Eukaryotic Dna replication

In eukaryotes, the process of DNA replication is the same as that of the bacterial/prokaryotic

DNA replication with some minor modifications. In eukaryotes, the DNA molecules are larger

than in prokaryotes and are not circular; there are also usually multiple sites for the initiation of

replication.

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.

Thus, each eukaryotic chromosome is composed of many replicating units or replicons stretches

of DNA with a single origin of replication. In comparison, the E. coli chromosome forms only a

single replication fork. In eukaryotes, these replicating forks, which are numerous all along the

DNA, form "bubbles" in the DNA during replication. The replication fork forms at a specific

point called autonomously replicating sequences (ARS).The ARS contains a somewhat

degenerate 11-bp sequences called the origin replication element (ORE). The ORE is located

adjacent to an 80-bp AT rich sequence that is easy to unwind.

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.

In yeast, ORE is called as DUE (DNA unwinding element). Multiple origins allow eukaryotes to

replicate their larger quantities' of DNA in a relatively short time, even though eukaryotic DNA

replication is considerably slowed by the presence of histone proteins associated with the DNA

to form chromatin.

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.

Eukaryotes have clamp loader complex, similar to β subunit of pol of prokaryotes, and a six-unit

clamp called the proliferating cell nuclear antigen. The RNA primers are removed during

Okazaki fragment completion by mechanisms similar to those in Prokaryotes. In eukaryotes,

RNase enzymes remove the RNA primers in okazaki fragments; a repair polymerase fills gaps

and a DNA ligase forms the final seal.

Helicases, topoisomerase and single strand binding proteins play roles similar to that in

prokaryotes. All the enzymatic processes are generally the same in prokaryotes and eukaryotes.

DNA replication developed in prokaryotes, and was refined as prokaryotes evolved into

eukaryotes. The completion of the replication of linear eukaryotic chromosome involves the

formation of specialized structures at the tips of chromosomes.

Termination of replication in eukaryotes is different from that of prokaryotes. Eukaryotes have

linear chromosome, and once the first primer is removed from the strand, there is no known way

to fill in the gap, since DNA cannot be extended in the 3' -5 ' direction and there is no 3 ' end

upstream as there would be in a circle. If this were actually the situation, the DNA strands would

get shorter every time they replicated and genes would be lost forever

To avoid this condition, the cell has devised a system. The ends of chromosomes do not have

genes and instead, they are composed of many repeats of short, GC rich sequences. The exact

sequence of the repeat in a telomere is species-specific.

These repeats are added to the 3' end of DNA, not by Semiconservative replication, but by an

enzyme called telomerase. This enzyme has small RNA of 159-200 bp length which act as

template. The telomerase adds many repeated copies of its characteristic sequence to the 3' ends

of chromosome

Priming for synthesis of the opposite strand can then occur within these telomeres. Interestingly,

somatic cells lack telomerase while the germ cell retains the enzyme. Clearly, a picture of the

"replication apparatus" of eukaryotic organisms is beginning to emerge, but still there are many

things which need to be explored

The Enzymes : It is now clear that eukaryotic cells have mnor ethan a dozen DNA polymerases. Two of these (α and γ) are important for the replication of eukaryotic chromosomes. The rate of synthesis of DNA in eukaryotic cells is only 50 nt/s - about one tenth the rate of bacterial DNA synthesis.

DNA polymerase alpha (α)This enzyme is composed of 4 subunits, one of which (167 kDa) carries the polymerase activity. It has a low processivity and does not appear to have any proofreading activity. It is probably responsible for lagging strand synthesis.

DNA polymerase delta (γ)This enzyme contains at least 4 and maybe as many as a dozen subunits. It has a proofreading activity. When associated with proliferating cell nuclear antigen (PCNA), it has a very high processivity. This enzyme is probably responsible for leading strand synthesis.

DNA polymerase beta & DNA polymerase epsilonBoth enzymes are involved in DNA repair.

DNA polymerase gammaThis enzyme is located in the mitochondrion where it is responsible for replication of mtDNA.