DNA Replication in Eukaryotes
DNA replication is initiated from specific sequences called origins of replication, and eukaryotic cells have multiple replication origins. To initiate DNA replication, multiple replicative proteins assemble on and dissociate from these replicative origins. The individual factors described below work together to direct the formation of the pre-replication complex (pre-RC), a key intermediate in the replication initiation process.
Association of the origin recognition complex (ORC) with a replication origin is required to recruit both cell division cycle 6 protein (Cdc6) and chromatin licensing and DNA replication factor 1 protein (Cdt1), which initiate the assembly of the pre-RC. Both Cdc6 and Cdt1 proteins associate with the already bound ORC independently from each other. The ORC, Cdc6, and Cdt1 together are required for the stable association of the minichromosome maintenance (Mcm 2-7) complex proteins with replicative origins during G
1 phase of the cell cycle.
Pre-replicative Complex
Eukaryotic origins of replication control the formation of a number of protein complexes that lead to the assembly of two bidirectional DNA replication forks. These events are initiated by the formation of the pre-replication complex (pre-RC) at the origins of replication. This process takes place in the G
1
stage of the cell cycle. The pre-RC formation involves the ordered assembly of many replication factors including the origin recognition complex (ORC), Cdc6 protein, Cdt1 protein, and minichromosome maintenance proteins (Mcm2-7). Once the pre-RC is formed, activation of the complex is triggered by two kinases, cyclin-dependent kinase 2 (CDK) and Dbf4-dependent kinase (DDK) that help transition the pre-RC to the initiation complex prior to the initiation of DNA replication. This transition involves the ordered assembly of additional replication factors to unwind the DNA and accumulate the multiple eukaryotic DNA polymerases around the unwound DNA.
Origin Recognition Complex
The first step in the assembly of the pre-replication complex (pre-RC) is the binding of the origin recognition complex (ORC) to the replication origin. In late mitosis, Cdc6 protein joins the bound ORC followed by the binding of the Cdt1 protein. ORC, Cdc6, and Cdt1 are all required to load the six protein minichromosome maintenance (Mcm 2-7) complex onto the DNA.

When the ORC binds to DNA at replication origins, it then serves as a scaffold for the assembly of other key initiation factors of the pre-replicative complex, which includes Cdc6, Cdt1, and minichromosome maintenance (Mcm 2-7) complex proteins.
A six-subunit complex called the origin recognition complex (ORC) serves as a platform for the assembly of pre-replication complexes. ORC binds yeast replicators throughout the cell cycle; in metazoans, the binding of the large subunit of ORC, ORC1, to chromatin is cell-cycle regulated.
During the mitosis–G1-phase transition, chromatin-bound ORC recruits CDC6 and CDT1, which facilitate the loading of a helicase complex consisting of MCM (minichromosome maintenance) proteins 2–7 (licensing). The resulting complex is termed the pre-replication complex (PreRC).
The PreRC is activated to create the pre-initiation complex (PreIC) by recruitment of additional factors including CDC45, SLD2–3, DPB11, the GINS complex (SLD1 and PSF1–3) and
MCM10. The initiation of DNA replication during S phase requires phosphorylation of components of the PreRC by cyclin-dependent kinases and DBF4- dependent kinase, and the

recruitment of DNA polymerases (Pol) to the PreRC in order to form the pre-initiation complex
(PreIC). After replication (PostRC), all the components of the pre-IC except for some ORC subunits leave the chromatin
Elongation
The formation of the pre-replicative complex (pre-RC) marks the potential sites for the initiation of DNA replication. Consistent with the minichromosome maintenance complex encircling double stranded DNA, formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead, the pre-RC that is formed during the G
1
of the cell cycle is only activated to unwind the DNA and initiate replication after the cells pass from the G
1
to the S phase of the cell cycle.
Once the initiation complex is formed and the cells pass into the S phase, the complex then becomes a replisome. The eukaryotic replisome complex is responsible for coordinating DNA replication. Replication on the leading and lagging strands is performed by DNA polymerase ε and DNA polymerase δ. Many replisome factors including Claspin, And1, replication factor C clamp loader and the fork protection complex are responsible for regulating polymerase functions and coordinating DNA synthesis with the unwinding of the template strand by Cdc45-
Mcm-GINS complex. As the DNA is unwound the twist number decreases. To compensate for this the writhe number increases, introducing positive supercoils in the DNA. These supercoils would cause DNA replication to halt if they were not removed. Topoisomerases are responsible for removing these supercoils ahead of the replication fork.
The replisome is responsible for copying the entire genomic DNA in each proliferative cell. The base pairing and chain formation reactions, which form the daughter helix, are catalyzed by
DNA polymerases. These enzymes move along single-stranded DNA and allow for the extension of the nascent DNA strand by "reading" the template strand and allowing for incorporation of the proper purine nucleobases, adenine and guanine, and pyrimidine nucleobases, thymine and cytosine. Activated free deoxyribonucleotides exist in the cell as deoxyribonucleotide triphosphates (dNTPs). These free nucleotides are added to an exposed 3'-hydroxyl group on the last incorporated nucleotide. In this reaction, a pyrophosphate is released from the free dNTP, generating energy for the polymerization reaction and exposing the 5' monophosphate, which is then covalently bonded to the 3' oxygen. Additionally, incorrectly inserted nucleotides can be removed and replaced by the correct nucleotides in an

