Regulating the licensing of DNA replication origins in metazoa Melvin L DePamphilis 1 , J Julian Blow 2 , Soma Ghosh 1 , Tapas Saha 1 , Kohji Noguchi 1 and Alex Vassilev 1 Eukaryotic DNA replication is a highly conserved process; the proteins and sequence of events that replicate animal genomes are remarkably similar to those that replicate yeast genomes. Moreover, the assembly of prereplication complexes at DNA replication origins (‘DNA licensing’) is regulated in all eukaryotes so that no origin fires more than once in a single cell cycle. And yet there are significant differences between species both in the selection of replication origins and in the way in which these origins are licensed to operate. Moreover, these differences impart advantages to multicellular animals and plants that facilitate their development, such as better control over endoreduplication, flexibility in origin selection, and discrimination between quiescent and proliferative states. Addresses 1 National Institute of Child Health and Human Development, National Institutes of Health, Building 6/3A-15, 9000 Rockville Pike, Bethesda, MD 20892-2753, USA 2 Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee, DD1 5EH, UK Corresponding author: DePamphilis, Melvin L (depamphm@mail.nih.gov) Current Opinion in Cell Biology 2006, 18:231–239 This review comes from a themed issue on Nucleus and gene expression Edited by Thoru Pederson and Robert Singer Available online 2nd May 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.04.001 Introduction Human development begins when an egg is fertilized by a sperm to produce a single cell containing a genome of 3.3 billion base pairs of DNA packaged into 46 individual chromosomes. Remarkably, some 5 trillion cell divisions later, an adult human appears that contains more than 20 trillion meters of DNA, the equivalent of 100 times the distance from the earth to the sun! And yet, with the exception of trophoblast giant cells and megakaryocytes, the human genome is replicated once and only once each time a cell divides. How is this amazing feat accom- plished? Although much remains unclear, four concepts have emerged that provide a framework for understand- ing the linkage between DNA replication and cell divi- sion in multicellular animals and plants (the metazoa). First, the protein kinases and ubiquitin ligases that reg- ulate cell division also regulate DNA replication. Second, the rate-limiting step in initiating DNA replication is the unwinding of the two DNA template strands at replica- tion origins by the DNA helicase activity of the mini- chromosome maintenance (MCM) complex, an event that is regulated through the activity of the MCM loading factor, Cdt1/RLF-B. Third, cell-cycle-dependent changes occur in the origin recognition complex (ORC) that not only help to prevent premature DNA licensing, but may serve to regulate origin selection. Finally, the transition from a quiescent state to a proliferative one appears to be regulated through stabilization of Cdc6, one of two proteins required for loading the MCM helicase. Here we describe mechanisms common to all metazoa that prevent relicensing of DNA replication origins before cell division has been completed. Specific differences between these mechanisms and those found in single cell organisms, such as yeast, are noted, and the biological significance of these differences is discussed. DNA replication licensing DNA replication begins with the assembly of a prerepli- cation complex (preRC) consisting of at least 14 different proteins [1]. First, ORC binds to DNA in newly repli- cated chromatin at some time during the S- to early G 1 - phases of the cell division cycle, a process that may be facilitated by Noc3 (Figure 1a). Cdc6 then binds to these ORC:chromatin sites during early G 1 -phase, an event that is followed quickly by binding of Cdt1/RLF-B. MCM helicases are then chaperoned in by Cdt1 to complete the preRC. DNA synthesis (S-phase) begins when preRCs are acted upon by at least 22 additional proteins that activate the MCM helicase and assemble two divergent replication forks at each replication origin [2]. Loading of the MCM helicase onto DNA is referred to as DNA replication licensing, because only these replication origins can initiate DNA synthesis. Regulation of licen- sing refers to those mechanisms that restrict DNA repli- cation to one and only one duplication of the genome prior to cell division, and therefore prevent endoredupli- cation (multiple rounds of DNA replication without an intervening mitosis). This restriction is brought about by inactivating existing preRCs during S, G 2 and early M- phase (Figure 1a), and by preventing the assembly of new preRCs until mitosis is complete and a nuclear membrane is present (Figure 1b) [3,4]. Regulating cell division The same events that regulate eukaryotic cell division also regulate licensing of DNA replication origins, thereby www.sciencedirect.com Current Opinion in Cell Biology 2006, 18:231–239