DNA Replication in Eukaryotes

The process of DNA replication involves duplication of DNA. It occurs by a semiconservative way in both prokaryotes and eukaryotes. There are specific chromosomal sequences found in the yeast Saccharomyces cerevisiae. Approximately 100 bp sequences are present in the yeast. These sequences can replicate autonomously. Such sequences are known as autonomously replicating sequences (ARS). There are three sequence elements known as A, B1, and B2. The origin recognition complex (ORC) is the initiator protein with a multi-subunit. The ORC binds to A and B1 in the yeast and recruits other proteins such as B2 DNA unwinding proteins. Between the B1 and B2 lies the replication origin. DNA replication occurs in the S phase of the cell cycle. The origin of replication is not used more than once in the cell cycle. There is a specific temporal ordering of initiation of replication. The replicator selection occurs in the G1 phase by assembling proteins on each of the replicator. They form pre-replicative complexes. Then the ORC recruits other proteins. Once the cell reaches the S phase of its cycle, the pre-replicative complexes get activated for initiating the replication. Cyclin-Dependent Kinase (CDK) enzyme activates the pre-RC to initiate the cell cycle. They also inhibit the formation of new pre-RCs. Except in the G1 stage, CDKs are present in all the phases of the cell cycle.

Image: DNA replication in eukaryotes

Eukaryotic replication enzymes:

Like prokaryotes, eukaryotes also require enzymes for replicating the DNA. There are more than 15 replicative enzymes in the eukaryotes. They are known as DNA polymerases. Pol α (Primase) is an enzyme that initiates new strands in replication by primase. It makes about ten nucleotides of an RNA which are further extended by Pol α. Formation of RNA-DNA primers occurs. Pol δ and Pol ε further extend these primers. Some of the functions of the replication enzymes of the eukaryotes are still not clear. There is no evidence about the role of specific enzymes in the synthesis of the leading and the lagging strand. Some other enzymes are involved in the replication of organelle DNA such as mitochondrial and chloroplast DNA. The mitochondrial DNA  replication follows a D-loop model. DNA polymerase γ is mainly involved in mitochondrial DNA replication. Another eukaryotic DNA polymerase is known as DNA Pol κ. The cohesion proteins hold the sister chromatids together until the anaphase of the nuclear division. The DNA Pol κ helps in the attachment of the cohesion proteins. DNA Pol δ works in synergy with an accessory protein known as proliferating cell nuclear antigen (PCNA), which is a functional equivalent of a β subunit of E.coli Polymerase III. PCNA holds the enzyme lightly to the template DNA.

The eukaryotic replication fork:

The DNA Pol α extends the initial RNA primer. Polymerase δ further replaces the Pol α. An enzyme is known as flap endonuclease (FEN1). It associates with the DNA Pol δ at the 3’ end of the Okazaki fragment. The primer from the 5’ end of the adjacent fragment gets degraded. However, the extreme 5’ end consists of a phosphate group which blocks the activity of the FEN 1. 
There are two models for studying the completion of the lagging strand in the eukaryotes.

a.     RNase H model: RNase H can remove the primer just to the last ribonucleotide. However, it cannot cleave the phosphodiester bond between the last ribonucleotide and the first deoxyribonucleotide. This ribonucleotide carries a 5’- monophosphate which can be removed by FEN-1. Thus the FEN-1 removes the last ribonucleotide and some of the DNA. The eukaryotic cell is capable of replicating DNA even in the absence of RNase H. Thus the discovery of the flap model came into existence.

b.    Flap model: The DNA Polymerase δ combines with the helicases for extending the Okazaki fragment. While the extension of the Okazaki fragment, the primer gets pushed aside. The extension of the exposed region occurs. A flap arises as a result of primer removal. FEN1 cuts this flap due to its endonuclease activity. FEN1 cleaves the phosphodiester bond at the branch point. Through the study of flap model, a few pieces of evidence revealed that the DNA Pol α may synthesize DNA in an error-prone way. DNA Pol α does not have 3’to 5’ proofreading activity. The helicase activity not only removes the primer but also eliminates the DNA that was originally synthesized by the DNA Pol α. On the other side, DNA Polymerase δ has a proofreading activity. Thus, it makes a highly accurate copy of a template.

There are certain problems in replicating the ends (telomeres) of the eukaryotic chromosomes. The parental DNA replicates and gives rise to two new DNA molecules. Each daughter DNA has an RNA primer at the telomere region. It is mainly at the 5’ end. The primase activity leads to the elimination of RNA primers. The primer removal leaves a single-strand stretch of DNA, thereby creating the gap at the 5’ end. DNA Polymerases cannot fill this gap. Thus the gaps may remain as they are. The leftover gaps may reduce the size of the chromosome after each replication. The process of aging is mainly due to the telomere shortening. The telomere replication has a specific mechanism. Eukaryotic chromosomes have tandem repeats at the telomeres.

Study of telomere replication in Tetrahymena:

The Tetrahymena is a protozoan. It has a repeated sequence of 5’-TTGGGG-3’ reading towards the DNA ends on the top strands. An enzyme known as telomerase contains protein and RNA subunits. The telomerase enzyme is responsible for maintaining the chromosome lengths. It adds repeats to the telomeres. Its RNA component has a sequence complementary to the telomere. The telomerase binds to the overhanging sequence and catalyzes the synthesis of three nucleotides TTG using its RNA component. The telomerase then slides towards the end of the chromosome. Its AAC region pairs with the newly synthesized TTG on the DNA. The rest of the telomere repeat is synthesized later on.

The Human DNA Replication:

The exact replication in human DNA is not extensively studied. However, few studies have described human DNA replication. The timing of the DNA replication varies in humans. Next to the lamin B2 gene on chromosome 19 is the origin of replication.

References:

[1] IGenetics, Peter Russell, second edition
[2] Eukaryotic DNA replication - Wikipedia

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