Localization of the large subunit of replication factor C near the 5' end of DNA primers.
Replication factor C (RFC) is a heteropentameric sliding clamp loader protein essential for processive synthesis of DNA by eukaryotic DNA polymerases delta and epsilon. To study the interaction of RFC with 3' and 5' ends of the DNA primer, we have developed chemical photocrosslinking assay using a synthetic DNA gap and DNA primer-template structures. We have found that the radioactively labeled primers containing a photoreactive group at their 5' end could crosslink with the largest RFC subunit (RFC140) on primer-templates and DNA gap structures, but that 3' end photoreactive primers could only crosslink with RFC140 within the DNA gap structure. Addition of replication protein A (RPA) to the reaction mixture resulted in the crosslinking of RPA subunits and inhibited crosslinking of RFC140 using 3' but not 5' photoreactive primers present at the gap. The results suggest specific contacts between RFC140 and the 5' end of the DNA primer. Together with previous data, these experiments allow us to propose a model for the DNA polymerase switch during eukaryotic DNA replication.
Kolpashchikov
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Institut Jacques Monod (CNRS, Universite Paris 6, Universite Paris 7), 75351 Paris Cedex 05, France.
Hughes
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Favre
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Baldacci
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Lavrik
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OI
D024506:5' Flanking Region; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry; D004257:DNA Polymerase II / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D005057:Eukaryotic Cells; D051379:Mice; D019920:Photoaffinity Labels; D010777:Photochemistry; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A; D051818:Replication Protein C; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D013698:Templates, Genetic
D003432:Cross-Linking Reagents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019920:Photoaffinity Labels; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D051716:Replication Protein A; C497168:Rpa1 protein, mouse; D000255:Adenosine Triphosphate; D004257:DNA Polymerase II; D019951:DNA Polymerase beta; D051818:Replication Protein C