Completing genome replication outside of S phase.

Mitotic DNA synthesis (MiDAS) is an unusual form of DNA replication that occurs during mitosis. Initially, MiDAS was characterized as a process associated with intrinsically unstable loci known as common fragile sites that occurs after cells experience DNA replication stress (RS). However, it is now believed to be a more widespread "salvage" mechanism that is called upon to complete the duplication of any under-replicated genomic region. Emerging data suggest that MiDAS is a DNA repair process potentially involving two or more pathways working in parallel or sequentially. In this review, we introduce the causes of RS, regions of the human genome known to be especially vulnerable to RS, and the strategies used to complete DNA replication outside of S phase. Additionally, because MiDAS is a prominent feature of aneuploid cancer cells, we will discuss how targeting MiDAS might potentially lead to improvements in cancer therapy.

Integrator facilitates RNAPII removal to prevent transcription-replication collisions and genome instability.

DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.

RAD51 bypasses the CMG helicase to promote replication fork reversal.

Replication fork reversal safeguards genome integrity as a replication stress response. DNA translocases and the RAD51 recombinase catalyze reversal. However, it remains unknown why RAD51 is required and what happens to the replication machinery during reversal. We find that RAD51 uses its strand exchange activity to circumvent the replicative helicase, which remains bound to the stalled fork. RAD51 is not required for fork reversal if the helicase is unloaded. Thus, we propose that RAD51 creates a parental DNA duplex behind the helicase that is used as a substrate by the DNA translocases for branch migration to create a reversed fork structure. Our data explain how fork reversal happens while maintaining the helicase in a position poised to restart DNA synthesis and complete genome duplication.

A new fluorescent probe for sensing Al3+ ions in the solution phase and CH3COO- in the solid state with aggregation induced emission (AIE) activity.

An AIE (aggregation induced emission) active probe DFP-AMQ was designed and synthesized as a hexa-coordinated N2O donor chelator for the selective sensing of Al3+ colorimetrically as well as fluorimetrically with a 27-fold fluorescence enhancement for CH3CN-H2O (9 : 1, v/v, pH 7.2, HEPES buffer). The fluorescence enhancement occurred through the blocking of ESIPT, chelation enhanced fluorescence effect (CHEF) arose, and as a result fluorescence enhancement was observed through 1 : 1 complexation with Al3+ ions. Detailed spectroscopic studies including UV-Vis, FTIR, 1H NMR, and HRMS studies were carried out to characterize the probable structure of DFP-AMQ including the complexation of DFP-AMQ with Al3+ ions. The spectrophotometric and spectrofluorimetric titrations revealed strong binding towards Al3+ and the K d values were obtained from UV-Vis (3.26 × 10-5 M-1) and fluorescence titration (2.02 × 10-5 M-1). The limit of detection of Al3+ by DFP-AMQ was 1.11 µM. The quantum yields of DFP-AMQ and [DFP-AMQ-Al]+ were calculated to be 0.008 and 0.211, respectively. Dynamic light scattering (DLS) studies showed that the sizes of the particles increased with increasing water percentage due to aggregation. SEM (scanning electron microscopy) studies revealed interesting morphological changes in microstructures in which DFP-AMQ demonstrated a rod-like shape, which was converted to a spherical-like shape in the presence of Al3+ and when DFP-AMQ aggregated in H2O it showed aggregated block-like shape. In the solid phase, DFP-AMQ with nitrate has no particular shape, but in the presence of acetate, it converts to stone-like shape. This probe (DFP-AMQ) could be employed for on-site Al3+ ion detection in the solid state.

Mitotic DNA synthesis in response to replication stress requires the sequential action of DNA polymerases zeta and delta in human cells.

Oncogene activation creates DNA replication stress (RS) in cancer cells, which can generate under-replicated DNA regions (UDRs) that persist until cells enter mitosis. UDRs also have the potential to generate DNA bridges in anaphase cells or micronuclei in the daughter cells, which could promote genomic instability. To suppress such damaging changes to the genome, human cells have developed a strategy to conduct 'unscheduled' DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated loci. Previous studies have shown that MiDAS proceeds via a POLD3-dependent pathway that shows some features of break-induced replication. Here, we define how human cells utilize both DNA gap filling (REV1 and Pol ζ) and replicative (Pol δ) DNA polymerases to complete genome duplication following a perturbed S-phase. We present evidence for the existence of a polymerase-switch during MiDAS that is required for new DNA synthesis at UDRs. Moreover, we reveal that, upon oncogene activation, cancer cell survival is significantly compromised when REV1 is depleted, suggesting that REV1 inhibition might be a feasible approach for the treatment of some human cancers.

