The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

It is generally recognized that dysregulation of the immune system plays a critical role in many diseases, including autoimmune diseases and cancer. T cells play a crucial role in maintaining self-tolerance, while loss of immune tolerance and T cell activation can lead to severe inflammation and tissue damage. T cell responses have a key role in the effectiveness of vaccination strategies and immunomodulating therapies. Immunomonitoring methods have the ability to elucidate immunological processes, monitor the development of disease and assess therapeutic effects. In this respect, it is of particular interest to evaluate antigen (Ag)-specific T cells by determining their frequency, type and functionality in cellular assays. Nevertheless, Ag-specific T cells are detected infrequently in most diseases using current techniques. Many efforts have been made to develop more sensitive, reproducible, and reliable methods for Ag-specific T cell detection. It has been found that analysis of cellular proliferation can be a useful tool to determine the presence and frequency of Ag-specific T cell and to provides insight into modulation of the T cell response by a specific antigen or therapy. However, the selection of a cut-off value for a positive response and therefore a more accurate interpretation of the data, continues to be a major concern. Here, we provide guidelines to select a proper cut-off for monitoring of Ag-specific CD4+ T cell responses. In vitro Ag-stimulation has been assessed with two methods; a dye-based proliferation assay and 3H-thymidine-based assay. Two cut-off approaches are compared; mean and variance of control wells, and the stimulation index. By evaluating the proliferative response to the in vitro Ag-stimulation using these two methods, we demonstrate the importance of taking into consideration the variability of the control wells to distinguish a positive from a false positive response.

Establishing consensus around the transcriptional interface between coronavirus (CoV) infection and human cellular signaling pathways can catalyze the development of novel anti-CoV therapeutics. Here, we used publicly archived transcriptomic datasets to compute consensus regulatory signatures, or consensomes, that rank human genes based on their rates of differential expression in MERS-CoV (MERS), SARS-CoV-1 (SARS1) and SARS-CoV-2 (SARS2)-infected cells. Validating the CoV consensomes, we show that high confidence transcriptional targets (HCTs) of MERS, SARS1 and SARS2 infection intersect with HCTs of signaling pathway nodes with known roles in CoV infection. Among a series of novel use cases, we gather evidence for hypotheses that SARS2 infection efficiently represses E2F family HCTs encoding key drivers of DNA replication and the cell cycle; that progesterone receptor signaling antagonizes SARS2-induced inflammatory signaling in the airway epithelium; and that SARS2 HCTs are enriched for genes involved in epithelial to mesenchymal transition. The CoV infection consensomes and HCT intersection analyses are freely accessible through the Signaling Pathways Project knowledgebase, and as Cytoscape-style networks in the Network Data Exchange repository.

The response to DNA replication stress in eukaryotes is under the control of the ataxia-telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase ε accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity and the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.

CRL4Cdt2 ubiquitin ligase plays an important role maintaining genome integrity during the cell cycle. A recent report suggested that Cdk1 negatively regulates CRL4Cdt2 activity through phosphorylation of its receptor, Cdt2, but the involvement of phosphorylation remains unclear. To address this, we mutated all CDK consensus phosphorylation sites located in the C-terminal half region of Cdt2 (Cdt2-18A) and examined the effect on substrate degradation. We show that both cyclinA/Cdk2 and cyclinB/Cdk1 phosphorylated Cdt2 in vitro and that phosphorylation was reduced by the 18A mutation both in vitro and in vivo. The 18A mutation increased the affinity of Cdt2 to PCNA, and a high amount of Cdt2-18A was colocalized with PCNA foci during S phase in comparison with Cdt2-WT. Poly-ubiquitination activity to Cdt1 was concomitantly enhanced in cells expressing Cdt2-18A. Other CRL4Cdt2 substrates, Set8 and thymine DNA glycosylase, begin to accumulate around late S phase to G2 phase, but the accumulation was prevented in Cdt2-18A cells. Furthermore, mitotic degradation of Cdt1 after UV irradiation was induced in these cells. Our results suggest that CDK-mediated phosphorylation of Cdt2 inactivates its ubiquitin ligase activity by reducing its affinity to PCNA, an important strategy for regulating the levels of key proteins in the cell cycle.

Since its approval, fluoroquinolones have become one of the most prescribed antibacterial agents. Because of its widespread use, serious concerns about the emergence of resistance in Streptococcus pneumoniae, Pseudomonas spp, and entrobacteriaceae, has arisen, especially because of cross-resistance between fluoroquinolones. Huge efforts has been done to identify pharmacokinetic (PK) parameters like maximum serum concentration (Cmax), area under the curve of serum concentrations (AUC) and pharmacodynamic (PD) parameters like the minimum inhibitory concentration (MIC) or the mutant prevention concentration (MPC), to optimize the use of the new fluoroquinolones, especially against these difficult to treat microorganisms. The new fluoroquinolones commercially available in Spain, levofloxacin and moxifloxacin, have significant differences in their PK (Cmax, half-life, volume of distribution, etc), PD (MIC, MPC,) and in their PK/PD parameters (AUC/MIC; AUC/MPC) that allow clinicians to establish clear preference for the utilization of one of them. Proper use of these new fluoroquinolones according to these PK/PD parameters will result in better management of respiratory infections with a reduction in the emergence of resistance. Based on data reviewed in this paper moxifloxacin use, with best PK/PD characteristics, should be preferred over levofloxacin. Should levofloxacin be used, alternative dosing strategies would be recommended to avoid selection of resistant variants.

