Deletion of Orc4 during oogenesis severely reduces polar body extrusion and blocks zygotic DNA replication

Origin Recognition Complex subunit 4 (ORC4) is a DNA binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted GV oocytes cultured in vitro arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. A reduced number of Orc4-depleted oocytes developed to the MII stage and after activation these oocytes arrested at the 2-cell stage, without undergoing DNA synthesis. This confirms that transcription of full length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint since cytokinesis progressed without DNA synthesis. Together the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.

BI-2536 Promotes Neuroblastoma Cell Death via Minichromosome Maintenance Complex Components 2 and 10

DNA replication is initiated with the recognition of the starting point of multiple replication forks by the origin recognition complex and activation of the minichromosome maintenance complex 10 (MCM10). Subsequently, DNA helicase, consisting of the MCM protein subunits MCM2-7, unwinds double-stranded DNA and DNA synthesis begins. In previous studies, replication factors have been used as clinical targets in cancer therapy. The results showed that MCM2 could be a proliferation marker for numerous types of malignant cancer. We analyzed samples obtained from patients with neuroblastoma, revealing that higher levels of MCM2 and MCM10 mRNA were associated with poor survival rate. Furthermore, we combined the results of the perturbation-induced reversal effects on the expression levels of MCM2 and MCM10 and the sensitivity correlation between perturbations and MCM2 and MCM10 from the Cancer Therapeutics Response Portal database. Small molecule BI-2536, a polo-like kinase 1 (PLK-1) inhibitor, is a candidate for the inhibition of MCM2 and MCM10 expression. To test this hypothesis, we treated neuroblastoma cells with BI-2536. The results showed that the drug decreased cell viability and reduced the expression levels of MCM2 and MCM10. Functional analysis further revealed enrichments of gene sets involved in mitochondria, cell cycle, and DNA replication for BI-2536-perturbed transcriptome. We used cellular assays to demonstrate that BI-2536 promoted mitochondria fusion, G2/M arrest, and apoptosis. In summary, our findings provide a new strategy for neuroblastoma therapy with BI-2536.

Dynamics of DNA replication during male gametogenesis in the malaria parasite Plasmodium falciparum

Malaria parasites undergo a single phase of sexual reproduction in their complex lifecycle, during which they cycle between mosquito and vertebrate hosts. Sexual reproduction occurs only at the point when parasites move into the mosquito host. It involves specialised, sexually committed cells called gametocytes, which develop very rapidly into mature gametes and then mate inside the mosquito midgut. The gamete development process is unique, involving unprecedentedly fast replication and cell division to produce male gametes. A single male gametocyte replicates its ~23Mb genome three times over to produce 8 genomes, segregates these into newly-assembled flagellated gamete cells and releases them to seek out female gametes, all within ~15 minutes. Here, for the first time, we use fluorescent labelling of de novo DNA synthesis to follow this process at the whole-cell and single-molecule levels, yielding several novel observations. Firstly, we confirm that no DNA replication occurs before gametogenesis is triggered, although the origin recognition complex protein Orc1 is abundant even in immature gametocytes. Secondly, between repeated rounds of DNA replication there is no detectable karyokinesis - in contrast to the repeated replicative rounds that occur in asexual schizonts. Thirdly, cytokinesis is clearly uncoupled from DNA replication, and can occur even if replication fails, implying a lack of cell cycle checkpoints. Finally the single-molecule dynamics of DNA replication are entirely different from those in asexual schizonts.

The Organization of Pericentromeric Heterochromatin in Polytene Chromosome 3 of the Drosophila melanogaster Line with the Rif11; SuURES Su(var)3-906 Mutations Suppressing Underreplication

Although heterochromatin makes up 40% of the Drosophila melanogaster genome, its organization remains little explored, especially in polytene chromosomes, as it is virtually not represented in them due to underreplication. Two all-new approaches were used in this work: (i) with the use of a newly synthesized Drosophila line that carries three mutations, Rif11, SuURESand Su(var)3-906, suppressing the underreplication of heterochromatic regions, we obtained their fullest representation in polytene chromosomes and described their structure; (ii) 20 DNA fragments with known positions on the physical map as well as molecular genetic features of the genome (gene density, histone marks, heterochromatin proteins, origin recognition complex proteins, replication timing sites and satellite DNAs) were mapped in the newly polytenized heterochromatin using FISH and bioinformatics data. The borders of the heterochromatic regions and variations in their positions on arm 3L have been determined for the first time. The newly polytenized heterochromatic material exhibits two main types of morphology: a banding pattern (locations of genes and short satellites) and reticular chromatin (locations of large blocks of satellite DNA). The locations of the banding and reticular polytene heterochromatin was determined on the physical map.

Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist

Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes.

Expression and Clinical Significance of Origin Recognition Complex Family in Breast Cancer

BackgroundThe aim of this study is to investigate the potential clinical and prognostic value, role and driving molecular mechanisms of the origin recognition complex family in breast cancer.Resultsata from Oncomine, TCGA, GEO and ULCAN showed that ORC1L and ORC6L were highly expressed in breast cancer tissues, while the expression of ORC5L was inconsistent and there was no significant difference in the expression of ORC2L, ORC3L and ORC4L. High expression of ORC1L and ORC6L were mainly Her2 overexpressed subtype, and their expression were negatively correlated with patient age and positively correlated with tumor size, but not with lymph node metastasis, distant metastasis, or tumor stage. Expression of ORC5L was also negatively correlated with age and positively correlated with lymph node metastasis, but not with breast cancer molecular subtype and tumor size. Expression of ORC1L and ORC5L had high diagnostic value, and ORC6L had the highest diagnostic value in breast cancer. ORC6L was an independent poor prognostic factor for overall survival of breast cancer patients. It was involved in cell cycle progression, cell senescence, epigenetic regulation and other biological functions, and may regulate signaling pathways such as NF-KB, TP53 and WNT in breast cancer. We also found that the expression of ORC6L was related to the increased infiltration of Th1/2 cell and Treg cell, and decreased infiltration of Mast cell and NK cell.ConclusionsORC1L and ORC6L are highly expressed in breast cancer tissues, of which ORC6L has high diagnostic value and is an independent poor prognostic factor for overall survival of breast cancer patients. ORC6L may be involved in the occurrence and progression of breast cancer by regulating cell cycle progression, promoting the activation of cancer signaling pathways, and influencing tumor immune cells infiltration.

ORC binds and remodels nucleosomes to specify MCM loading onto DNA

ABSTRACTSaccharomyces cerevisiae has been a faithful guide for study of eukaryotic DNA replication, as the numerous initiation and elongation proteins are conserved from yeast to human. However, there is a gap in our knowledge of why yeast uses a consensus DNA sequence at replication origins, while higher eukaryotes do not. The current study closes this gap. By direct single-molecule visualization, we show that the Origin Recognition Complex (ORC) searches for and stably binds nucleosomes, and that nucleosomes funtionalize ORC to load MCM helicase onto DNA, regardless of DNA sequence. Furthermore, we discover that ORC can remodel nucleosomes and expel H2A-H2B histone dimers, a heretofore unexpected function. Thus ORC helps create a chromatin environment permissive to origin function. The finding that ORC binding to nucleosomes leads to MCM loading at any DNA sequence is likely to generalize, and that higher eukaryotes follow this same paradigm for origin selection

Systemic Analysis of the Dna Replication Regulators Origin Recognition Complex in Lung Adenocarcinomas Identifies Prognostic and Expression Significance

Background: Origin recognition complex (ORC) 1, ORC2, ORC3, ORC4, ORC5 and ORC6, form a replication-initiator complex to mediate DNA replication, which play a key role in carcinogenesis, while their role in lung adenocarcinomas (LUAD) remains poorly understood.Methods: We confirmed the transcriptional and post-transcriptional levels, DNA alteration, DNA methylation, miRNA network, protein structure, PPI network, functional enrichment, immune infiltration and prognostic value of ORCs in LUAD based on Oncomine, GEPIA, HPA, cBioportal, TCGA, GeneMANIA, Metascape, KM-plot, GENT2, and TIMER database. Results: ORC mRNA and protein were both enhanced obviously based on Oncomine, Ualcan, GEPIA, TCGA and HPA database. Furthermore, ORC1 and ORC6 have significant prognostic values for LUAD patients based on GEPIA database. Protein structure, PPI network, functional enrichment and immune infiltration analysis indicated that ORC complex cooperatively accelerate the LUAD development by promoting DNA replication, cellular senescence and metabolic process. Conclusion: the ORC complex has an important prognostic and expression significance for LUAD patients.