The nucleolar protein GNL3 prevents resection of stalled replication forks.

Faithful DNA replication requires specific proteins that protect replication forks and so prevent the formation of DNA lesions that may damage the genome. Identification of new proteins involved in this process is essential to understand how DNA lesions accumulate in cancer cells and how they tolerate them. Here, we show that human GNL3/nucleostemin, a GTP-binding protein localized mostly in the nucleolus and highly expressed in cancer cells, prevents nuclease-dependent resection of nascent DNA in response to replication stress. We demonstrate that inhibiting origin firing reduces resection. This suggests that the heightened replication origin activation observed upon GNL3 depletion largely drives the observed DNA resection probably due to the exhaustion of the available RPA pool. We show that GNL3 and DNA replication initiation factor ORC2 interact in the nucleolus and that the concentration of GNL3 in the nucleolus is required to limit DNA resection. We propose that the control of origin firing by GNL3 through the sequestration of ORC2 in the nucleolus is critical to prevent nascent DNA resection in response to replication stress.

CircORC2 is involved in the pathogenesis of slow transit constipation via modulating the signalling of miR-19a and neurotensin/motilin.

In this study, we aimed to investigate the role of circORC2 in modulating miR-19a and its downstream signalling during the pathogenesis of STC. In this study, three groups of patients, that is healthy control (HC) group, normal transit constipation (NTC) group (N = 42) and slow transit constipation (STC) group, were, respectively, recruited. RT-PCR and Western blot analysis were exploited to investigate the changes in the expression levels of miR-19a and circORC2 in these patients, so as to establish a circORC2/miR-19a signalling pathway. The basic information of the patients showed no significant differences among different patient groups. Compared with the HC group, concentrations of neurotensin (NST) and motilin (MLN) were both significantly reduced in the NTC and STC groups, especially in the STC group. Also, miR-19a level was highest, whereas circORC2 level was lowest in the STC group. Furthermore, circORC2 was validated to sponge the expression of miR-19a, and the transfection of circORC2 reduced the expression of miR-19a. Meanwhile, MLN and NST mRNAs were both targeted by miR-19a, and the transfection of circORC2 dramatically up-regulated the expression of MLN and NST. On the contrary, the transfection of circORC2 siRNA into SMCs and VSMCs exhibited the opposite effect of circORC2. Collectively, the results of this study established a regulatory relationship among circORC2, miR-19a and neurotensin/motilin, which indicated that the overexpression of circORC2 could up-regulate the levels of neurotensin and motilin, thus exerting a beneficial effect during the treatment of STC.

Initiation of Drosophila chorion gene amplification requires Claspin and mus101, whereas Claspin, but not mus101, plays a major role during elongation.

BACKGROUND: Claspin and TopBP1 are checkpoint mediators that are required for the phosphorylation of Chk1 by ATR to maintain genomic stability. Here, we investigated the functions of Drosophila Claspin and mus101 (TopBP1 ortholog) during chorion (eggshell component) gene amplification, which occurs in follicle cells in the absence of global genomic DNA replication. RESULTS: Unlike Drosophila mei-41 (ATR ortholog) mutant embryos, Claspin and mus101 mutant embryos showed severe eggshell defects resulting from defects in chorion gene amplification. EdU (5-ethynyl-2'-deoxyuridine) incorporation assay during initiation and elongation stages revealed that Claspin and mus101 were required for initiation, while only Claspin had a major role in the efficient progression of the replication forks. Claspin proteins were enriched in the amplification foci both in the initiation and elongation stage-follicle cell nuclei in a mei-41-independent manner. The focal localization of ORC2, a component of the origin recognition complex, was not significantly affected in the Claspin mutant, whereas it was reduced in the mus101 mutant. CONCLUSIONS: Drosophila Claspin plays a major role in the initiation and elongation stages of chorion gene amplification by localizing to the amplification foci in a mei-41-independent manner. Drosophila mus101 is also involved in chorion gene amplification, mostly functioning in initiation, rather than elongation. Developmental Dynamics 246:466-474, 2016. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Presence of the Paternal Pronucleus Assists Embryo in Overcoming Cycloheximide Induced Abnormalities in Zygotic Mitosis.

