Sulfolobus Replication Factor C Stimulates the Activity of DNA Polymerase B1

Abstract

bReplication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3=-5= exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication.A ll forms of cellular life replicate their chromosomal DNA in a strikingly similar fashion, employing clamp loader proteins to assemble ring-shaped sliding clamps in ATP-dependent reactions at new RNA-primed sites, where the clamp tethers DNA polymerase for highly processive DNA synthesis (1). Although clamp loader proteins of different origins differ at the amino acid sequence level and in subunit composition, they share both overall structures and molecular mechanisms in clamp-loading processes (2). In Escherichia coli, the clamp loader, known as the ␥ complex, consists of five subunits, i.e., ␦, ␦=, and three copies of /␥ (3). In eukarya, the clamp loader, referred to as replication factor C (RFC), has four different small subunits (RFC S ) and one large subunit (RFC L ) (2). Archaea, the third domain of life, replicate DNA in a eukaryotic-like fashion. While most of the archaeal clamp loaders are a pentameric complex of one large subunit and four identical small subunits (4-6), RFCs from some archaea exhibit variations in subunit composition. For example, RFC from Methanosarcina acetivorans shows a stoichiometry of one large subunit to three small subunits to one even smaller subunit (7).PCNA loading by the RFC heteropentamer (1 RFC L /4 RFC S ) from the thermoacidophilic crenarchaeon Sulfolobus solfataricus has been extensively studied (8,9). Unlike eukaryotes and euryarchaea, which possess a homotrimeric PCNA, crenarchaea have an unusual heterotrimeric PCNA comprising PCNA1, PCNA2, and PCNA3 (8). The S. solfataricus RFC has been shown to interact with the PCNA1 and PCNA2 subunits through RFC S and with PCNA3 through RFC L (8). Opening of the PCNA ring by RFC at the PCNA3-PCNA1 interface allows the passage of DNA into the PCNA ring (9). In addition to its role in PCNA loading, RFC also interacts with the eukaryotic-type primase (10). As the result of this interaction, primer synthesis by the primase is inhibited while the ATPase activity of RFC is stimulated, suggesting that RFC may serve roles in regulating primer synthesis and transfer of primers to DNA polymerase.In the present study, we show that Sulfolobus RFC interacts physically with DNA polymera...

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