The dynamics of eukaryotic replication initiation: origin specificity, licensing, and firing at the single-molecule level.
Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2-7: the core of the replicative helicase. Here, we use single-molecule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2-7 loading specificity and that Mcm2-7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2-7 hexamers propagate bidirectionally, monotonically, and processively as constituents of active replisomes.
Department of Biological Sciences, Columbia University, New York, NY 10032, USA.
Daniel
D
Duzdevich
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Megan D
MD
Warner
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Simina
S
Ticau
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Nikola A
NA
Ivica
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Stephen P
SP
Bell
Department of Biochemistry and Molecular Biophysics, and the Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA. Electronic address: ecg2108@columbia.edu.
Eric C
EC
Greene
D000465:Algorithms; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D007700:Kinetics; D064110:Minichromosome Maintenance Proteins / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D051738:Origin Recognition Complex / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C064055:ABFI protein, S cerevisiae; C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D064110:Minichromosome Maintenance Proteins