The most important replication proteins, their structural and functional assembly with each other and with DNA, and the whole mechanism that ensures complete and accurate duplication of DNA, are highly conserved during eukaryotic evolution. Prior to initiation of DNA replication, a multiprotein assembly called pre-replication complex (pre-RC) forms at replication origins. Formation of the pre-RC is initiated by the eukaryotic initiator Origin Recognition Complex (ORC). ORC recognizes origin DNA and recruits Cdc6, Cdt1, and MCM complex to origins (review Blow & Dutta, 2005). The selection of particular locations within the eukaryotic chromosome for initiation is poorly understood. The whole process of origin recognition includes complex interplay between factors affecting ORC assembly on replication origins and structural constrains of bound DNA. Given that replication origins of higher eukaryots do not have common consensus sequences, specificity of protein-DNA interactions does not play a central role in origin recognition. However, origin transfer studies show that origins have some genetic elements comprised in different modules which are essential for origin activity and are functionally interchangeable between origins (review Aladjem, 2007). The quite obscure step of origin selection is followed by origin remodeling which is promoted by pre-RC. Origin remodeling opens DNA for replication proteins and prepares the origin to be activated and to accommodate the double replisome. The complete sequence of events from origin recognition to activation involves multiple protein-protein and protein-DNA interactions. Identification of all abovementioned components and their interactions will ultimately lead to understanding of the complex mechanism which governs origin selection and ensures accurate initiation in eukaryotic cells.
2. Eukaryotic replication origins
Replication origins are DNA sites at which replication initiates during the S phase. Using different approaches, these sites were identified in simple eukaryotes and in metazoa. In yeast, specific origin sequences were identified by their ability to confer autonomous replication to small circular plasmids. The same assay employed in multicellular systems reviled that virtually any DNA fragment not smaller than 10kb had origin activity (Krysan, 1993). Real replication origins of metazoa were identified by alternative procedures, but they are not numerous and not yet well understood, due to extreme genome complexity and cell-to-cell heterogeneity in origin selection (Hamlin et al., 2008). Recent development of genome-scale methods for identifying hundreds or thousands of new origins may compensate for this lack of data and thus provide information regarding the recognition features of complex origins (Eaton et al., 2010; MacAlpin et al., 2010).
The best understood replication origins belong to budding yeast. They are composed of AT-rich sequences of about 150bp, termed ARS elements. ARS elements were discovered because of their capacity to confer high-frequency transformation of plasmids in
Different origin mapping procedures have revealed that replication origins of metazoa belong to two categories. Some of them contain unique high-frequency initiation sites, while the others have extensive zones with numerous initiation sites (Aladjem, 2007) and diffuse initiation pattern. Within such zones each site is activated only in a fraction of cell cycles which means that, once initiated at one site, replication forks just pass through the others. In beta-globin genes (HBB), the replication origin contains two adjacent initiation sites which are activated in different cell cycles (Wang et al., 2004). It therefore seems that combined data suggest that replication origins in metazoa generally contain few or more nonrandom initiation sites which could be activated in different cell cycles. Some of these sites are strong and initiate DNA replication at high frequency while the others are not and initiate DNA at low frequency. It, however, seems that initiation sites do not contain any common DNA motif, which is consistent with the apparent lack of sequence specificity of metazoa ORCs
Analysis of HBB
Sequences rich in A+T are abundant in eukaryotic origins and could have roles in facilitating DNA unwinding. Asymmetric AT stretches are present in the hamster DHFR origin and in the human LMNB2, HBB and DHFR origins. Such sequences are recognized by proteins, such as SpORC4 that has the relevant AT hooks (Kong & DePamphilis, 2002). Interestingly, even its human homologue displays a similar preference for asymmetric AT stretches (Stefanovic et al., 2003). The other AT rich elements that could be important for initiation of DNA replication are matrix attachment sites. Matrix attachment regions are required for maintenance of plasmid replication in human cells and could be part of metazoan replication origins. In addition, different AT elements could build unorthodox structures similar to one detected in the asymmetric AT-rich stretch of the LMNB2 origin (Kusic et al., 2005).
In some promoters CpG islands are correlated with open chromatin structure and it is believed that they could have similar roles in replication origins. Human nascent DNA is 10-fold enriched in CpG islands (Delgado et al., 1998), whereas removal of a CpG island significantly decreases the efficiency of ectopic LMNB2 origin (Paixao et al., 2004).
Unusual DNA structures could form from origin elements that are not AT rich. Palindromes were found in hamster DHFR and human HBB and LMNB2 origins. Different unorthodox structures were detected in the hamster DHFR origin (Bianchi et al., 1990) including one bent element important for DHFR ectopic activity, which indicated a correlation between origin topology and its function (Altamn & Fanning, 2004).
In summary, replication origins in metazoa are determined by a complex, poorly understood set of structural and topological features of DNA in which no single-sequence module has a key role.
3. Origin recognition complex
The Origin Recognition Complex (ORC) was first discovered and purified from budding yeast, based on its ability to specifically bind to replication origins (Bell & Stillman, 1992). Shortly thereafter, the corresponding genes were cloned and orthologues of Orc1-Orc5 were identified in organisms such as
ORC-like proteins are not just confined to eukaryotes. Studies of archeal Orc1/cdc6 proteins, as well as DnaA have provided important structural information about ORC-DNA interactions. DnaA, like ORC, acts as an initiator of DNA replication, and whereas DnaA and the archeal Orc1/cdc6 proteins share little sequence identity, structural studies have shown that they do have a high degree of similarity in some of their functional domains (Mott & Berger, 2007). Moreover, a study of
ORC function is tightly controlled by ATP binding and hydrolysis. Three of the ORC subunits (Orc1, 4, and 5) are members of AAA+ family of ATPases. Recent studies suggest that ORC2 and ORC3 represent more distant relatives of the AAA+ proteins that lack the key conserved elements of the ATP-binding site (Speck et al., 2005). In
Although formally a member of ORC, Orc6 does not share similar structural features or a common evolutionary origin with Orc1-5 (Duncker et al., 2009). Nevertheless, its association with the other five subunits is required to promote the initiation of DNA replication and it is considered an ORC protein.
It appears that all six ORC subunits remain associated with chromatin throughout the cell cycle in
Assembly of the human recombinant ORC subunits was investigated
4. Mechanisms of replication initiation site selection
Selection of DNA replication origins may be regulated by various factors and may be achieved at different levels. Replication initiates at many sites along linear chromosomes, which ensures complete genome duplication within a single S phase, but the number of activated origins does not match the number of prereplication complexes previously assembled on DNA (review Gilbert, 2010). From the large pool of all assembled pre-RCs only a subset is chosen for subsequent initiation while the rest remain dormant. Two-step mechanism or mechanisms that select preRCs for initiation or govern the pre-RC assembly remain unknown.
In budding yeast, ORC binds the corresponding ARS element in a sequence specific manner. One component of the recognition site is the 11-bp ACS. As shown by analysis of modified DNA substrates, DNA-bound ORC primarily interacts with the A-rich strand of the ACS. It is not yet clear which subunit of ORC determines DNA binding, but protein-DNA cross-linking studies show four out of six ORC subunits (Orc1, Orc2, Orc4 and Orc5) in close association with origin DNA (Lee & Bell, 1997).
ARS function appears to be governed primarily by AT content and length (Dai et al., 2005). Whether the replicator length is needed to include the required DNA elements or to provide spacing between them is not clear. DNA appears to wrap around ORC (Gaczynska et al., 2004), suggesting a possible spacing length requirement between ORC-binding sites. Intervening deletion mutations could affect replicator function by either shortening the spacing length between elements or by removing elements.
In the metazoan model system ORC-DNA interactions were first explored in
Interestingly, human ORC and human Orc4 exhibit similar DNA binding properties, such as preference for negatively supercoiled DNA (Kusic or Tomic unpublished), preference for AT rich DNA and the ability to distinguish between different AT-rich DNA structures. HsOrc4 protein also exhibits preference for triple stranded DNA (Kusic et al., 2010) and the ability to stimulate formation of noncanonical oligonucleotide structures (Stefanovic et al., 2008). Such HsOrc4 properties could play part in origin selection through directing ORC to DNA sequences able to adopt unorthodox structures.
Pre-RC factors other than ORC may also contribute to origin recognition. In budding yeast, Cdc6 ATPase activity contributes to stabile and specific binding of the ORC-Cdc6 complex to the origin (Speck & Stillman, 2007), whereas in fission yeast Cdt1 and Cdc6 proteins facilitate SpORC-DNA interactions (Houchens et al., 2008).
In addition to origin structure and preRC components, chromatin structure could significantly affect replication origin selection. As revealed by ChIP-seq for ORC in budding yeast, many consensus sequences are not bound by ORC (Eaton et al., 2010). A genome-wide analysis of nucleosome architecture of replication origins in budding yeast, aligned by their ORC-binding sites, suggested a model in which the underlying DNA sequence at replication origins occludes nucleosomes. This creates a permissive environment for ORC binding, after which ORC positions nucleosomes in regular array on both sides (Berbenetz et al., 2010). In addition, only a subset of nucleosome free regions (NFR) with specific flanking sequence features – which allow the ORC to position nucleosomes with sufficient space for MCM protein loading – can promote binding of ORC. Accordingly, by genome-scale mapping of
Transcription factors may also play a role in localization of ORC. Thus, at the chorion loci of
5. Assembly of Pre-RC complexes
Originally identified by
In pre-RCs formed
A similarity of ORC subunits and Cdc6 to sliding clamp loaders and the ring shaped structure of MCM have led to the proposal of a model for MCM origin loading (Speck & Majka, 2009). Loading initiates by association of ORC with origin DNA in the ATP-bound state. ORC-ATP recruits Cdc6, stimulates its association with ATP and subsequent recruitment of Cdt1 and the MCM proteins. According to the model, this leads to the opening of the MCM ring thus exposing a previously hidden DNA-binding site. MCM binding to DNA triggers Cdc6 ATP hydrolysis which leads to two events: the release of Cdc6 and Cdt1, and closing of the MCM ring around the DNA.
As suggested by extensive conservation of replication factors, the basic mechanism of DNA replication is evolutionally conserved. However, regulation of origin firing in higher eukaryotes is much more complex than in lower eukaryotes. Consequently, in order to understand what specifies the metazoan origins one must look far beyond simple linear sequences and take into account combinatorial interaction of multiple components that make up the initiation machinery and insert it in the cell cycle regulatory network. The main entity that initiates preRC formation, protein complex ORC, does not have the ability to select origins in metazoa based solely on its own affinity for specific DNA sequences. In this function it could be aided by other pre-RC proteins, DNA topology, and even unorthodox DNA structures. Characteristics and state of chromatin structure in specific regions of the genome, nucleosome positioning, binding of transcription factors and degree of DNA supercoiling may restrict the area in which initiation could occur. Altogether these features could play a critical role in initiation of DNA replication by the mechanism that requires many precise small steps leading to a single goal.
This work was supported by grants from the Ministry of Science and Technological Development, Serbia (173008) and the International Centre for Genetic Engineering and Biotechnology, Italy (CRP/YUG08-01).