DNA polymerase switching: I. Replication factor C displaces DNA polymerase alpha prior to PCNA loading.
An important not yet fully understood event in DNA replication is the DNA polymerase (pol) switch from pol alpha to pol delta. Indirect evidence suggested that the clamp loader replication factor C (RF-C) plays an important role, since a replication competent protein complex containing pol alpha, pol delta and RF-C could perform pol switching in the presence of proliferating cell nuclear antigen (PCNA). By using purified pol alpha/primase, pol delta, RF-C, PCNA and RP-A we show that: (i) RF-C can inhibit pol alpha in the presence of ATP prior to PCNA loading, (ii) RF-C decreases the affinity of pol alpha for the 3'OH primer ends, (iii) the inhibition of pol alpha by RF-C is released upon PCNA loading, (iv) ATP hydrolysis is required for PCNA loading and subsequent release of inhibition of pol alpha, (v) under these conditions a switching from pol alpha/primase to pol delta is evident. Thus, RF-C appears to be critical for the pol alpha to pol delta switching. Based on these results, a model is proposed in which RF-C induces the pol switching by sequestering the 3'-OH end from pol alpha and subsequently recruiting PCNA to DNA.
Maga
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Istituto di Genetica Biochimica ed Evoluzionistica IGBE-CNR, Pavia, I-27100, Italy.
Stucki
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M
Spadari
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Hübscher
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U
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D004256:DNA Polymerase I / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D007700:Kinetics; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology; D014779:Virus Replication
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D019915:DNA Primase; D004256:DNA Polymerase I; D004258:DNA Polymerase III; D051818:Replication Protein C