RECONSTITUTION OF MAMMALIAN DNA-REPLICATION

This chapter focuses on efforts to perform the series of reactions necessary to carry out mammalian chromosomal DNA replication using purified enzymes in vitro. This work fits into a larger framework of genetic and cell biological experiments, using prokaryotes, viruses, and eukaryotes. The particular features of DNA replication include initiation at replication origins, and the components and propagation of the replication fork. DNA replication begins when T antigen recognizes and binds simian virus 40 (SV40's) origin of replication. T antigen binds at positions designated as site I and site II. Although T antigen function is unique to SV40, the details of its action are important because it probably has common features with proteins that initiate DNA replication at all eukaryotic replication origins. Significant structural features of the SV40 origin have been determined by genetic analysis and site-directed mutagenesis. Analysis of the interaction specificity of a protein with other replication proteins can be used to verify its unique participation at a particular step in DNA replication, compared to proteins with the same catalytic activity. Phosphorylation plays an important role in the regulation of T antigen transcription and replication activities. © 1995 Academic Press Inc.