| Abstract: |
This chapter discusses purified systems that can replicate closed-circular, double-stranded superhelical (form I) DNAs in vitro. It also discusses problems unique to the replication of DNAs under topological constraint. Studies on the reconstituted pBR322 DNA replication system are used as a guide in the chapter. Recent studies on the replication of form I DNAs indicate that new classes of proteins are required to replicate these DNAs. The activities of these new enzymes help to solve some of the problems peculiar to the replication of superhelical DNAs. The replication of the two parental DNA strands without the introduction of a discontinuity in either strand presents a unique problem for the initiation of DNA replication of form I DNAs. Two pathways of initiation have been revealed. In the case of form I DNAs containing the origins of DNA replication from either the chromosome of E. coli (oriC) or bacteriophage λ (λdv), special proteins interact with specific DNA sequences to form protein-DNA complexes subsequently recognized by other priming proteins that gain access to the DNA template at the site of the initial specific protein-DNA complex. In the initiation of pBR322 DNA replication, a different pathway operates. The formation of a RNA-DNA hybrid by a transcript manufactured by RNA polymerase serves to prime leading-strand DNA synthesis after being processed by RNase and allows a “primosome” assembly site sequence on the lagging strand template to assume an active configuration and catalyze the assembly of a primosome that subsequently primes lagging-strand DNA synthesis. © 1986, Academic Press, Inc. All rights reserved. |
