16 Regulating Initiation Events in Metazoa

Abstract

At least 5 trillion cell divisions and 20 trillion meters of DNA synthesis are required to produce the hundreds of tissues that comprise an adult human (Appendix, Table 1). And yet, endoreduplication (multiple rounds of DNA replication in the absence of an intervening mitosis) is a rare event. In mammals, for example, it occurs only in the terminally differentiated trophoblast giant cells and in megakaryocytes, a fact that highlights one of the most important universal features of eukaryotic DNA replication: The genome is replicated once and only once each time a cell divides. This is accomplished by inactivating pre-replication complexes (pre-RCs) after S phase begins, while preventing assembly of new pre-RCs until mitosis is complete and a nuclear membrane is present. These events are driven by changes in cyclin-dependent protein kinase (CDK), anaphase-promoting complex (APC) ubiquitin ligase, and Skp1-Cullin-F-Box (SCF) ubiquitin ligase activities (Table 1) (Blow and Hodgson 2002; Diffley 2004).

Pre-RC assembly in eukaryotes involves at least 15 different proteins that bind sequentially to chromatin (Chapter 3). First, a six-subunit origin recognition complex (ORC) binds to newly replicated chromatin sometime during the S to early G₁ phases of the cell division cycle, a process that may be facilitated by Noc3. Cdc6 then binds to these ORC: chromatin sites during early G₁ phase, an event that is soon followed by binding of Cdt1/RLF-B. Cdt1 brings with it the six-subunit Mcm2-7 DNA helicase to form a pre-RC. DNA synthesis (S phase) begins when pre-RCs are acted upon by at least 22...