Proliferating cell nuclear antigen recruits cyclin-dependent kinase inhibitor Xic1 to DNA and couples its proteolysis to DNA polymerase switching.
The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.
Chuang
Li-Chiou
LC
University of Texas Health Science Center at San Antonio, Department of Molecular Medicine, Institute of Biotechnology, San Antonio, Texas 78245-3207, USA.
Yew
P Renee
PR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D004247:DNA / Q000737:chemistry*; D004256:DNA Polymerase I / Q000378:metabolism; D019915:DNA Primase / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005775:Gastrula / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D051818:Replication Protein C / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000737:chemistry; D014981:Xenopus; D029867:Xenopus Proteins / Q000378:metabolism*
D002843:Chromatin; D017931:DNA Primers; D004277:DNA, Single-Stranded; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D025801:Ubiquitin; D029867:Xenopus Proteins; C493963:Xicl protein, Xenopus; D050760:Cyclin-Dependent Kinase Inhibitor p27; D012313:RNA; D004247:DNA; D005982:Glutathione Transferase; D019915:DNA Primase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D051818:Replication Protein C