In order to maintain eukaryotic genomic integrity, precise duplication of DNA must occur once, and only once, during each cell division cycle. This is achieved, during the G1-S transition, through tightly regulated initiation events at multiple replication origins scattered along each chromosome (Machida, Hamlin, and Dutta 2005). The basic mechanism of initiation occurs in several steps and results in the establishment of bidirectional replication forks at DNA origins:
(i) Origin recognition: label the origin with ORC.
(ii) Assembly of the pre-replicative initiation complex: load the DNA helicase to form the pre-RC.
(iii) DNA unwinding: activate the DNA helicase.
(iv) Elongative assembly: load the replisome including DNA polymerase and single stranded DNA binding protein.
A replication origin is a specific site on the chromosome where initiative proteins bind and open the DNA helix to allow DNA synthesis to begin (Figure 1.5). These sites on the DNA are commonly called the origin recognition complex (ORC) (Bell and Stillman 1992). Usually, replication begins at an origin and proceeds in a bidirectional manner to complete a single replicon.
University College London 51 The replication origins in mammalian cells are not distributed at regular intervals
throughout the chromosomes but tend to be organised into clusters and are all activated simultaneously (Prasanth et al. 2004; Siddiqui and Stillman 2007). Approximately thirty thousand origins are postulated to exist in the mammalian genome; however, the number of licensed origins exceeds the number of origins activated in each cell cycle due to the assembly of pre-RCs at both active and silent origins. This acts to ensure that the entire genome is replicated even if some origins fail to initiate (Bell and Dutta 2002).
In higher eukaryotes the organisation of origins appears more complex and difficult to define compared to the models constructed in yeast. Human ORC is capable of binding functionally to any DNA sequence, which is consistent with the observation that replication can occur at multiple sites. Although the mechanism of ORC recruitment varies between eukaryotes, the subsequent steps involved in assembling the pre-RC after ORC binding are conserved among all eukaryotes. The replication origin forms the foundation on to which the pre-replicative complexes are assembled during late M and early G1 phases. Replication licensing factors: ORC, Cdc6, Cdt1 and Mcm2-7 assemble into pre-RCs, which render replication origins “licensed” for DNA synthesis (Blow and Hodgson 2002). This is a highly coordinated process with sequential loading of each factor onto the ORC subunit under the control of ATPase activity (Siddiqui and Stillman 2007).
Six paralogous proteins constitute the MCM complex, with Mcm2 to Mcm7 first identified in budding yeast; where defects in both initiation and elongation phases in
University College London 52 Figure 1.5
University College London 53 DNA replication were noted in mutated forms (Lei et al. 1997; Tye 1999; Forsburg
2004). Studies show that the six subunit MCM complex lacks helicase activity in
vitro, suggesting that Mcm4/6/7 constitutes the core helicase while Mcm2/3/5 are
regulatory subunits (Ishimi 1997; You, Komamura, and Ishimi 1999; Ishimi and Komamura-Kohno 2001). The model is supported by studies showing that the ATPase activity of the Mcm2-7 complex requires the coordinated action of all six subunits with Mcm4/6/7 involved in ATP hydrolysis and Mcm2/3/5 regulating its activity (Schwacha and Bell 2001). Consequently, the activation of MCM helicase activity, probably involves the re-arrangement of some of the units to form a ring-like molecule that encircles and unwinds the DNA (Davey et al. 2002). Each pre-RC constituent is essential for the initiation of DNA replication with reduced levels of any component leading to the loss of replicative capacity and an inability to progress into S-phase. Once MCM complexes are loaded onto chromatin, the other components of the pre-RC are dispensable for replication initiation. Therefore, the primary function of ORC, Cdc6 and Cdt1 in replication is to load the Mcm2-7 complex (Donovan et al. 1997; Hua and Newport 1998; Harvey and Newport 2003).
The transition from G1 to S phase involves the conversion of pre-RCs into replication forks. Initiation requires origin unwinding, stabilisation of single stranded DNA and loading of the replicative polymerases. These processes require the function of a second set of replication factors and the activity of at least two kinases – cyclin dependent kinases (CDKs) and Dbf4 dependent kinase (DDK); also termed Cdc7 kinase (Jiang et al. 1999; Grishina and Lattes 2005). These factors are temporally regulated throughout S phase and induce a conformational change in the pre-RC,
University College London 54 resulting in the recruitment of additional initiator proteins which collectively promote
origin unwinding and recruitment of DNA polymerases.
The earliest initiation factor recruited to pre-RCs is Mcm10, which is involved in several critical functions. Mcm10 interacts with Mcm2-7, as well as ORC2 and Cdc7- Dbf4, and has been suggested to facilitate phosphorylation of Mcm7 by Cdc7-Dbf4 (Mendez and Stillman 2003; Takeda and Dutta 2005). The formation of an active helicase leads to the recruitment of additional factors, including Cdc45 and the four subunit GINS complex, which is also dependent on Cdc7 kinase activity (Gambus et al. 2006). Phosphorylation of the MCM complex by CDKs and DDK results in the recruitment of Cdc45 to the origin (Wohlschlegel et al. 2002a; Sawyer et al. 2004). In addition Mcm10 is required for the loading of Cdc45 onto the chromatin and like Mcm2-7 is essential for elongation of DNA synthesis. As well as activating the MCM complex the pre-initiation complex acts on the final part of DNA replication initiation by loading polymerase-alpha-primase and the other DNA polymerases delta and epsilon onto the DNA (Mimura and Takisawa 1998; Aparicio, Stout, and Bell 1999).
Once activated, the MCM helicase unwinds double-stranded DNA at origins to generate a single-stranded DNA template required to recruit the DNA synthesis machinery containing RPA, PCNA and DNA polymerase α-primase (Sclafani and Holzen 2007). Following entry into S phase, the licensing system is shut down to prevent re-initiation events at origins that have already been ‘fired’. The key event in suppressing relicensing of origins is the inactivation of the MCM loading factor Cdt1 through two mechanisms (Blow and Dutta 2005). First, Cdt1 undergoes cell cycle dependent proteolysis during S and G2 (Li et al. 2003). Second, residual Cdt1 is
University College London 55 inhibited by the binding of a small regulatory protein called geminin, which is
expressed at high levels during the S, G2 and M phases (Tada et al. 2001; Wohlschlegel et al. 2002b).