RAD51 protects human cells from transcription-replication conflicts.

Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements. Many episodes of physiological RS likely arise due to conflicts between the DNA replication and transcription machineries operating simultaneously at the same loci. One role of the RAD51 recombinase in human cells is to protect replication forks undergoing RS. Here, we have identified a key role for RAD51 in preventing transcription-replication conflicts (TRCs) from triggering replication fork breakage. The genomic regions most affected by RAD51 deficiency are characterized by being replicated and transcribed in early S-phase and show significant overlap with loci prone to cancer-associated amplification. Consistent with a role for RAD51 in protecting against transcription-replication conflicts, many of the adverse effects of RAD51 depletion are ameliorated by inhibiting early S-phase transcription. We propose a model whereby RAD51 suppresses fork breakage and subsequent inadvertent amplification of genomic loci prone to experiencing TRCs.

MicroRNA-449a Inhibits Triple Negative Breast Cancer by Disturbing DNA Repair and Chromatid Separation.

Chromosomal instability (CIN) can be a driver of tumorigenesis but is also a promising therapeutic target for cancer associated with poor prognosis such as triple negative breast cancer (TNBC). The treatment of TNBC cells with defects in DNA repair genes with poly(ADP-ribose) polymerase inhibitor (PARPi) massively increases CIN, resulting in apoptosis. Here, we identified a previously unknown role of microRNA-449a in CIN. The transfection of TNBC cell lines HCC38, HCC1937 and HCC1395 with microRNA-449a mimics led to induced apoptosis, reduced cell proliferation, and reduced expression of genes in homology directed repair (HDR) in microarray analyses. EME1 was identified as a new target gene by immunoprecipitation and luciferase assays. The reduced expression of EME1 led to an increased frequency of ultrafine bridges, 53BP1 foci, and micronuclei. The induced expression of microRNA-449a elevated CIN beyond tolerable levels and induced apoptosis in TNBC cell lines by two different mechanisms: (I) promoting chromatid mis-segregation by targeting endonuclease EME1 and (II) inhibiting HDR by downregulating key players of the HDR network such as E2F3, BIRC5, BRCA2 and RAD51. The ectopic expression of microRNA-449a enhanced the toxic effect of PARPi in cells with pathogenic germline BRCA1 variants. The newly identified role makes microRNA-449a an interesting therapeutic target for TNBC.

High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing.

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.

RTEL1 suppresses G-quadruplex-associated R-loops at difficult-to-replicate loci in the human genome.

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.

TRAIP drives replisome disassembly and mitotic DNA repair synthesis at sites of incomplete DNA replication.

The faithful segregation of eukaryotic chromosomes in mitosis requires that the genome be duplicated completely prior to anaphase. However, cells with large genomes sometimes fail to complete replication during interphase and instead enter mitosis with regions of incompletely replicated DNA. These regions are processed in early mitosis via a process known as mitotic DNA repair synthesis (MiDAS), but little is known about how cells switch from conventional DNA replication to MiDAS. Using the early embryo of the nematode Caenorhabditis elegans as a model system, we show that the TRAIP ubiquitin ligase drives replisome disassembly in response to incomplete DNA replication, thereby providing access to replication forks for other factors. Moreover, TRAIP is essential for MiDAS in human cells, and is important in both systems to prevent mitotic segregation errors. Our data indicate that TRAIP is a master regulator of the processing of incomplete DNA replication during mitosis in metazoa.

The RIF1-PP1 Axis Controls Abscission Timing in Human Cells.

