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

Reduction of peritoneal carcinomatosis by intraperitoneal administration of phospholipids in rats

<p>Abstract</p> <p>Background</p> <p>Intraperitoneal tumor cell attachment after resection of gastrointestinal cancer may lead to a developing of peritoneal carcinosis. Intraabdominal application of phospholipids shows a significant decrease of adhesion formation even in case of rising tumor cell concentration.</p> <p>Methods</p> <p>In experiment A 2*10⁶colonic tumor cells (DHD/K12/Trb) were injected intraperitonely in female BD-IX-rats. A total of 30 rats were divided into three groups with treatments of phospholipids at 6% or 9% and the control group. In experiment B a total of 100 rats were divided into ten groups with treatments of phospholipids at 9% and the control group. A rising concentration of tumor cells (10,000, 50,000, 100,000, 250,000 and 500,000) were injected intraperitonely in female BD-IX-rats of the different groups. After 30 days, the extent of peritoneal carcinosis was determined by measuring the tumor volume, the area of attachment and the Peritoneal Cancer Index (PCI).</p> <p>Results</p> <p>In experiment A, we found a significant reduction (control group: tumor volume: 12.0 ± 4.9 ml; area of tumor adhesion: 2434.4 ± 766 mm²; PCI 28.5 ± 10.0) of peritoneal dissemination according to all evaluation methods after treatment with phospholipids 6% (tumor volume: 5.2 ± 2.2 ml; area of tumor adhesion: 1106.8 ± 689 mm²; PCI 19.0 ± 5.0) and phospholipids 9% (tumor volume: 4.0 ± 3.5 ml; area of tumor adhesion: 362.7 ± 339 mm²; PCI 13.8 ± 5.1). In experiment B we found a significant reduction of tumor volume in all different groups of rising tumor cell concentration compared to the control. As detected by the area of attachment we found a significant reduction in the subgroups 1*10⁴, 25*10⁴and 50*10⁴. The reduction in the other subgroups shows no significance. The PCI could be reduced significantly in all subgroups apart from 5*10⁴.</p> <p>Conclusion</p> <p>In this animal study intraperitoneal application of phospholipids resulted in reduction of the extent of peritoneal carcinomatosis after intraperitoneal administration of free tumor cells. This effect was exceptionally noticed when the amount of intraperitoneal tumor cells was limited. Consequently, intraperitoneal administration of phospholipids might be effective in reducing peritoneal carcinomatosis after surgery of gastrointestinal tumors in humans.</p

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

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Eukaryotic replisomes are multiprotein complexes. Here the authors reveal two distinct stressed replisomes, associated with DONSON and FANCM, displaying a bias in replication timing and chromatin domain

A Chromosome Co-Entrapment Assay to Study Topological Protein-DNA Interactions.

Chromosome organization, DNA replication, and transcription are only some of the processes relying on dynamic and highly regulated protein-DNA interactions. Here, we describe a biochemical assay to study the molecular details of associations between ring-shaped protein complexes and chromosomes in the context of living cells. Any protein complex embracing chromosomal DNA can be enriched by this method, allowing for the underlying loading mechanisms to be investigated

Dynamic Thiolation–thioesterase Structure of a Non-ribosomal Peptide Synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties1. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations2. In solution, the large size and internal dynamics of multidomain fragments (\u3e35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation–thioesterase (T–TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4′-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T–TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T–TE interaction described here provides a model for NRPS, PKS and FAS function in general as T–TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries