Regulation of eukaryotic Mcm2-7 activity

The transfer of genetic material from one cell generation to the next requires precise genome duplication. Aberrant DNA replication can lead to genomic instability and contribute to diseases arising from an unregulated cell cycle, such as cancer. Replicative DNA polymerases require a single-stranded (ssDNA) template from which to produce newly synthesized DNA. In eukaryotes, ssDNA is generated by the heterohexameric minichromosome maintenance 2 through 7 (Mcm2-7) replicative helicase that unwinds duplex DNA. Strict temporal separation of helicase loading and activation at multiple replication origins ensures once per cell cycle replication. The processes involved in activating Mcm2-7 to unwind DNA during S phase are poorly understood. Through in vivo and in vitro analyses, the current study examines the factors involved in modulating S. cerevisiae Mcm2-7 activity. Mec1, a member of the PIKK (phosphoinositide three-kinase-related kinase) family of proteins, is involved in the response to replicative stress and DNA damage. It also plays a role during an unperturbed cell cycle and is required to phosphorylate Mcm2-7 prior to helicase activation. We characterized alleles of S. cerevisiae mec1 that alter the conserved FATC domain. Mutants of Mec1 resulted in temperature sensitive growth, sensitivity to hydroxyurea and reduced kinase activity in vitro. These mutants were also less stable than wild-type Mec1 and demonstrated reduced nuclear localization. We also identified rpn3-L140P, which encodes a component of the 19S proteasomal regulatory particle of the 26S proteasome, as a suppressor of the temperature-sensitive growth caused by mec1-W2368A. As Cdt1 is required for the nuclear import and origin loading of Mcm2-7, we also sought to investigate the interaction between these two components in more detail. Using reconstituted Mcm2-7·Cdt1 complexes from bacterial-expressed proteins, we demonstrated that these complexes exhibit lower ATPase and helicase activity than Mcm2-7. We also showed that Mcm2-7 dissociates into subcomplexes, and that Mcm3, 5 and 7 bound origins in the absence of Cdt1. We propose that the reduced ATPase activity of Mcm2-7 by Cdt1 binding is induced by structural changes in the Mcm2-7 ring. We also suggest that Cdt1 helps to stabilize the Mcm2-7 hexamer. To investigate the role of phosphorylation on Mcm2-7, we utilized a phosphomimetic mutant of Mcm4 that when incorporated into Mcm2-7 can bypass the requirement for DDK. While phosphomimetic Mcm4 demonstrated slightly lower ATPase activity than the wildtype protein, phosphomimetic Mcm2-7 complexes exhibited wildtype ATPase, helicase and DNA binding activity. Taken together, our work identifies the functional role of the C-terminal residues of Mec1 and the protein’s turnover by the proteosome. Our studies also provide new insights into the factors and processes involved in the activation of Mcm2-7 to unwind DNA

A quantitative model of the initiation of DNA replication in Saccharomyces cerevisiae predicts the effects of system perturbations.

BackgroundEukaryotic cell proliferation involves DNA replication, a tightly regulated process mediated by a multitude of protein factors. In budding yeast, the initiation of replication is facilitated by the heterohexameric origin recognition complex (ORC). ORC binds to specific origins of replication and then serves as a scaffold for the recruitment of other factors such as Cdt1, Cdc6, the Mcm2-7 complex, Cdc45 and the Dbf4-Cdc7 kinase complex. While many of the mechanisms controlling these associations are well documented, mathematical models are needed to explore the network's dynamic behaviour. We have developed an ordinary differential equation-based model of the protein-protein interaction network describing replication initiation.ResultsThe model was validated against quantified levels of protein factors over a range of cell cycle timepoints. Using chromatin extracts from synchronized Saccharomyces cerevisiae cell cultures, we were able to monitor the in vivo fluctuations of several of the aforementioned proteins, with additional data obtained from the literature. The model behaviour conforms to perturbation trials previously reported in the literature, and accurately predicts the results of our own knockdown experiments. Furthermore, we successfully incorporated our replication initiation model into an established model of the entire yeast cell cycle, thus providing a comprehensive description of these processes.ConclusionsThis study establishes a robust model of the processes driving DNA replication initiation. The model was validated against observed cell concentrations of the driving factors, and characterizes the interactions between factors implicated in eukaryotic DNA replication. Finally, this model can serve as a guide in efforts to generate a comprehensive model of the mammalian cell cycle in order to explore cancer-related phenotypes

Cytokine response patterns, exposure to viruses, and respiratory infections in the first year of life

