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Single‐Molecule Studies of Eukaryotic DNA Replication
Author(s) -
Bell Stephen P,
Ticau Simina,
Friedman Larry J,
Gelles Jeff
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.111.1
Subject(s) - helicase , replisome , control of chromosome duplication , random hexamer , dnab helicase , origin recognition complex , dna , pre replication complex , dna replication , eukaryotic dna replication , microbiology and biotechnology , chemistry , biophysics , biology , biochemistry , gene , rna
The earliest step in eukaryotic DNA replication is the loading of the Mcm2–7 replicative DNA helicase onto origin DNA. This event marks all potential origins of replication and establishes the head‐to‐head configuration required for bidirectional replication initiation. Helicase loading requires the coordinate action of the Mcm2–7 helicase, and three helicase‐loading proteins: the origin‐recognition complex (ORC), Cdc6 and Cdt1. In an ATP‐hydrolysis‐dependent process, these proteins open the Mcm2–7 ring, insert the double stranded DNA into the central channel, and close the Mcm2–7 ring. The final outcome of this process is a head‐to‐head Mcm2–7 double hexamer. We have developed a reconstituted single‐molecule helicase‐loading assay using Colocalization Single Molecule Spectroscopy (CoSMoS). We use co‐localization of the fluorescently‐labeled DNA and proteins as a real‐time assay for protein‐DNA binding and helicase loading. This assay recapitulates all the know requirements of Mcm2–7‐loading. Using this assay, we found that initial association and loading of the two Mcm2–7 complexes at the origin DNA occurs sequentially. Furthermore, each of the two Mcm2–7 loading events is driven by distinct Cdc6 and Cdt1 molecules that are sequentially recruited and released. In contrast to Cdc6 and Cdt1, a single ORC complex is sufficient to load both of the oppositely‐oriented helicases of the double hexamer. We have extended this assay by developing a FRET‐based method to monitor opening and closing of the Mcm2–7 ring during helicase loading. We are using this assay to determine when these events occurs during the loading reaction and what proteins mediate these changes. We will discuss the implications of our findings for the mechanism of Mcm2–7 loading. Support or Funding Information This work was supported by NIH grants GM52339 (S.P.B.) and R01 GM81648 (J.G.) and a grant from the G. Har‐ old and Leila Y. Mathers Foundation (J.G.). S.T. was supported in part by an NIH Pre‐Doctoral Training Grant (GM007287). S.P.B. is an investigator with the Howard Hughes Medical Institute.