Abstract
DNA polymerase alpha-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim(2)) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim(2) indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2 x 10(-8) m and 1.3 x 10(-8) m, respectively.
Highlights
In eukaryotic cells the duplication of the genome is a highly accurate and tightly coordinated process
Two cell-free initiation reactions have served as model systems to investigate these processes, primer synthesis in the cell-free SV40 DNA replication system and primer synthesis on natural single-stranded DNA templates bound by replication protein A (RPA) [17, 18]
It is thought that the coordinated oligoribonucleotide synthesis by pol-prim on M13 ssDNA in the presence of RPA and T antigen (Tag) in vitro resembles the initiation of Okazaki fragments on the lagging strand in vivo
Summary
In eukaryotic cells the duplication of the genome is a highly accurate and tightly coordinated process (reviewed in Ref. 1). Two cell-free initiation reactions have served as model systems to investigate these processes, primer synthesis in the cell-free SV40 DNA replication system and primer synthesis on natural single-stranded (ss) DNA templates bound by replication protein A (RPA) [17, 18] Using these cell-free systems, it was shown that the initiation of SV40 DNA replication at the origin is species-specific and requires the activity of the p180 subunit of primate pol-prim (19 –21). The leading strand initiation of SV40 DNA replication in vitro requires double-stranded (ds) plasmid DNA with a viral origin of replication, the multifunctional viral Tag, the cellular topoisomerase I, and two cellular complexes, pol-prim and the eukaryotic ssDNA-binding protein, RPA [1, 3, 26, 27]. Since only the four-subunit pol-prim can start DNA replication de novo at the SV40 origin of replication, these data suggest that the initiation of leading and lagging strand synthesis are mechanistically different
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