DNA Replication Origins

The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.Timely duplication of the genome is an essential step in the reproduction of any cell, and it is not surprising that chromosomal DNA synthesis is tightly regulated by mechanisms that determine precisely where and when new replication forks are assembled. The first model for a DNA synthesis regulatory circuit was described about 50 years ago (Jacob et al. 1963), based on the idea that an early, key step in building new replication forks was the binding of a chromosomally encoded initiator protein to specialized DNA regions, termed replication origins (Fig. 1). The number of replication origins in a genome is, for the most part, dependent on chromosome size. Bacterial and archaeal genomes, which usually consist of a small circular chromosome, frequently have a single replication origin (Barry and Bell 2006; Gao and Zhang 2007). In contrast, eukaryotic genomes contain significantly more origins, ranging from 400 in yeast to 30,000–50,000 in humans (Cvetic and Walter 2005; Méchali 2010), because timely duplication of their larger linear chromosomes requires establishment of replication forks at multiple locations. The interaction of origin DNA and initiator proteins (Fig. 1) ultimately results in the assembly of prereplicative complexes (pre-RCs), whose role is to load and activate the DNA helicases necessary to unwind DNA before replication (Remus and Diffley 2009; Kawakami and Katayama 2010). Following helicase-catalyzed DNA unwinding, replisomal proteins become associated with the single-stranded DNA, and new replication forks proceed bidirectionally along the genome until every region is duplicated (for review, see O’Donnell 2006; Masai et al. 2010).Open in a separate windowFigure 1.Revised versions of the replicon model for all domains of life. For cells of each domain type, trans-acting initiators recognize replication origins to assemble prereplicative complexes required to unwind the DNA and load DNA helicase. Eukaryotic initiators are preassembled into hexameric origin recognition complexes (ORCs) before interacting with DNA. In prokaryotes, single initiators (archaeal Orc1/Cdc6 or bacterial DnaA) bind to recognition sites and assemble into complexes on DNA. In all cases, the DNA helicases (MCMs or DnaB) are recruited to the origin and loaded onto single DNA strands. In bacteria, DNA-bending proteins, such as Fis or IHF, may modulate the assembly of pre-RC by bending the origin DNA. Two activities of DnaA are described in the figure. The larger version binds to recognition sites, and the smaller version represents DnaA required to assist DnaC in loading DnaB helicase on single-stranded DNA.Initiator proteins from all forms of life share structural similarities, including membership in the AAA⁺ family of proteins (ATPases associated with various cellular activities) (Duderstadt and Berger 2008; Wigley 2009) that are activated by ATP binding and inactivated by ATP hydrolysis (Duderstadt and Berger 2008; Duncker et al. 2009; Kawakami and Katayama 2010). Despite these similarities, initiators assemble into prereplicative complexes in two fundamentally different ways (Fig. 2). In prokaryotes, initiator monomers interact with the origin at multiple repeated DNA sequence motifs, and the arrangement of these motifs (see below) can direct assembly of oligomers that mediate strand separation (Erzberger et al. 2006; Rozgaja et al. 2011). In eukaryotes, a hexameric origin recognition complex (ORC) binds to replication origins and then recruit additional factors (as Cdc6 and Cdt1) that will themselves recruit the hexameric MCM2-7 DNA helicase to form a prereplicative complex (for review, see Diffley 2011). This process occurs during mitosis and along G₁ and is called “DNA replication licensing,” a crucial regulation of eukaryotic DNA replication (for review, see Blow and Gillespie 2008). Importantly, this complex is still inactive, and only a subset of these preassembled origins will be activated in S phase. This process is, therefore, fundamentally different from initiation of replication in bacteria. Moreover, because sequence specificity appears more relaxed in large eukaryotic genomes, prokaryotic mechanisms that regulate initiator–DNA site occupation must be replaced by alternative mechanisms, such as structural elements or the use of epigenetic factors.Open in a separate windowFigure 2.Functional elements in some well-studied prokaryotic replication origins. (A) Bacterial oriCs. The DNA elements described in the text are (arrows) DnaA recognition boxes or (boxes) DNA unwinding elements (DUEs). When recognition site affinities are known, colored arrows designate high- (K_d > 100 nm) and low- (K_d < 100 nm) affinity sites. (B) Archaeal oriCs. Arrows and boxes designate DNA elements as in A, but the initiator protein is Orc1/Cdc6 rather than DnaA. (Thick arrows) Long origin recognition boxes (ORBs); (thin arrows) shorter versions (miniORBs). Both ORBs and miniORBs are identified in Pyrococcus. DUEs are not yet well defined for Helicobacter or Sulfolobus genera and are not labeled in this figure.Here, we describe replication origins on prokaryotic and eukaryotic genomes below, with a particular focus on the attributes responsible for orderly initiator interactions and origin selection specificity, as well as on the shift from origin sequence-dependent regulation to epigenetic regulation. You are also referred to other related articles in this collection and several recent reviews covering the topics of DNA replication initiation in more detail (Méchali 2010; Beattie and Bell 2011; Blow et al. 2011; Bryant and Aves 2011; Ding and MacAlpine 2011; Dorn and Cook 2011; Kaguni 2011; Leonard and Grimwade 2011; Sequeira-Mendes and Gomez 2012).