energetically favorable reaction. This property is vital to proper proofreading and repair of errors that occur during DNA replication.
Figure 2.
The eukaryotic replisome complex coordinates DNA replication. Replication on the leading and lagging strands is performed by Pol ε and Pol δ, respectively. Many replisome factors (including the FPC [fork protection complex], Claspin, And1, and RFC [the replication factor C clamp loader]) are charged with regulating polymerase functions and coordinating DNA synthesis with unwinding of the template strand by Cdc45-MCM [mini-chromosome maintenance]-GINS [go-ichi-ni-san]. The replisome also associates with checkpoint proteins as
DNA replication and genome integrity surveillance mechanisms.
Replicative DNA Polymerases fter the replicative helicase has unwound the parental DNA duplex, exposing two single-stranded
DNA templates, replicative polymerases are needed to generate two copies of the parental genome. DNA polymerase function is highly specialized and accomplish replication on specific templates and in narrow localizations. At the eukaryotic replication fork, there are three distinct replicative polymerase complexes that contribute to DNA replication: Polymerase α, Polymerase
δ, and Polymerase ε. These three polymerases are essential for viability of the cell.
Because DNA polymerases require a primer on which to begin DNA synthesis, polymerase α
(Pol α) acts as a replicative primase. Pol α is associated with an RNA primase and this complex accomplishes the priming task by synthesizing a primer that contains a short 10 nucleotide

stretch of RNA followed by 10 to 20 DNA bases.
[3]
Importantly, this priming action occurs at replication initiation at origins to begin leading-strand synthesis and also at the 5' end of each
Okazaki fragment on the lagging strand.
However, Pol α is not able to continue DNA replication and must be replaced with another polymerase to continue DNA synthesis. Polymerase switching requires clamp loaders and it has been proven that normal DNA replication requires the coordinated actions of all three DNA polymerases: Pol α for priming synthesis, Pol ε for leading-strand replication, and the Pol δ, which is constantly loaded, for generating Okazaki fragments during lagging-strand synthesis.
[68]
 Polymerase α (Pol α)
: Forms a complex with a small catalytic subunit (PriS) and a large noncatalytic (PriL) subunit. First, synthesis of an RNA primer allows DNA synthesis by
DNA polymerase alpha. Occurs once at the origin on the leading strand and at the start of each Okazaki fragment on the lagging strand. Pri subunits act as a primase, synthesizing an
RNA primer. DNA Pol α elongates the newly formed primer with DNA nucleotides. After around 20 nucleotides, elongation is taken over by Pol ε on the leading strand and Pol δ on
 the lagging strand.
Polymerase δ (Pol δ)
: Highly processive and has proofreading, 3'->5', exonuclease activity.
The main polymerase involved in lagging strand synthesis.
 Polymerase ε (Pol ε)
: Highly processive and has proofreading, 3'->5', exonuclease activity.
Highly related to pol δ, and is the main polymerase involved in leading strand synthesis
Proliferating Cell Nuclear Antigen
DNA polymerases require additional factors to support DNA replication. DNA polymerases have a semiclosed 'hand' structure, which allows the polymerase to load onto the DNA and begin translocating. This structure permits DNA polymerase to hold the single-stranded DNA template, incorporate dNTPs at the active site, and release the newly formed double-stranded DNA.
However, the structure of DNA polymerases does not allow a continuous stable interaction with the template DNA.
To strengthen the interaction between the polymerase and the template DNA, DNA sliding clamps associate with the polymerase to promote the processivity of the replicative polymerase.
In eukaryotes, the sliding clamp is known as the proliferating cell nuclear antigen (PCNA).
PCNA-dependent stabilization of DNA polymerases has a significant effect on DNA replication because PCNAs are able to enhance the polymerase processivity up to 1,000-fold. PCNA is an

essential cofactor and has the distinction of being one of the most common interaction platforms in the replisome to accommodate multiple processes at the replication fork, and so PCNA is also viewed as a regulatory cofactor for DNA polymerases.
Replication Factor C
PCNA fully encircles the DNA template strand and must be loaded onto DNA at the replication fork. At the leading strand, loading of the PCNA is an infrequent process, because DNA replication on the leading strand is continuous until replication is terminated. However, at the lagging strand, DNA polymerase δ needs to be continually loaded at the start of each Okazaki fragment. This constant initiation of Okazaki fragment synthesis requires repeated PCNA loading for efficient DNA replication.
PCNA loading is accomplished by the replication factor C (RFC) complex. The PCNA is opened by RFC by and then loaded onto DNA in the proper orientation to facilitate its association with the polymerase. Clamp loaders can also unload PNCA from DNA; a mechanism needed when replication must be terminated.
Termination
Synthesis of the lagging strand requires a 5´
3´ 5´
3´ short primer which will be removed. At the extreme end of a chromosome, there is no
Telomerase way to synthesize this region when the last primer is removed. Therefore, the lagging
5´
3´ 5´
3´ strand is always shorter than its template
DNA synthesis by at least the length of the primer. This is the so-called " end-replication problem ".
5´
3´ 5´
To extend the length of a telomere, the
3´
Bacteria do not have the end-replication problem, because its DNA is circular. In telomerase first extends its longer strand. Then, eukaryotes, the chromosome ends are using the same mechanism as synthesizing the called telomeres which have at least two lagging strand, the shorter strand is extended. functions:

 to protect chromosomes from fusing with each other. to solve the end-replication problem

A depiction of telomerase progressively elongating telomeric DNA.