequencing and test a number of mechanisms for replication slowdown, including transcription-replication conflicts. We expect this approach to be widely applicable to other biological systems.

Rtt109 slows replication speed by histone N-terminal acetylation.

The wrapping of DNA around histone octamers challenges processes that use DNA as their template. In vitro, DNA replication through chromatin depends on histone modifiers, raising the possibility that cells modify histones to optimize fork progression. Rtt109 is an acetyl transferase that acetylates histone H3 before its DNA incorporation on the K56 and N-terminal residues. We previously reported that, in budding yeast, a wave of histone H3 K9 acetylation progresses ∼3–5 kb ahead of the replication fork. Whether this wave contributes to replication dynamics remained unknown. Here, we show that the replication fork velocity increases following deletion of RTT109, the gene encoding the enzyme required for the prereplication H3 acetylation wave. By using histone H3 mutants, we find that Rtt109-dependent N-terminal acetylation regulates fork velocity, whereas K56 acetylation contributes to replication dynamics only when N-terminal acetylation is compromised. We propose that acetylation of newly synthesized histones slows replication by promoting replacement of nucleosomes evicted by the incoming fork, thereby protecting genome integrity.

Abstract 2391: Centromeric chromatin alteration is associated with increase in alphoid array replication fork speed in ovarian cancer cell lines

Abstract The centromere is well known as the center point of cell division, with aberrant centromeric function leading to mitotic dysfunction and chromosomal abnormalities. These abnormalities may in turn lead to diseases, including cancer and premature aging. Binding of CENPB to the CENPB box is orchestrated epigenetically, and has emerged as an important factor in the regulation of the centromeric-chromatin structural landscape. CENP-B depletion that resulted in euchromatization of the alphoid centromere array, also is associated with elevated centromeric DNA replication activity. Observation in two ovarian cancer cells from NCI-60 collection; OVCAR3 and SKOV3, identified as having low and high expression of CENPB RNA respectively, indicates the different level of centromeric DNA instability. Centromeric DNA damage fraction is more abundant and replication fork velocity is elevated in less CENPB expresser cells, indicates alphoid array alteration. We conclude that CENP-B is required for maintenance of centromeric chromatin structure and centromeric DNA integrity. Citation Format: Indri Erliandri, Christophe Redon, Jung-Hyun Kim, Haiqing Fu, Mirit Aladjem, William Reinhold, Vladimir Larionov. Centromeric chromatin alteration is associated with increase in alphoid array replication fork speed in ovarian cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2391.

How do CD8+ T cells complete DNA replication in less than 4 hours?

Abstract We demonstrate that activated CD8+ T cells from Pmel mice replicate their genome within 4 hours which is significantly faster than most cancer cells. Many genotoxic chemotherapeutic agents target the rapid proliferation of cancer cells. However, activated T cells also expand rapidly and this can lead to lymphopenia in patients receiving these chemotherapeutic agents. Understanding how CD8+ T cells complete DNA replication in less than 4 hours could lead to the development of treatment schedules that spare CD8+ T cells. Three possible mechanisms may underlie the rapid DNA replication in CD8+ T cells: 1) faster replication fork velocity, 2) higher density of origin firing, or 3) modified replication timing program, allowing for simultaneous replication of early- and late-replicating regions of the genome. Using DNA combing, we show that the replication fork velocity and inter-origin distance is similar in unperturbed CD8+ T cells and B16 cancer cells. Repli-seq analysis, a NGS genome-wide replication timing analysis, suggest that regions of the genome replicated late in S phase in fibroblasts are replicated earlier in CD8+ T cells, suggesting that the rapid DNA replication in CD8+ T cells is a consequence of a modified, truncated replication timing program. We also show for the first time that actively replicating CD8+ T cells have dormant origins suggesting that this mechanism of genome stability is maintained in activated CD8+ T cells.

Separase activity distribution can be a marker of major molecular response and proliferation of CD34+ cells in TKI-treated chronic myeloid leukemia patients

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

Thymineless Death by the Fluoropyrimidine Polymer F10 Involves Replication Fork Collapse and Is Enhanced by Chk1 Inhibition

We are developing the fluoropyrimidine polymer F10 to overcome limitations of 5-fluorouracil (5-FU) that result from inefficient metabolism to 5-fluoro-2′-deoxyuridine-5′-mono- and tri-phosphate, the deoxyribonucleotide metabolites that are responsible for 5-FU's anticancer activity. F10 is much more cytotoxic than 5-FU to colorectal cancer (CRC) cells; however, the mechanism of enhanced F10 cytotoxicity remains incompletely characterized. Using DNA fiber analysis, we establish that F10 decreases replication fork velocity and causes replication fork collapse, while 1000-fold excess of 5-FU is required to achieve similar endpoints. Treatment of HCT-116 cells with F10 results in Chk1 phosphorylation and activation of intra–S-phase checkpoint. Combining F10 with pharmacological inhibition of Chk1 with either PF-477736 or prexasertib in CRC cells enhanced DNA damage relative to single-agent treatment as assessed by γH2AX intensity and COMET assay. PF-477736 or prexasertib co-treatment also inhibited upregulation of Rad51 levels in response to F10, resulting in reduced homologous repair. siRNA knockdown of Chk1 also increased F10-induced DNA damage assessed and sensitized CRC cells to F10. However, Chk1 knockdown did not inhibit Rad51 upregulation by F10, indicating that the scaffolding activity of Chk1 imparts activity in DNA repair distinct from Chk1 enzymatic activity. Our results indicate that F10 is cytotoxic to CRC cells in part through DNA damage subsequent to replication fork collapse. F10 is ~1000-fold more potent than 5-FU at inducing replication-mediated DNA damage which correlates with the increased overall potency of F10 relative to 5-FU. F10 efficacy can be enhanced by pharmacological inhibition of Chk1.

Analysis of Fission Yeast Single DNA Molecules on the Megabase Scale Using DNA Combing.

DNA combing enables the quantitative analysis of DNA replication, DNA recombination, DNA-protein interaction, and DNA methylation along genomic single DNA molecules at 1kb resolution. However, DNA combing has been restricted to short 200-500kb long DNA fragments, which introduces significant bias in data analysis. An improved DNA combing methodology that allows to routinely image Mb-scale single DNA molecules and occasionally up to full-length fission yeast chromosomes is presented in this chapter. DNA combing of Mb-scale single DNA molecules can be applied to accurately measure the dynamic properties of DNA replication such as the rate of origin firing, replication fork velocity, fork directionality and the frequency of fork blockage. In addition, Mb-scale single DNA molecules enable the quantitative analysis of complex genomic rearrangements including gross chromosomal translocations, repetitive DNA sequences, large deletions, and duplications, which are difficult to investigate with deep sequencing strategies.

Abstract 4886: The BRIP1 DNA helicase is a 17q dosage sensitive cooperative driver in neuroblastoma

Abstract Neuroblastoma (NB) is an aggressive pediatric tumor arising from sympathetic neuronal progenitors. NBs have a low mutation burden while copy number alterations are highly recurrent: MYCN amplification is present in half of high risk tumors often accompanied by 1p deletions while MYCN non-amplified aggressive NB frequently exhibit 11q deletions. Remarkably, both high risk groups show almost invariably chromosome 17q gain and we also reported that the mouse syntenic chromosome 11 region was gained during MYCN driven tumor formation. We propose that one or more dosage sensitive genes on 17q act as cooperative drivers during NB development. Using an integrated bioinformatics analysis, we identified several candidate drivers implicated in DNA repair including BRIP1, also known as FANCJ and located on 17q23.2. BRIP1 acts as a DNA helicase in unwinding of stable G-quadruplex (G4) structures in single stranded DNA during replication ensuring timely progression through S-phase. We show that BRIP1 knock down causes increased replicative stress in MYCN overexpressing NB cells as evidenced as shown by increased RPA32 levels and reduced replication fork velocity. Overexpression of BRIP1 in dβh-MYCN-eGFP transgenic zebrafish caused accelerated tumor formation supporting its role as cooperative driver gene. Gene expression profiling after BRIP1 knock down confirmed enrichment for gene sets implicated in DNA replication and repair and are indicative for perturbation of G4 enriched genes. We also identified further additional 17q dosage sensitive genes implicated in replication fork dynamics including BRCA1, BRCA2, EME1 and TOP2A. We propose that 17q gain acts as an amplifier for expression of multiple genes implicated in control of replicative stress and replication fork dynamics. Finally, we explored whether this replicative stress resistance phenotype could represent a novel therapeutic vulnerability for NB cells or other MYC(N) driven tumor entities. To this end, we tested several compounds for synergistic interaction with G4 stabilizing ligands such as TMPYP4, pyridostatin and BRACO-19 with promising results. In conclusion, we identified BRIP1 as 17q cooperative driver in NB through relief from G4 induced replication fork stalling in rapidly dividing tumor cells. Further, NB cells exhibit replicative stress resistance through upregulation of multiple critical regulators of replication fork dynamics, offering a new venue for therapeutic interventions. Citation Format: Suzanne Vanhauwaert, Kaat Durinck, Els Janssens, Givani Dewyn, Bram De Wilde, Genevieve Laureys, Daniel Carter, Chueng Belamy, Katleen De Preter, Christophe Vanneste, Frank Speleman. The BRIP1 DNA helicase is a 17q dosage sensitive cooperative driver in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4886. doi:10.1158/1538-7445.AM2017-4886

Model-based analysis of DNA replication profiles: predicting replication fork velocity and initiation rate by profiling free-cycling cells.

