Replication of SV40 in vitro using proteins derived from a human cell extract.
In the presence of large T antigen and plasmids containing a functional origin of replication, extracts from a human cell line will support multiple rounds of simian virus 40 (SV40) replication in vitro. Fractionation of this extract has led to the identification of several factors, some of which have been purified to homogeneity. The characterisation of these proteins has led to the separation of SV40 replication in vitro into multiple stages. Two proteins, the cell cycle-regulated proliferating cell nuclear antigen and replication factor-C, have been shown to be essential for coordinating leading and lagging strand synthesis in this system. Another protein, replication factor-A, is a multi-subunit protein of 70, 34 and 11K (K = 10(3) Mr) polypeptides which, because of its high affinity for DNA, is thought to function as a eukaryotic single-stranded DNA binding protein. Interactions between other cellular factors are also described that effect the initiation of DNA replication, but are not required in a more purified system. In addition a model for a hypothetical replication fork is described, which suggests a role for both alpha- and delta-polymerases in this system, and may be applicable to higher eukaryotes.
Fairman
M P
MP
Cold Spring Harbor Laboratories, New York 11724.
Prelich
G
G
Tsurimoto
T
T
Stillman
B
B
D000956:Antigens, Viral; D004257:DNA Polymerase II / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis; D018809:Proliferating Cell Nuclear Antigen; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D013539:Simian virus 40 / Q000502:physiology*; D014779:Virus Replication
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II