Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase

In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA1,2. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer3. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites4,5. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.

Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.

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From structure to mechanism-understanding initiation of DNA replication.

A multi-stage optimization of passively designed high-rise residential buildings in multiple building operation scenarios

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro. Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase, which requires the leading-strand polymerase Pol ɛ. Here the authors present a structural analysis of a CMG Pol ɛ on a DNA fork, providing insight on the steps leading productive helicase engagement to the DNA junction.

Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome

A high-resolution cryo-EM structure of the heptameric Cdt1–Mcm2–7 complex of the replicative helicase from budding yeast suggests a ‘spring-action’ DNA-unwinding mechanism. The minichromosome maintenance complex (MCM) hexameric complex (Mcm2–7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1–Mcm2–7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1–MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10–15-A gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.

Open-ringed structure of the Cdt1-Mcm2-7 complex as a precursor of the MCM double hexamer

As a major energy consumer and CO2 emitter, buildings have an undeniably important role to play in cutting carbon emissions and combating climate change. Over the recent decades, green buildings have gained increasing attention and popularity from various stakeholders in the construction industry. Green building design practice builds upon the conventional building design practice but adds the concerns of environmental impacts and occupants’ well-being in the design philosophy.
Many researchers advocate utilizing optimization for green building design due to its capability in obtaining improved design solutions and providing building designers a better understanding of the design space. A comprehensive and in-depth review on previous relevant optimization models has revealed the following two limitations which might undermine their application in practice. Firstly, the focus of optimization in most of these models was on the reduction of cost and energy consumption while occupants’ comfort level in terms of indoor environmental quality was seldom considered. Secondly, for those models which have set comfort level of indoor environmental quality as a design objective, only thermal comfort was taken into account and thus they failed to address other essential factors governing indoor environmental quality such as visual comfort and indoor air quality. 
Aiming at addressing the limitations of previous related studies, this research has developed an improved optimization model for green building design with a more comprehensive set of design objectives, namely minimization of cost, minimization of energy consumption, and maximization of occupants’ comfort level in terms of indoor environmental quality. The importance of the three design objectives and the necessity for including them in the model were verified through a series of semi-structured interviews with respondents from different stakeholder groups in relation to green building design and construction. The three design objectives are evaluated in the developed model in terms of (i) cost according to life cycle cost; (ii) energy consumption analyzed by a widely-adopted building energy performance simulation program – EnergyPlus; and (iii) comfort level of indoor environmental quality by adopting an empirical-based multivariate-logistic regression model identified from literatures. Non-dominated Sorting Genetic Algorithm II, a powerful multi-objective optimization technique, was selected as the optimization engine in the developed model. The developed model was then implemented into to a prototype tool in the MATLAB environment which can be utilized by building designers to determine the appropriate design solutions. Through a hypothetical office building design problem, the applicability of the model was demonstrated. Finally, the developed model was validated through demonstration and face-to-face discussion with experts.

Hao Wu

Papers

Open-ringed structure of the Cdt1-Mcm2-7 complex as a precursor of the MCM double hexamer

A multi-objective optimization model for green building design

History of Chinese women's costume