This chapter discusses the characterization of the xenopus replication licensing system. In eukaryotes the entire genome is replicated precisely once during each S phase of the cell cycle. Cell-free extracts of Xenopus eggs support chromosomal DNA replication in vitro, apparently under the same cell cycle controls that exist in vivo. When DNA is added to the cell-free system it is assembled into chromatin, and then into structures resembling normal interphase nuclei, processes that is necessary before the initiation of DNA replication can occur. The precise duplication of chromosomal DNA in the Xenopus cell-free system requires many thousands of replication origins to each fire once and only once. To achieve this, two distinct signals are required for initiation to occur at any given replication origin.
|Name||Methods in Enzymology|