Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I.
Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.
Riva
Federica
F
Istituto di Genetica Molecolare del CNR, Dipartimento di Biologia Animale, sezione Istochimica e Citometria, Università di Pavia, Piazza Botta 10, 27100 Pavia, Italy.
Savio
Monica
M
Cazzalini
Ornella
O
Stivala
Lucia A
LA
Scovassi
Ivana A
IA
Cox
Lynne S
LS
Ducommun
Bernard
B
Prosperi
Ennio
E
D000906:Antibodies / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D042846:CDC2-CDC28 Kinases / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics*; D002460:Cell Line; D019925:Cyclin A / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D004247:DNA / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D003851:Deoxyribonucleases / Q000378:metabolism; D005333:Fetus; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics
D000906:Antibodies; D019925:Cyclin A; C000607892:LIG1 protein, human; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D004258:DNA Polymerase III; D003851:Deoxyribonucleases; D011088:DNA Ligases; D000072481:DNA Ligase ATP