1. Flexible DNA Path in the MCM Double Hexamer Loaded on DNA.
- Author
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Hizume K, Kominami H, Kobayashi K, Yamada H, and Araki H
- Subjects
- DNA Replication, DNA, Fungal biosynthesis, DNA, Fungal chemistry, DNA, Fungal isolation & purification, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Microscopy, Atomic Force, Minichromosome Maintenance Proteins chemistry, Minichromosome Maintenance Proteins genetics, Minichromosome Maintenance Proteins isolation & purification, Minichromosome Maintenance Proteins metabolism, Nucleic Acid Conformation, Origin Recognition Complex chemistry, Origin Recognition Complex genetics, Origin Recognition Complex isolation & purification, Osmolar Concentration, Protein Multimerization, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins isolation & purification, Transcription Factors chemistry, Transcription Factors genetics, Transcription Factors metabolism, DNA, Fungal metabolism, Models, Molecular, Origin Recognition Complex metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The formation of the pre-replicative complex (pre-RC) during the G1 phase, which is also called the licensing of DNA replication, is the initial and essential step of faithful DNA replication during the subsequent S phase. It is widely accepted that in the pre-RC, double-stranded DNA passes through the holes of two ring-shaped minichromosome maintenance (MCM) 2-7 hexamers; however, the spatial organization of the DNA and proteins involved in pre-RC formation is unclear. Here we reconstituted the pre-RC from purified DNA and proteins and visualized the complex using atomic force microscopy (AFM). AFM revealed that the MCM double hexamers formed elliptical particles on DNA. Analysis of the angle of binding of DNA to the MCM double hexamer suggests that the DNA does not completely pass through both holes of the MCM hexamers, possibly because the DNA exited from the gap between Mcm2 and Mcm5. A DNA loop fastened by the MCM double hexamer was detected in pre-RC samples reconstituted from purified proteins as well as those purified from yeast cells, suggesting a higher-order architecture of the loaded MCM hexamers and DNA strands.
- Published
- 2017
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