Purification and functional characterization of bovine RP-A in an in vitro SV40 DNA replication system.
The single-stranded DNA binding protein RP-A is required in SV40 DNA in vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replication in vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase alpha-primase complex.
Nasheuer
H P
HP
Institute for Biochemistry, LMU München, Federal Republic of Germany.
von Winkler
D
D
Schneider
C
C
Dornreiter
I
I
Gilbert
I
I
Fanning
E
E
D000645:Ammonium Sulfate; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000502:physiology; D002417:Cattle; D002474:Cell-Free System; D004265:DNA Helicases / Q000502:physiology; D004257:DNA Polymerase II / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D051716:Replication Protein A; D013539:Simian virus 40
D000952:Antigens, Polyomavirus Transforming; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D016203:CDC2 Protein Kinase; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D004265:DNA Helicases; D000645:Ammonium Sulfate