ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA.
Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC-Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.
Speck
Christian
C
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Chen
Zhiqiang
Z
Li
Huilin
H
Stillman
Bruce
B
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000648:ultrastructure; D009154:Mutation / Q000235:genetics; D051738:Origin Recognition Complex / Q000378:metabolism* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000648:ultrastructure; D013329:Structure-Activity Relationship
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D017931:DNA Primers; D046912:Multiprotein Complexes; C497214:ORC1 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA