Switch on the engine: how the eukaryotic replicative helicase MCM2–7 becomes activated

© 2014, Springer-Verlag Berlin Heidelberg.A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2–7 (MCM2–7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2–7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2–7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process

Development of a high-speed current injection and voltage measurement system for electrical impedance tomography-based stretchable sensors

In common EIT systems, the voltage data are serially measured by means of multiplexers, and are hence collected at slightly different times, which affects the real-time performance of the system. They also tend to have complicated hardware, which increases power consumption. In this paper, we present our design of a 16-electrode high-speed EIT system that simultaneously implements constant current injection and differential potential measurements. This leads to a faster, simpler-to-implement and less-noisy technique, when compared with traditional EIT approaches. Our system consists of a Howland current pump with two multiplexers for a constant DC current supply, and a data acquisition card. It guarantees a data collection rate of 78 frames/s. The results from our conductive stretchable fabric sensor show that the system successfully performs voltage data collection with a mean signal-to-noise ratio (SNR) of 55 dB, and a mean absolute deviation (MAD) of 0.5mV. The power consumption can be brought down to 3mW; therefore, it is suitable for battery-powered applications. Finally, pressure contacts over the sensor are properly reconstructed, thereby validating the efficiency of our EIT system for soft and stretchable sensor applications

Anisotropy and Ising-like transition of the S=5/2 two-dimensional Heisenberg antiferromagnet Mn-formate di-Urea

Recently reported measurements of specific heat on the compound Mn-formate di-Urea (Mn-f-2U) by Takeda et al. [Phys. Rev. B 63, 024425 (2001)] are considered. As a model to describe the overall thermodynamic behavior of such compound, the easy-axis two-dimensional Heisenberg antiferromagnet is proposed and studied by means of the 'pure quantum self-consistent harmonic approximation' (PQSCHA). In particular it is shown that, when the temperature decreases, the compound exhibits a crossover from 2D-Heisenberg to 2D-Ising behavior, followed by a 2D-Ising-like phase transition, whose location allows to get a reliable estimate of the easy-axis anisotropy driving the transition itself. Below the critical temperature T_N=3.77 K, the specific heat is well described by the two-dimensional easy-axis model down to a temperature T*=1.47 K where a T^3-law sets in, possibly marking a low-temperature crossover of magnetic fluctuations from two to three dimensions.Comment: 3 pages, 2 figures, 47th Annual Conference on Magnetism and Magnetic Materials (Tampa, FL, USA, 11-15/11/2002

Spectral shapes of solid neon

We present a Path Integral Monte Carlo calculation of the first three moments of the displacement-displacement correlation functions of solid neon at different temperatures for longitudinal and transverse phonon modes. The Lennard-Jones potential is considered. The relevance of the quantum effects on the frequency position of the peak and principally on the line-width of the spectral shape is clearly pointed out. The spectrum is reconstructed via a continued fraction expansion; the approximations introduced using the effective potential quantum molecular dynamics are discussed.Comment: 3 pages, 2 figures, 3 table

Heisenberg antiferromagnet on the square lattice for S>=1

Theoretical predictions of a semiclassical method - the pure-quantum self-consistent harmonic approximation - for the correlation length and staggered susceptibility of the Heisenberg antiferromagnet on the square lattice (2DQHAF) agree very well with recent quantum Monte Carlo data for S=1, as well as with experimental data for the S=5/2 compounds Rb2MnF4 and KFeF4. The theory is parameter-free and can be used to estimate the exchange coupling: for KFeF4 we find J=2.33 +- 0.33 meV, matching with previous determinations. On this basis, the adequacy of the quantum nonlinear sigma model approach in describing the 2DQHAF when S>=1 is discussed.Comment: 4 pages RevTeX file with 5 figures included by psfi

An ORC/Cdc6/MCM2-7 Complex Is Formed in a Multistep Reaction to Serve as a Platform for MCM Double-Hexamer Assembly

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing

Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA.

The regulated loading of the replicative helicase minichromosome maintenance proteins 2–7 (MCM2–7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2–7 hexamers into one double hexamer around dsDNA. Although the MCM2–7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2–7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC–Cdc6–Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2–7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2–7 complex formation prior to MCM2–7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation

Kinetic energy of solid neon by Monte Carlo with improved Trotter- and finite-size extrapolation

The kinetic energy of solid neon is calculated by a path-integral Monte Carlo approach with a refined Trotter- and finite-size extrapolation. These accurate data present significant quantum effects up to temperature T=20 K. They confirm previous simulations and are consistent with recent experiments.Comment: Text and figures revised for minor corrections (4 pages, 3 figures included by psfig

Quantum fluctuations in one-dimensional arrays of condensates

The effects of quantum and thermal fluctuations upon the fringe structure predicted to be observable in the momentum distribution of coupled Bose-Einstein condensates are studied by the effective-potential method. For a double-well trap, the coherence factor recently introduced by Pitaevskii and Stringari [Phys. Rev. Lett. 87, 180402 (2001)] is calculated using the effective potential approach and is found in good agreement with their result. The calculations are extended to the case of a one-dimensional array of condensates, showing that quantum effects are essentially described through a simple renormalization of the energy scale in the classical analytical expression for the fringe structure. The consequences for the experimental observability are discussed.Comment: RevTeX, 4 pages, 5 eps figures (published version with updated references