Abscission is the final step of cell division when the cytokinetic furrow ingresses completely, leading to midbody formation and plasma membrane fission [1]. In human cells, the Aurora-B-driven abscission checkpoint delays cytokinesis until any residual chromatin spanning the midbody is removed [2-5]. If this does not occur efficiently, uneven segregation of daughter genomes can occur. The mechanism by which the abscission checkpoint becomes satisfied to permit cytokinesis is poorly defined. Here, we identify RIF1 and its binding partner, protein phosphatase 1 (PP1), as being critical for regulation of abscission timing in human cells. We show that RIF1 promotes cytokinesis through recruitment of PP1 to the midbody, which then counteracts Aurora B kinase activity, leading to dephosphorylation of a regulator of abscission timing, CHMP4C [6-10]. Although RIF1 binds to unresolved DNA bridges that persist into telophase [11], we show that this cytokinetic function of the RIF1-PP1 axis is not limited to instances where cell division is perturbed by the presence of bridges. Nevertheless, we show that altering the balance of the opposing Aurora B kinase and PP1 phosphatase activities makes cells unresponsive to DNA bridges and sensitizes cells to agents that induce bridge formation. Our data define a new mechanism for regulation of abscission timing and emphasize how antagonism between kinases and phosphatases is a widespread mechanism for determining the timing of mitotic transactions. Because cancer cells experiencing oncogene-induced replication stress generate excessive mitotic DNA bridging [12], targeting this new regulatory pathway could be a promising therapeutic strategy.

Synthesis and characterization of biopolymer based hybrid hydrogel nanocomposite and study of their electrochemical efficacy.

A highly competent material, based on poly lactic acid (PLLA) grafted hydroxypropyl guar gum (HPG-g-PLLA) and polypyrrole/carboxylated multiwalled carbon nanotube (PPy/C-MWCNT) composite of various binary composition and copolymer of one of these nanocomposites have been synthesized successfully by in-situ polymerization. The environmentally affable nanocomposites have been characterized by spectroscopy, microscopy and thermogravimetry. Cytotoxicity of bio-nanocomposite has been inquired by cell viability study, which reveals its eco-friendly nature. The electrochemical properties of the biomaterials have been appraised by cyclic voltammetric studies. The PPy/C-MWCNT composite having 1 wt% C-MWCNT appears as the optimum composition from electrochemical studies. The hydrogel nanocomposite (HPG-g-PLLA5/0.5) copolymer behaves as a super ordinate material than pure PPy and PPy/C-MWCNT in every aspect of electrochemical properties like current density, stability, processibility and reversibility. Moreover the hydrogel nanocomposite, making electrode fabrication more simple and binder-free, nullifies all the interfacial complications arising from binders as well.

Site-Selective Interaction of Human Serum Albumin with 4-Chloro-7-nitro-1,2,3-benzoxadiazole Modified Olanzapine Derivative and Effect of ß-Cyclodextrin on Binding: In the Light of Spectroscopy and Molecular Docking.

Here, we present a detailed investigation on the interaction of 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD) embedded olanzapine derivative (OLA-NBD) with a model transport protein, human serum albumin (HSA). The thermodynamic parameters, ΔHo, ΔSo, and ΔGo, as evaluated by considering the van't Hoff relationship imply the major contribution of electrostatic/ionic interactions for the HSA-OLA-NBD association. The OLA-NBD induced quenching of HSA emission occurs through static quenching mechanism, indicating a 1:1 association, as portrayed from Benesi-Hildebrand plot, with ∼104 M-1 association constant value, and it is in good harmony with the value estimated from anisotropy experiment. The invariance of the time-resolved decay behavior of HSA with added OLA-NBD concentration, along with matching dependency of the binding constant (Kb) value on temperature, also supports the occurrence of static quenching. The effect of ß-cyclodextrin on HSA-OLA-NBD binding is characterized by a smaller Kb value revealing that the OLA-NBD molecules are gradually removed from ß-CD by HSA to achieve its medicinal outcome of drug delivery. The outcome from circular dichroism (CD) illustrates the variation of HSA secondary structure upon interaction with OLA-NBD. Concurrently, HSA-OLA-NBD association kinetics is also explored by applying the fluorescence technique. The possible interaction zone of OLA-NBD in HSA is investigated from AutoDock-based docking simulation study.

Nitric Oxide Sensing through 1,2,3,4-Oxatriazole Formation from Acylhydrazide: A Kinetic Study.

A simple molecular probe displays highly selective turn-on response toward NO by the unprecedented NO-induced formation of a 1,2,3,4-oxatriazole ring exhibiting no interference from various endogenous biomolecules including DHA, AA, etc. Kinetics of the reactions between NO and the probe provide a mechanistic insight into the formation of 1,2,3,4-oxatriazole which showed that, though initially 1,2,3,4-oxatriazole is formed and extractable in solid form, it exists in equilibrium with the ring opened azide form which ultimately hydrolyzed and converted to carboxylic acid and nitrate. The reaction displays second-order dependence on [NO] and first-order on [Probe]. The probe is water-soluble, cell permeable, and noncytotoxic and appropriates for live cell imaging. This constitutes the first report where there is a direct evidence of NO-induced ring closing reaction of an acyl hydrazide moiety leading to the formation of 1,2,3,4-oxatriazole.

