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Localization of the large subunit of replication factor C near the 5′ end of DNA primers

✍ Scribed by Dmitry M. Kolpashchikov; Patrick Hughes; Alain Favre; Giuseppe Baldacci; Olga I. Lavrik

Publisher
John Wiley and Sons
Year
2001
Tongue
English
Weight
200 KB
Volume
14
Category
Article
ISSN
0952-3499

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✦ Synopsis

Abstract

Replication factor C (RFC) is a heteropentameric sliding clamp loader protein essential for processive synthesis of DNA by eukaryotic DNA polymerases δ and ϵ. 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. Copyright © 2001 John Wiley & Sons, Ltd.

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