Keywords: interferon-␥; respiratory syncytial virus; daycare; sibling Viral respiratory illnesses, many of which are contracted through contact with siblings or attendance at daycare, are the most common triggers for wheezing and asthma exacerbations among young children. In both the Tucson Children's Respiratory Study (1, 2) and the Italian Studies of Respiratory Disorders in Childhood and the Environment project (3), daycare attendance was shown to be a risk factor for viral infections and viral-associated wheezing in the first 2 years of life, but protective against viral infections and asthma later in life. Celedon and colleagues found that daycare attendance during the first year of life was associated with lower rates of asthma at 6 years of age, but only among children without a maternal history of asthma (4). Similarly, children with older siblings in the home are more likely to experience viral illnesses and wheezing in the first 2 years of life, but have decreased rates of wheezing, and decreased rates of atopic sensitization later in childhood (2, 3, 5, 6). Despite the convincing evidence linking exposure to other children with rates of respiratory illnesses and wheezing, the effects of increased exposure on specific viral infections have not been ascertained. While exposure is an important determinant of lower respiratory tract illnesses, it does not explain why some children entering daycare experience a dramatic increase in viral infections, while others are relatively healthy. In addition, recent genetic studies suggest that clinical outcomes of viral infections in infancy might also be influenced by polymorphisms in cytokine genes (7-9). These observations suggest the hypotheses that variations and/or subtle defects in the antiviral immune response also affect the clinical expression of viral respiratory infections. Furthermore, stressing the immune system with increased viral exposure may uncover relatively minor immune defects that are not apparent in children with less exposure to viruses. To test these hypotheses, we conducted a prospective birth cohort study to evaluate interactions between exposure to other children, the development patterns of cytokine responses in peripheral blood cells, and the etiology and severity of respiratory viral infections during the first year of life. Other study results pertinent to this cohort have been previously published as both original articles and abstracts (10-13)

Expression quantitative trait locus fine mapping of the 17q12–21 asthma locus in African American children: a genetic association and gene expression study

Background: African ancestry is associated with a higher prevalence and greater severity of asthma than European ancestries, yet genetic studies of the most common locus associated with childhood-onset asthma, 17q12–21, in African Americans have been inconclusive. The aim of this study was to leverage both the phenotyping of the Children's Respiratory and Environmental Workgroup (CREW) birth cohort consortium, and the reduced linkage disequilibrium in African Americans, to fine map the 17q12–21 locus. Methods: We first did a genetic association study and meta-analysis using 17q12–21 tag single-nucleotide polymorphisms (SNPs) for childhood-onset asthma in 1613 European American and 870 African American children from the CREW consortium. Nine tag SNPs were selected based on linkage disequilibrium patterns at 17q12–21 and their association with asthma, considering the effect allele under an additive model (0, 1, or 2 effect alleles). Results were meta-analysed with publicly available summary data from the EVE consortium (on 4303 European American and 3034 African American individuals) for seven of the nine SNPs of interest. Subsequently, we tested for expression quantitative trait loci (eQTLs) among the SNPs associated with childhood-onset asthma and the expression of 17q12–21 genes in resting peripheral blood mononuclear cells (PBMCs) from 85 African American CREW children and in upper airway epithelial cells from 246 African American CREW children; and in lower airway epithelial cells from 44 European American and 72 African American adults from a case-control study of asthma genetic risk in Chicago (IL, USA). Findings: 17q12–21 SNPs were broadly associated with asthma in European Americans. Only two SNPs (rs2305480 in gasdermin-B [GSDMB] and rs8076131 in ORMDL sphingolipid biosynthesis regulator 3 [ORMDL3]) were associated with asthma in African Americans, at a Bonferroni-corrected threshold of p<0·0055 (for rs2305480_G, odds ratio [OR] 1·36 [95% CI 1·12–1·65], p=0·0014; and for rs8076131_A, OR 1·37 [1·13–1·67], p=0·0010). In upper airway epithelial cells from African American children, genotype at rs2305480 was the most significant eQTL for GSDMB (eQTL effect size [β] 1·35 [95% CI 1·25–1·46], p<0·0001), and to a lesser extent showed an eQTL effect for post-GPI attachment to proteins phospholipase 3 (β 1·15 [1·08–1·22], p<0·0001). No SNPs were eQTLs for ORMDL3. By contrast, in PBMCs, the five core SNPs were associated only with expression of GSDMB and ORMDL3. Genotype at rs12936231 (in zona pellucida binding protein 2) showed the strongest associations across both genes (for GSDMB, eQTLβ 1·24 [1·15–1·32], p<0·0001; and for ORMDL3 (β 1·19 [1·12–1·24], p<0·0001). The eQTL effects of rs2305480 on GSDMB expression were replicated in lower airway cells from African American adults (β 1·29 [1·15–1·44], p<0·0001). Interpretation: Our study suggests that SNPs regulating GSDMB expression in airway epithelial cells have a major role in childhood-onset asthma, whereas SNPs regulating the expression levels of 17q12–21 genes in resting blood cells are not central to asthma risk. Our genetic and gene expression data in African Americans and European Americans indicated GSDMB to be the leading candidate gene at this important asthma locus.6 month embargo; published: 01 May 2020This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]