Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system.
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.
Weisshart
K
K
Institut für Molekulare Biotechnologie e.V., Abteilung Biochemie, Beutenbergstrasse 11, D-07745 Jena, Germany.
Förster
H
H
Kremmer
E
E
Schlott
B
B
Grosse
F
F
Nasheuer
H P
HP
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D001665:Binding Sites; D002474:Cell-Free System; D004256:DNA Polymerase I / Q000737:chemistry* / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D011487:Protein Conformation; D051716:Replication Protein A; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D051716:Replication Protein A; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I