The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

ABSTRACT

The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, namely, its size distribution, RNA primer content, and replication fork polarity, were determined. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase α-primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging-strand DNA synthesis are evaluated in view of our results.

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The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

ACKNOWLEDGMENTS

We thank Jerard Hurwitz and Sara Lavi for providing anti-RPA antibodies, Marc Wold and Olga Lavrik for making results available prior to publication, and Mark Wold and Rolf Knippers for critical readings of the manuscript.

This work was supported by grants from the U.S.-Israel Binational Science Foundation, the Israel Cancer Society, and the German-Israeli Foundation for Research and Development to G.K.