DNA replication is highly regulated, ensuring faithful inheritance of genetic information through each cell cycle. In metazoans, this process is initiated at many thousands of DNA replication origins whose cell type-specific distribution and usage are poorly understood. We exhaustively mapped the genome-wide location of replication origins in human cells using deep sequencing of short nascent strands and identified ten times more origin positions than we expected; most of these positions were conserved in four different human cell lines. Furthermore, we identified a consensus G-quadruplex-forming DNA motif that can predict the position of DNA replication origins in human cells, accounting for their distribution, usage efficiency and timing. Finally, we discovered a cell type-specific reprogrammable signature of cell identity that was revealed by specific efficiencies of conserved origin positions and not by the selection of cell type-specific subsets of origins.

Merkel Cell Polyomavirus (MCPyV) genomes are clonally integrated in tumor tissues of approximately 85% of all Merkel cell carcinoma (MCC) cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosuppressed patients. All integrated viral genomes recovered from MCC tissue or MCC cell lines harbor signature mutations in the early gene transcript encoding for the large T-Antigen (LT-Ag). These mutations selectively abrogate the ability of LT-Ag to support viral replication while still maintaining its Rb-binding activity, suggesting a continuous requirement for LT-Ag mediated cell cycle deregulation during MCC pathogenesis. To gain a better understanding of MCPyV biology, in vitro MCPyV replication systems are required. We have generated a synthetic MCPyV genomic clone (MCVSyn) based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Here, we demonstrate that transfection of recircularized MCVSyn DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with virus particle formation. However, serial transmission of infectious virus was not observed. This in vitro culturing system allows the study of viral replication and will facilitate the molecular dissection of important aspects of the MCPyV lifecycle.

Evolutionary change in gene regulation is a key mechanism underlying the genetic component of organismal diversity. Here, we study evolution of regulation at the posttranslational level by examining the evolution of cyclin-dependent kinase (CDK) consensus phosphorylation sites in the protein subunits of the pre-replicative complex (RC). The pre-RC, an assembly of proteins formed during an early stage of DNA replication, is believed to be regulated by CDKs throughout the animals and fungi. Interestingly, although orthologous pre-RC components often contain clusters of CDK consensus sites, the positions and numbers of sites do not seem conserved. By analyzing protein sequences from both distantly and closely related species, we confirm that consensus sites can turn over rapidly even when the local cluster of sites is preserved, consistent with the notion that precise positioning of phosphorylation events is not required for regulation. We also identify evolutionary changes in the clusters of sites and further examine one replication protein, Mcm3, where a cluster of consensus sites near a nucleocytoplasmic transport signal is confined to a specific lineage. We show that the presence or absence of the cluster of sites in different species is associated with differential regulation of the transport signal. These findings suggest that the CDK regulation of MCM nuclear localization was acquired in the lineage leading to Saccharomyces cerevisiae after the divergence with Candida albicans. Our results begin to explore the dynamics of regulatory evolution at the posttranslational level and show interesting similarities to recent observations of regulatory evolution at the level of transcription.

BACKGROUND: Eukaryotic replication origins exhibit different initiation efficiencies and activation times within S-phase. Although local chromatin structure and function influences origin activity, the exact mechanisms remain poorly understood. A key to understanding the exact features of chromatin that impinge on replication origin function is to define the precise locations of the DNA sequences that control origin function. In S. cerevisiae, Autonomously Replicating Sequences (ARSs) contain a consensus sequence (ACS) that binds the Origin Recognition Complex (ORC) and is essential for origin function. However, an ACS is not sufficient for origin function and the majority of ACS matches do not function as ORC binding sites, complicating the specific identification of these sites.
RESULTS: To identify essential origin sequences genome-wide, we utilized a tiled oligonucleotide array (NimbleGen) to map the ORC and Mcm2p binding sites at high resolution. These binding sites define a set of potential Autonomously Replicating Sequences (ARSs), which we term nimARSs. The nimARS set comprises 529 ORC and/or Mcm2p binding sites, which includes 95% of known ARSs, and experimental verification demonstrates that 94% are functional. The resolution of the analysis facilitated identification of potential ACSs (nimACSs) within 370 nimARSs. Cross-validation shows that the nimACS predictions include 58% of known ACSs, and experimental verification indicates that 82% are essential for ARS activity.
CONCLUSIONS: These findings provide the most comprehensive, accurate, and detailed mapping of ORC binding sites to date, adding to the emerging picture of the chromatin organization of the budding yeast genome.