A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint
H. Sanchez González (Kavli institute of nanoscience Delft, TU Delft - BN/Nynke Dekker Lab)
Z. Liu (Kavli institute of nanoscience Delft, TU Delft - BN/Nynke Dekker Lab)
E.N.W. van Veen (TU Delft - BN/Nynke Dekker Lab, Kavli institute of nanoscience Delft)
T. van Laar (Kavli institute of nanoscience Delft, TU Delft - BN/Nynke Dekker Lab)
John F.X. Diffley (Francis Crick Institute)
N.H. Dekker (Kavli institute of nanoscience Delft, TU Delft - BN/Nynke Dekker Lab)
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Abstract
Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.
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