Eukaryotic DNA replication

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Although the mechanisms of DNA synthesis in eukaryotes and prokaryotes are similar, DNA replication in eukaryotes is much more complicated. Though DNA synthesis in prokaryotes such as E. coli is regulated, DNA replication is initiated before the end of the cell cycle. Eukaryotic cells can only initiate DNA replication at a specific point in the cell cycle, the beginning of S phase.

DNA replication in eukaryotes occurs only in the S phase of the cell cycle. However, pre-initiation occurs in the G1 phase. Due to the sheer size of chromosomes in eukaryotes, eukaryotic chromosomes contain multiple origins of replication. Some origins are well characterized, such as the autonomously replicating sequences (ARS) of yeast while other eukaryotic origins, particularly those in metazoa, can be found in spans of thousands of basepairs. However, the assembly and initiation of replicaton is similar in both the protozoa and metazoa.

The first step in DNA replication is the formation of the pre-initiation replication complex (the pre-RC). The formation of this complex occurs in two stages. The first stage requires that there is no CDK activity. This can only occur in early G1. The formation of the pre-RC is known as licensing, but a licensed pre-RC cannot initiate replication. Initiation of replication can only occur during the S-phase. Thus, the separation of licensing and activation ensures that the origin can only fire once per cell cycle.

DNA replication in eukaryotes is not very well characterized. However, researchers believe that it begins with the binding of the origin recognition complex (ORC) to the origin. This complex is a hexamer of related proteins and remains bound to the origin, even after DNA replication occurs. Furthermore, ORC is the functional analogue of DnaA. Following the binding of ORC to the origin, Cdc6/Cdc18 and Cdt1 coordinate the loading of the MCM (minichromosome maintenance functions) complex to the origin by first binding to ORC and then binding to the MCM complex. The MCM complex is thought to be the major DNA helicase in eukaryotic organisms, and is a hexamer (mcm2-7). Once binding of MCM occurs, a fully licensed pre-RC exists.

Activation of the complex occurs in S-phase and requires Cdk2-Cyclin E and Ddk. The activation process begins with the addition of Mcm10 to the pre-RC, which displaces Cdt1. Following this, Ddk phosphorylates Mcm3-7, which activates the helicase. It is believed that ORC and Cdc6/18 are phosphorylated by Cdk2-Cyclin E. Ddk and the Cdk complex then recruits another protein called Cdc45, which then recruits all of the DNA replication proteins to the replication fork. At this stage the origin fires and DNA synthesis begins.

Activation of a new round of replication is prevented through the actions of the cyclin dependent kinases and a protein known as geminin. Geminin binds to Cdt1 and sequesters it. It is a periodic protein that first appears in S-phase and is degraded in late M-phase, possibly through the action of the anaphase promoting complex (APC). In addition, phosphorylation of Cdc6/18 prevent it from binding to the ORC (thus inhibiting loading of the MCM complex) while the phosphorylation of ORC remains unclear. Cells in the G0 stage of the cell cycle are prevented from initiating a round of replication because the Mcm proteins are not expressed.

Numerous polymerases can replicate DNA in eukaryotic cells. Currently, six families of polymerases (A, B, C, D, X, Y) have been discovered. At least four different types of DNA polymerases are involved in the replication of DNA in animal cells (POLA, POLG, POLD1 and POLE). POL1 functions by extending the primer in the 5' -> 3' direction and tightly associates with primase. However, it lacks the ability to proofread DNA. POLD1 has a proofreading ability and is able to replicate the entire length of a template only when associated with PCNA. POLE is able to replicate the entire length of a template in the absence of PCNA and is able to proofread DNA while POLG replicates mitochondrial DNA via the D-Loop mechanism of DNA replication. All primers are removed by RNaseH1 and Flap Endonuclease I. The general mechanisms of DNA replication on the leading and lagging strand, however, are the same as to those found in prokaryotic cells.