Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication

Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication

Massachusetts Institute of Technology, Department of Biology, Room 68-622, 77 Massachusetts Avenue, Cambridge, MA 02139

Communicated by Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA (received for review March 26, 1999)

Abstract

Chromosomes are replicated in characteristic, temporal patterns during S phase. We have compared the timing of association of replication proteins at early- and late-replicating origins of replication. Minichromosome maintenance proteins assemble simultaneously at early- and late-replicating origins. In contrast, Cdc45p association with late origins is delayed relative to early origins. DNA polymerase α association is similarly delayed at late origins and requires Cdc45p function. Activation of the S phase checkpoint inhibits association of Cdc45p with late-firing origins. These studies suggest that Cdc45p is poised to serve as a key regulatory target for both the temporal and checkpoint-mediated regulation of replication origins.

Footnotes

↵ * To whom reprint requests should be addressed. E-mail: spbell{at}mit.edu.
ABBREVIATIONS:

ARS,

autonomously replicating sequence;

RC,

replicative complex;

pre-RC,

pre-replicative complex;

FACS,

fluorescence-activated cell sorting;

MCM,

minichromosome maintenance;

CDK,

cyclin-dependent kinase;

Polα,

DNA polymerase α;

Polɛ,

DNA polymerase ɛ;

RPA,

replication protein A