In the previous post we discussed about the prokaryotic DNA replication. The replication in prokaryotic cell is much simpler than that of the eukaryotic cell. The eukaryotic DNA is much larger, more condensed and linear as compared to prokaryotic DNA. Also the eukaryotic replication occurs in a separate phase of the cell cycle known as S (synthesis) phase and has to be well regulated unlike prokaryotic cell where the DNA replication can take place continuously during growth. However, as in prokaryotes, the DNA replication process in eukaryotes, can be divided into three stages: Initiation, Elongation and Termination.
In this post we shall see about initiation of DNA replication in eukaryotes, next post we shall discuss the elongation and termination.
Initiation of DNA replication:
As seen in the last post, the replication begins at a particular site in the genome known as origin. The replication origin have conserved DNA sequences. As the eukaryotic genome is much larger than the prokaryotic genome, there are hundreds of origin in eukaryotic genome. Hence the replication can begin simultaneously and complete the replication faster. Origin has the sequence to which the ORC binds.
The origin recognition complex (ORC):
ORC or the origin recognition complex is a complex of six subunits arranged in a C-shaped structure encircling the DNA at origin (fig 1A). This complex binds and recruits other factors to establish a pre-replicative complex (pre-RC).
(Just for info: Read more on Origin Recognition Complex).
Cell division cycle 6 protein or Cdc6 binds the ORC/origin DNA complex, during late M phase of the cell cycle (fig 1B). ATP binding is required for ORC/Cdc6/DNA complex formation.
Cdt1/MCM2–7 heptamer then binds the ORC/Cdc6 complex and loads MCM2–7 onto the dsDNA. MCM2–7, the minichromosome maintenance 2–7 consists of six spirally arranged subunits which forms the core of the replicative DNA helicase. Cdt1 or the chromatin licensing and DNA replication factor 1 stabilises a single Mcm2-7 hexamer and loads it onto the chromosome.
The binding of Cdt1/MCM2–7 to ORC/Cdc6 complex leads to the formation of OCCM Complex (fig 1C).
The formation of this complex is accompanied with ATP hydrolysis and the release of Cdc6 and Cdt1. The release of these two proteins leads to the formation of the ORC/MCM2–7 (OM) intermediate.
To the OM intermediate another Cdc6 molecule binds to form an ORC/Cdc6/MCM2–7 (OCM) complex (fig 1D), which can now bind another MCM2–7 hexamer.
As another molecule of MCM2–7 hexamer is brought to the OCM complex, the two hexamers are positioned to form a head-to-head MCM2–7 double hexamer (DH) and Cdc6, ORC and Cdt1 are released as still in G1 phase (fig 1E).
The DH encircles the ds DNA and results in the formation of the pre-replication complex (pre-RC) formation. The process of formation of the pre-RC is also called as DNA licensing. The pre-RC DH is inactive and unable to carry out the unwinding activities.
Pre-initiation complex (pre-IC) formation:
During S phase, the kinase called DDK phosphorylates and activates the MCM2–7 DHs. The complex of the proteins with activated MCM2-7 is called as preinitiation complex (pre-IC) formation (fig 1F).
The phosphorylation of DH exposes a Sld3-binding site on the Mcm2–7 complex. This favours the binding of the Sld3/Sld7 complex alongwith Cdc45 to replication origins.
S-phase-specific cyclin-dependent kinase (CDK) phosphorylates Sld2 and Sld3. This allows the protein DNA replication regulator Dpb11 to get recruited at the origin, which interacts with both the phosphoproteins, Sld2 and Sld3.
(Just for info: Read this research paper about interaction between Sld2 and Dpb11 in Saccharomyces cerevisiae.)
These three proteins Sld 2, Sld3 and Dpb11 form a platform for the binding of the accessory factors Cdc45 and GINS with Mcm2–7. The association of Cdc45 and GINS results in the formation of the Cdc45/MCM2–7/GINS (CMG) complex and Sld2, Sld3, and Dpb11 get disassociated.
Hence through a highly regulated process, the MCM2-7 double hexamer, which encircled the duplex DNA, is converted into two CMG particles, each encircling single-stranded DNA.
(Just for info: Have a look at the structure of CMG complex).
The completely assembled CMG complex bound with another protein Mcm10 is highly active in ATP hydrolysis-driven 3′–5′ DNA unwinding i.e. the helicase activity, and is the main component at the eukaryotic DNA replication fork (Fig 1G).
As the origin is unwound, the heterotrimeric single‐stranded DNA‐binding protein RPA or Replication protein A (RPA) stabilises The ss DNA. RPA can be defined as eukaryotic single-stranded DNA binding (SSB) protein.
(Just of info: Read more on Human RPA).
Eventually Pol ε interacts directly with the CMG through GINS and Pol α is loaded with help of the protein ctf4 on to the helicase. Pol α is associated with DNA primase to form a four subunit, the DNA Polymerase α-Primase complex, which can synthesize RNA primer and further DNA extensions.
(Just for info: Read the paper on additional functions of the DNA Polymerase α-Primase Complex.)
After the initial DNA synthesis by pol α- primase complex, the leading and lagging strand DNA synthesis is then taken over by the polymerase ε and polymerase δ, respectively.
(To know what are leading and lagging strands, read our previous post DNA Replication: Prokaryotes.)
We shall discuss about the further elongation of the lagging and the leading strands by the polymerases and the termination of the eukaryotic DNA in the next post.
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Also read other posts by The Biotech Notes:
Bacteriophage Reproduction: Lytic Cycle.
Riera et al. (2017) From structure to mechanism- understanding initiation of DNA replication. Genes Dev. 31(11): 1073-1088. doi: 10.1101/gad.298232.117
Bhagavan & Ha (2015) Chapter 22 – DNA Replication, Repair, and Mutagenesis. Essentials of Medical Biochemistry (Second Edition) With Clinical Cases. 401-417. https://doi.org/10.1016/B978-0-12-416687-5.00022-1
Tanaka et al. (2011) Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast. EMBO J. 30(10): 2019-30. doi: 10.1038/emboj.2011.115. Epub 2011 Apr 12.
Kamimura et al. (1998) Sld2, Which Interacts with Dpb11 in Saccharomyces cerevisiae, Is Required for Chromosomal DNA Replication. Mol Cell Biol. 1998 Oct; 18(10): 6102–6109.
Georgescu et al. (2017) Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation. PNAS 114 (5): E697-E706; first published January 17, 2017 https://doi.org/10.1073/pnas.1620500114.
Watase et al. (2012) Mcm10 Plays a Role in Functioning of the Eukaryotic Replicative DNA Helicase, Cdc45-Mcm-GINS. Current Biology 22(4) 343-349.