Sunitinib-induced acute severe hypothyroidism in a case of metastatic gastrointestinal stromal tumor: A case report.

Thyroid abnormalities are found nearly 70% cases receiving sunitinib therapy. Mostly, patients suffer transient hypothyroidism rarely presents with overt acute symptoms requiring levothyroxine replacement. Onset is variable in published literature. We report a case of metastatic gastrointestinal stromal tumor receiving sunitinib with normal baseline thyroid function. The patient developed symptoms of acute severe hypothyroidism with high thyroid stimulating hormone level on the 4th week of therapy. The patient responded with oral levothyroxine. clinical and biochemical parameter resolved rapidly. Patient receiving sunitinib warns baseline and subsequent surveillance of thyroid function (both clinical and biochemical). This rare dreadful condition reverts promptly with thyroxine replacement.

Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism.

Telomeres resemble common fragile sites (CFSs) in that they are difficult-to-replicate and exhibit fragility in mitosis in response to DNA replication stress. At CFSs, this fragility is associated with a delay in the completion of DNA replication until early mitosis, whereupon cells are proposed to switch to a RAD52-dependent form of break-induced replication. Here, we show that this mitotic DNA synthesis (MiDAS) is also a feature of human telomeres. Telomeric MiDAS is not restricted to those telomeres displaying overt fragility, and is a feature of a wide range of cell lines irrespective of whether their telomeres are maintained by telomerase or by the alternative lengthening of telomeres (ALT) mechanism. MiDAS at telomeres requires RAD52, and is mechanistically similar to CFS-associated MiDAS, with the notable exception that telomeric MiDAS does not require the MUS81-EME1 endonuclease. We propose a model whereby replication stress initiates a RAD52-dependent form of break-induced replication that bypasses a requirement for MUS81-EME1 to complete DNA synthesis in mitosis.

A turn-on fluorogenic chemosensor for Fe3+ and a Schottky barrier diode with frequency-switching device applications.

A novel highly sensitive and selective fluorescent chemosensor L has been synthesized and characterized by various physicochemical techniques. In 3 : 7 water : MeCN (v/v) at pH 7.2 (10 mM HEPES buffer, µ = 0.05 M LiCl), it selectively recognizes Fe3+ through 1 : 1 complexation resulting in a 106-fold fluorescence enhancement and a binding constant of 8.10 × 104 M-1. The otherwise non-fluorescent spirolactam form of the probe results a dual-channel (absorbance and fluorescence) recognition of Fe3+via CHEF (chelation enhanced fluorescence) through the opening of the spirolactam ring. We have also carried out fluorescence titration and anisotropy (r) studies in pure water in the presence of SDS (sodium dodecyl sulphate). Based on the dependence of FI (fluorescence intensity) and r on [SDS] it was proposed that the probe is trapped between two SDS monolayers which again interact among themselves by ππ stacking. As a result, there is an increase in FI up to [SDS] ∼ 7 mM - a phenomenon reminiscent of aggregation-induced enhancement of emission (AIEE). Beyond this concentration of SDS (7 mM), micelle formation takes place and the ππ stacked polymer now becomes a monomer and is trapped inside the micellar cavity. As a result, there is a decrease in FI at [SDS] > 7 mM. But for anisotropy, it increases with [SDS] beyond 7 mM. Ligand, metal, and SDS interactions are well established through different optical and morphological studies. [L-Fe(NO3)]2+ thin films on FTO (Fluorine-doped Tin Oxide) glass substrates have been designed with the help of the spin-coating deposition technique. The deposited film of thickness 1.6 × 10-5 cm is well characterized by optical band gap calculation with a direct band gap, εg ∼ 1.6 eV. FESEM was also performed for the [L-Fe(NO3)]2+/FTO film. The current-voltage characteristics were measured by the two-probe technique. Light-dependent exciton generation was carried out by taking the top and bottom contacts with graphite paste on FTO and on the [L-Fe(NO3)]2+ films for the measurement of switching behavior. The response ratio curve for the light-induced frequency-switching phenomena has been obtained. The frequency taped here is the oscillation frequency of the photo-generated electron and the hole in an exiton. Thus, the light-induced frequency-switching behavior and Schottky barrier diode characteristics of the material were established.