After fertilization, the maternal and paternal chromosomes independently proceed through pronuclear formation. These chromatin reconfigurations occur within a shared cytoplasm thus exposing both gametes to the same factors. Here, we report that continuous cycloheximide [40 µg/mL] treatment of parthenogenotes, androgenotes, and ICSI embryos reveals ORC2 pronuclear instability in the maternal (MPN) but not the paternal pronucleus (PPN). When released from CHX after 8 h, the MPN can recover ORC2 and proceed through replication, however, parthenogenotes encounter severe mitotic defects while both ICSI embryos and androgenotes are able to recover and develop at significantly higher rates. Taken together, these data suggest cycloheximide treatment promotes an environment that asymmetrically affects the stability of ORC2 on the MPN, and the ability of the MPN to develop. Furthermore, the presence of the PPN in the zygote can ameliorate both effects. These data suggest further evidence for crosstalk between the two pronuclei during the first cell cycle of the embryo. J. Cell. Biochem. 117: 1806-1812, 2016. © 2016 Wiley Periodicals, Inc.

ORC4 surrounds extruded chromatin in female meiosis.

Six proteins, ORC1-6, make up the origin recognition complex (ORC) that initiates licensing of DNA replication origins. We have previously reported that subunit ORC2 is localized between the separating maternal chromosomes at anaphase II just after fertilization and is present in zygotic pronuclei at G1. Here, we found that ORC1, 3, and 5 all localize between the chromosomes at anaphase II, but could not be detected in zygotic G1. ORC6 localized to the periphery of the nucleoli at all zygotic stages. We identified an unexpected potential role for ORC4 in polar body formation. We found that in both female meiotic divisions, ORC4 surrounds the set of chromosomes, as a sphere-like structure, that will eventually be discarded in the polar bodies, but not the chromosomes that segregate into the oocyte. None of the other five ORC proteins are involved in this structure. In Zygotic G1, ORC4 surrounds the nuclei of the polar bodies, but was not detectable in the pronuclei. When the zygote entered mitosis ORC4 was only detected in the polar body. However, ORC4 appeared on both sets of separating chromosomes at telophase. At this point, the ORC4 that was in the polar body also migrated into the nuclei, suggesting that ORC4 or an associated protein is modified during the first embryonic cell cycle to allow it to bind DNA. Our results suggest that ORC4 may help identify the chromosomes that are destined to be expelled in the polar body, and may play a role in polar body extrusion. ORC4 surrounds the chromatin that will be extruded in the polar body in both female meiotic divisions, then makes a transition from the cytoplasm to the chromosomes at zygotic anaphase, suggesting multiple roles for this replication licensing protein.

Resonance assignments of the ORC2-WH domain of the human ORC protein.

ORC2 is a small subunit of the origin recognition complex (ORC), which is important for gene replication. The ORC2 WH domain recognizes dsDNA sequences with its flexible ß-sheet hairpins as anchors. Here, we report near-complete NMR backbone and side chain resonance assignments of the WH domain and study the backbone relaxation of the WH domain. These studies will contribute to further understanding of the structure-function relationship of the ORC protein.

Investigating the role of Rts1 in DNA replication initiation.

Background: Understanding DNA replication initiation is essential to understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with ORC2 (a DNA replication licensing factor). Deletion of RTS1 synthetically rescued the temperature-sensitive (ts-) phenotype of ORC2 mutants. Methods: We deleted RTS1 in multiple ts-replication factor Saccharomyces cerevisiae strains, including ORC2.  Dilution series assays were carried out to compare qualitatively the growth of double mutant ∆rts1 ts-replication factor strains relative to the respective single mutant strains.   Results: No synthetic rescue of temperature-sensitivity was observed. Instead we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that RTS1 deletion in several ts-replication factor strains led to increased temperature-sensitivity. Conclusions: We find no evidence that Rts1 is involved in the dephosphorylation of DNA replication initiation factors.

Reversible regulation of ORC2 SUMOylation by PIAS4 and SENP2.

The small ubiquitin-related modifier (SUMO) system is essential for smooth progression of cell cycle at the G2/M phase. Many centromeric proteins are reversibly SUMOylated to ensure proper chromosome segregation at the mitosis. SUMOylation of centromeric Origin Recognition Complex subunit 2 (ORC2) at the G2/M phase is essential in maintaining genome integrity. However, how ORC2 SUMOylation is regulated remains largely unclear. Here we show that ORC2 SUMOylation is reversibly controlled by SUMO E3 ligase PIAS4 and De-SUMOylase SENP2. Either depletion of PIAS4 or overexpression of SENP2 eliminated SUMOylation of ORC2 at the G/M phase and consequently resulted in abnormal centromeric histone H3 lysine 4 methylation. Cells stably expressing SENP2 protein or small interfering RNA for PIAS4 bypassed mitosis and endoreduplicated their genome to become polyploidy. Furthermore, percentage of polyploid cells is reduced after coexpression of ORC2-SUMO2 fusion protein. Thus, the proper regulation of ORC2 SUMOylation at the G2/M phase by PIAS4 and SENP2 is critical for smooth progression of the mitotic cycle of cells.