Investigating the Mechanism of Activation of the Mcm2-7 Replicative Helicase

DNA replication initiation is co-ordinated with progression through the cell cycle via a two-step mechanism. The first step, termed origin licensing, involves the assembly of a pre-replicative complex (pre-RC) at origins of replication, in which the Mcm2-7 replicative helicase is loaded onto DNA in an inactive form. Origin unwinding and DNA synthesis is only initiated during the second step of this process, origin firing, which requires the recruitment of multiple ‘firing factors’, such as Sld3/7 and Cdc45, as well as the activity of two essential cell cycle regulated kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). Whilst studies of the mechanism of origin licensing have been greatly furthered by the availability of an in vitro pre-RC assembly assay, the subsequent activation of the replicative helicase has not been well characterised, and how the firing factor proteins catalyse the complex topological changes required for Mcm2-7 helicase activation is unknown. In this study, I used an in vitro biochemical approach to investigate the function of DDK and Sld3/7 in origin firing. I reconstituted the recruitment of Sld3/7 to the pre- RC in vitro, which is thought to be the first step during Mcm2-7 helicase activation. I observed recruitment of Sld3/7 to be dependent on phosphorylation of the loaded Mcm2-7 complex by DDK, and was subsequently able to map the Mcm2-7 binding activity of Sld3/7 to a central domain of Sld3. By isolating a number of point mutants in Sld3 that were specifically defective in Mcm2-7 binding, I showed that the Sld3-Mcm2-7 interaction is essential for replicative helicase activation. Furthermore, I showed that the central portion of Sld3 contains a Cdc45 interacting site, which is also required for efficient replication initiation. Subsequently, I showed that Sld3 can interact with both Mcm6 and Mcm4 in a phosphorylation-dependent manner. Sld3 itself was shown to contain a novel phosphopeptide binding activity, and can interact with numerous phosphorylated residues throughout the N-terminal half of Mcm6. Elimination of these phosphorylation sites resulted in defects in both Sld3/7 recruitment to the pre-RC and replicative helicase activation. Thus, the novel DDK-dependent Sld3-Mcm2-7 interaction described in this study helps to explain the function of DDK during the early stages of origin firing. Additionally, the observation that Sld3 can interact directly with phosphorylated residues on Mcm2-7 indicates that Sld3 is a reader of DDK activity. As both an essential CDK substrate and a DDK reader, Sld3 thus functions as a point of intersection for the activities of CDK and DDK during Mcm2-7 helicase activation

Contribution of anadromous fish to the diet of European catfish in a large river system

Many anadromous fish species, when migrating from the sea to spawn in fresh waters, can potentially be a valuable prey for larger predatory fish, thereby efficiently linking these two ecosystems. Here, we assess the contribution of anadromous fish to the diet of European catfish (Silurus glanis) in a large river system (Garonne, southwestern France) using stable isotope analysis and allis shad (Alosa alosa) as an example of anadromous fish. Allis shad caught in the Garonne had a very distinct marine delta(13)C value, over 8 per thousand higher after lipid extraction compared to the mean delta(13)C value of all other potential freshwater prey fish. The delta(13)C values of European catfish varied considerably between these two extremes and some individuals were clearly specializing on freshwater prey, whereas others specialized on anadromous fish. The mean contribution of anadromous fish to the entire European catfish population was estimated to be between 53% and 65%, depending on the fractionation factor used for delta(13)C

Terminating the replication helicase.

A feature of the cell cycle is that the events of one cycle must be reset before the next one begins. A study now shows that the replication machinery is removed from fully replicated DNA by a conserved ubiquitin- and CDC48 (also known as p97)-dependent pathway. This explains how eukaryotic chromosomes are returned to the unreplicated state

Mechanism and timing of Mcm2–7 ring closure during DNA replication origin licensing

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.National Institutes of Health (U.S.) (Grant R01 GM52339)National Institutes of Health (U.S.) (Pre-Doctoral Training Grant GM007287)National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051

CUL-2^LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis

Replisome disassembly is the final step of DNA replication in eukaryotes, involving the ubiquitylation and CDC48-dependent dissolution of the CMG helicase (CDC45-MCM-GINS). Using Caenorhabditis elegans early embryos and Xenopus laevis egg extracts, we show that the E3 ligase CUL-2(LRR-1) associates with the replisome and drives ubiquitylation and disassembly of CMG, together with the CDC-48 cofactors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg extracts requires CUL2 neddylation, and our data identify chromatin recruitment of CUL2(LRR1) as a key regulated step during DNA replication termination. Interestingly, however, CMG persists on chromatin until prophase in worms that lack CUL-2(LRR-1), but is then removed by a mitotic pathway that requires the CDC-48 cofactor UBXN-3, orthologous to the human tumour suppressor FAF1. Partial inactivation of lrr-1 and ubxn-3 leads to synthetic lethality, suggesting future approaches by which a deeper understanding of CMG disassembly in metazoa could be exploited therapeutically

Physiological and psychological individual differences influence resting brain function measured by ASL perfusion

Effects of physiological and/or psychological inter-individual differences on the resting brain state have not been fully established. The present study investigated the effects of individual differences in basal autonomic tone and positive and negative personality dimensions on resting brain activity. Whole-brain resting cerebral perfusion images were acquired from 32 healthy subjects (16 males) using arterial spin labeling perfusion MRI. Neuroticism and extraversion were assessed with the Eysenck Personality Questionnaire-Revised. Resting autonomic activity was assessed using a validated measure of baseline cardiac vagal tone (CVT) in each individual. Potential associations between the perfusion data and individual CVT (27 subjects) and personality score (28 subjects) were tested at the level of voxel clusters by fitting a multiple regression model at each intracerebral voxel. Greater baseline perfusion in the dorsal anterior cingulate cortex (ACC) and cerebellum was associated with lower CVT. At a corrected significance threshold of p < 0.01, strong positive correlations were observed between extraversion and resting brain perfusion in the right caudate, brain stem, and cingulate gyrus. Significant negative correlations between neuroticism and regional cerebral perfusion were identified in the left amygdala, bilateral insula, ACC, and orbitofrontal cortex. These results suggest that individual autonomic tone and psychological variability influence resting brain activity in brain regions, previously shown to be associated with autonomic arousal (dorsal ACC) and personality traits (amygdala, caudate, etc.) during active task processing. The resting brain state may therefore need to be taken into account when interpreting the neurobiology of individual differences in structural and functional brain activity

Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen.

Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation