Abstract
The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1 is a molecular target for reversible phosphorylation by cyclin/Cdk complexes and PP1 during nucleosome assembly and DNA synthesis in vitro. Purified p60 can be directly phosphorylated by purified cyclin A/Cdk2, cyclin E/Cdk2, and cyclin B1/Cdk1, but not by cyclin D/Cdk4 complexes in vitro. Cyclin B1/Cdk1 triggers hyperphosphorylation of p60 in the presence of additional cytosolic factors. CAF-1 containing hyperphosphorylated p60 prepared from mitotic cells is inactive in nucleosome assembly and becomes activated by dephosphorylation in vitro. These data provide functional evidence for a requirement of the cell cycle machinery for nucleosome assembly by CAF-1 during DNA replication.
Highlights
The replication of eukaryotic chromosomes is integrated into the regulation of the cell division cycle [1]
Nucleosome assembly by chromatin assembly factor 1 (CAF-1) can be experimentally uncoupled from ongoing DNA replication by adding CAF-1 to an in vitro reaction after DNA strand synthesis has been inhibited by aphidicolin, suggesting that newly replicated DNA is marked for subsequent nucleosome assembly by CAF-1 [19]
We first analyzed the influence of the Cdk inhibitor roscovitine on nucleosome assembly by CAF-1 during complementary DNA strand synthesis
Summary
The replication of eukaryotic chromosomes is integrated into the regulation of the cell division cycle [1]. Nucleosome assembly by CAF-1 can be experimentally uncoupled from ongoing DNA replication by adding CAF-1 to an in vitro reaction after DNA strand synthesis has been inhibited by aphidicolin, suggesting that newly replicated DNA is marked for subsequent nucleosome assembly by CAF-1 [19]. This marking depends on a replication factor, proliferating cell nuclear antigen (PCNA) [20]. Successful coupling of these two processes required additional, as yet unknown factors present in the unfractionated cytosolic extract [20]
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