Eukaryotic cells initiate DNA synthesis by sequential firing of hundreds of origins. This ordered replication is described by replication profiles, which measure the DNA content within a cell population. Here, we show that replication dynamics can be deduced from replication profiles of free-cycling cells. While such profiles lack explicit temporal information, they are sensitive to fork velocity and initiation capacity through the passive replication pattern, namely the replication of origins by forks emanating elsewhere. We apply our model-based approach to a compendium of profiles that include most viable budding yeast mutants implicated in replication. Predicted changes in fork velocity or initiation capacity are verified by profiling synchronously replicating cells. Notably, most mutants implicated in late (or early) origin effects are explained by global modulation of fork velocity or initiation capacity. Our approach provides a rigorous framework for analyzing DNA replication profiles of free-cycling cells.

Abstract 3590: A Neil1 DNA glycosylase germline variant induces genomic instability and cellular transformation

Abstract NEIL1 DNA glycosylase is a bifunctional enzyme that recognizes oxidized pyrimidines and formamidopyrimidines, and appears to remove damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene has a minor allele frequency of 1% and is a G to A base substitution that encodes the G83D NEIL1 protein. Interestingly, this mutation is also present in cholangiocarcinomas. Unlike WT NEIL1, the G83D variant is not able to recognize oxidized bases efficiently due to a shift in the void filling residues that stabilize the DNA duplex once a damaged base has been extruded into the glycosylase substrate binding pocket. The goal of our study was to determine if the G83D NEIL1 variant exhibits a cancer-related phenotype in cells. We found that unlike wild-type (WT) NEIL1, expression of G83D in MCF10A immortal human breast epithelial cells induces genomic instability and cellular transformation. Expression of G83D but not WT NEIL1, in MCF10A cells leads to replication fork stalling and slow replication fork velocity. Our results suggest that G83D is unable to remove oxidative DNA damage at the replication fork, resulting in genomic instability and cellular transformation. Our results are consistent with the idea that individuals who harbor the G83D NEIL1 variant are at increased risk for cancer. Citation Format: Joann B. Sweasy, Heather Galick, Scott Kathe, Susan Wallace. A Neil1 DNA glycosylase germline variant induces genomic instability and cellular transformation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3590.

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.

Abstract B29: Inhibition of histone deacetylases 1 and 2 (HDAC1,2) perturbs DNA replication and DNA repair in cancer cells: Implications in mechanism-based therapeutic strategies

Abstract Class I family enzymes, histone deacetylases 1 and 2 (HDAC1,2), are targets of FDA-approved small molecule inhibitors currently used in cancer therapy. HDAC inhibitors impair DNA repair and replication in cancer cells to trigger genotoxic stress and cause cell death. How HDAC1,2 function in genome maintenance processes, DNA replication and repair, in mammalian cells is far from fully understood. Using small molecules to selectively inhibit HDAC1,2 activities, we have systematically defined their direct functions in DNA replication and repair. Our studies show that HDAC1,2 inhibition reduces replication fork velocity and activates the replication stress associated DNA damage independent of its effect on transcription. HDAC1,2 inhibition increases histone H4K16 acetylation (H4K16ac) and nuclease sensitivity of nucleosomes associated with newly replicated DNA, suggesting that HDAC1,2 are required for chromatin compaction during DNA replication. Thus, mechanistically, HDAC1,2 activities are required for maintaining nascent chromatin structure and dynamics required for efficient progression of the replication fork. In addition to H4K16ac, we have found that HDAC1,2 inhibition activities increases H3K27ac and H4K91ac present on the histone tail and core regions, respectively. Increase in these histone acetyl marks perturbs the acetyl-methyl or the acetyl-ubiquityl switch during DNA repair leading to impaired EZH2-mediated H3K27me3-dependent DNA repair signaling and the H4K91ubiquityl-dependent 53BP1-mediated DNA repair signaling in chemoresistant EZH2 gain-of-function diffuse large B-cell lymphoma cells. We are currently investigating the therapeutic benefits of HDAC1,2 inhibition in mouse models of aggressive chemoresistant diffuse large B-cell lymphoma and in primary patient samples. Overall, our studies have uncovered novel functions for HDAC1,2 in genome maintenance processes and provide insight into the mode-of-action of HDAC1,2 inhibitors in overcoming chemoresistance in cancer cells (via targeting the DNA repair process) and in inducing DNA damage and death in the rapidly cycling cancer cells (via targeting DNA replication). Note: This abstract was not presented at the conference. Citation Format: Danielle Johnson, Simon Jones, Steven Quayle, Jeff Shearstone, Srividya Bhaskara. Inhibition of histone deacetylases 1 and 2 (HDAC1,2) perturbs DNA replication and DNA repair in cancer cells: Implications in mechanism-based therapeutic strategies. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B29.

Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication.

UVA radiation (320–400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydrogen peroxide (H2O2), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen.

DNA replication stress underlies renal phenotypes in CEP290-associated Joubert syndrome.

Juvenile ciliopathy syndromes that are associated with renal cysts and premature renal failure are commonly the result of mutations in the gene encoding centrosomal protein CEP290. In addition to centrosomes and the transition zone at the base of the primary cilium, CEP290 also localizes to the nucleus; however, the nuclear function of CEP290 is unknown. Here, we demonstrate that reduction of cellular CEP290 in primary human and mouse kidney cells as well as in zebrafish embryos leads to enhanced DNA damage signaling and accumulation of DNA breaks ex vivo and in vivo. Compared with those from WT mice, primary kidney cells from Cep290-deficient mice exhibited supernumerary centrioles, decreased replication fork velocity, fork asymmetry, and increased levels of cyclin-dependent kinases (CDKs). Treatment of Cep290-deficient cells with CDK inhibitors rescued DNA damage and centriole number. Moreover, the loss of primary cilia that results from CEP290 dysfunction was rescued in 3D cell culture spheroids of primary murine kidney cells after exposure to CDK inhibitors. Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes.

Interlinked Sister Chromosomes Arise in the Absence of Condensin during Fast Replication in B. subtilis

SummaryCondensin—an SMC-kleisin complex—is essential for efficient segregation of sister chromatids in eukaryotes [1, 2, 3, 4]. In Escherichia coli and Bacillus subtilis, deletion of condensin subunits results in severe growth phenotypes and the accumulation of cells lacking nucleoids [5, 6]. In many other bacteria and under slow growth conditions, however, the reported phenotypes are much milder or virtually absent [7, 8, 9, 10]. This raises the question of what role prokaryotic condensin might play during chromosome segregation under various growth conditions. In B. subtilis and Streptococcus pneumoniae, condensin complexes are enriched on the circular chromosome near the single origin of replication by ParB proteins bound to parS sequences [11, 12]. Using conditional alleles of condensin in B. subtilis, we demonstrate that depletion of its activity results in an immediate and severe defect in the partitioning of replication origins. Multiple copies of the chromosome remain unsegregated at or near the origin of replication. Surprisingly, the growth and chromosome segregation defects in rich medium are suppressed by a reduction of replication fork velocity but not by partial inhibition of translation or transcription. Prokaryotic condensin likely prevents the formation of sister DNA interconnections at the replication fork or promotes their resolution behind the fork.

Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression

BackgroundHistone deacetylases (HDACs) play a critical role in the maintenance of genome stability. Class I HDACs, histone deacetylase 1 and 2 (Hdac1 and Hdac2) are recruited to the replication fork by virtue of their interactions with the replication machinery. However, functions for Hdac1 and Hdac2 (Hdacs1,2) in DNA replication are not fully understood.ResultsUsing genetic knockdown systems and novel Hdacs1,2-selective inhibitors, we found that loss of Hdacs1,2 leads to a reduction in the replication fork velocity, and an increase in replication stress response culminating in DNA damage. These observed defects are due to a direct role for Hdacs1,2 in DNA replication, as transcription of genes involved in replication was not affected in the absence of Hdacs1,2. We found that loss of Hdacs1,2 functions increases histone acetylation (ac) on chromatin in S-phase cells and affects nascent chromatin structure, as evidenced by the altered sensitivity of newly synthesized DNA to nuclease digestion. Specifically, H4K16ac, a histone modification involved in chromatin decompaction, is increased on nascent chromatin upon abolishing Hdacs1,2 activities. It was previously shown that H4K16ac interferes with the functions of SMARCA5, an ATP-dependent ISWI family chromatin remodeler. We found SMARCA5 also associates with nascent DNA and loss of SMARCA5 decreases replication fork velocity similar to the loss or inhibition of Hdacs1,2.ConclusionsOur studies reveal important roles for Hdacs1,2 in nascent chromatin structure maintenance and regulation of SMARCA5 chromatin-remodeler function, which together are required for proper replication fork progression and genome stability in S-phase.