DNA replication is the process of producing two identical replicas from one original DNA molecule
Eukaryotic DNA replication is a conserved mechanism that restricts DNA replication once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the duplication of a cell and is necessary for the maintenance of the eukaryotic genome
DNA replicates is the action of DNA polymerases synthesizing a DNA strand complementary to the original template strand. To synthesize a DNA, the double-stranded DNA is unwound by DNA helicases ahead of polymerase, forming a replication fork containing two single-stranded templates.
In G1 phase of the cell cycle, many of the DNA replication regulatory processes are initiated. In eukaryotes, the vast majority of DNA synthesis occurs during S phase of the cell cycle, and the entire genome must be unwound and duplicated to form two daughter copies. During G2 , any damaged DNA or replication errors are corrected. Finally, one copy of the genomes is Segregated to each daughter cell at mitosis or M phase. These daughter copies each contain one strand from the parental duplex DNA and one mascent antiparallel stand
The mechanism is conserved from prokaryotes to eukaryotes and is known as semiconservative DNA replication. The process of semiconservative replication for the site of DNA replication is a fork-like DNA structure, the replication fork, where the DNA helix is open or unwound, exposing unpaired DNA nucleotides into doubles stranded DNA.
3 Major steps in Eukaryotes DNA replication
1. Initiation
Initiation of eukaryotic DNA replication is the first stage of DNA synthesis where the DNA double helix is unwound and an initial priming event by DNA polymerase a occurs on the leading strand. The private event on the lagging strand establishes a replication fork. Priming of the DNA helix consists of synthesis of RNA primer to allow DNA synthesis by DNA polymerase a. Priming occurs once at the origin on the leading strand and at the start of each Okazaki Fragments on the lagging strand.
Pre-Replicating complex
Diagram below summarize the steps of formation of Pre-Replicative Complex.
The first step is the assembly of the Pre-Replicative Complex (pre-RC) is the binding of the origin Recognition Complex (ORC) to the replication origin.
Cdc6 protein
Binding of the cell division cycle 6 (Cdc6) protein to the origin recognition complex (ORC) is an essential step in the Assembly of the Pre-Replication complex (pre-RC) at the origins of replication. Cdc6 binds to the ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires ORC10 AT Pass. Cdc6 requires RC in order to associate with Chromatin and is in done required for the minichromosome maintenance proteins (Mcm2-7) to bind the Chromatin. The ORC-cdc6 complex form a ring-shaped structure and is analogous to other ATP-dependent protein machines. The level and activity of Cdc6 regulate the frequency with which the origins of replication are utilized during the cell cycle.
Cd1t protein
in fission yeast and Xenopus, Chromatin licensing and DNA replication factor 1 (Cdt1) protein is required for the licensing of chromatin for DNA replication. Cdt1 is essential for DNA replication and perform its role during formation of the pre- complex by loading the minichromosome maintenance protein (Mcm) onto the chromosome
Minichromosome maintenance protein complex
The Assembly of the minichromosome maintenance proteins function together as a complex in the cell. The assembly of the Mcm proteins onto chromatin requires the coordinated function of the origin recognition Complex ORC, Cdc6, and Cdt1. Vans the MCM proteins have been loaded on to the chromatin, ORC and Cdc6 can be removed from the chromatin without preventing subsequent DNA replication. This suggests that the primary role of the pre replication complex is to correctly load the MCM proteins. The Mcm proteins support roles both in the initiation and elongation steps of DNA synthesis.
Initiation complex
During the G1 stage of the cell cycle, the replication initiation factors, origin recognition complex (ORC), Cdc6, Cdt1, and minichromosome maintenance (Mcm) protein Complex, Bind sequential to DNA to form the pre-replication Complex (pre-RC).
DDK and CDK kinases
At the onset of S phase, the pre-replicative complex must be activated by two S phase-specific kinases in order to form an initiation complex at the origin of replication. One kinase is the Cdc7- Dbf4 kinase called Dbf4 dependent kinase (DDK) and the other is cyclin-dependent Kinase (CDK).
2. Elongation
During elevation, an enzyme called DNA polymerase adds DNA nucleotides to the 3’ end of the newly synthesized polynucleotide strand. The template strand specifies which of the four DNA nucleotides (A, T, C or G) is added at each position along the new chain. Only the nucleotide complementary to the template nucleotide at that position is added to the new strand.
Replication fork
The replication fork is the junction between the newly separated templates strands, known as the leading and lagging strands, and the double stranded DNA.
3. Termination
Eukaryotic chromosomes have multiple origins of replication, which initiate replication almost simultaneously. Each origin of replication forms a bubble of duplicated DNA on either side of the origin of replication. Eventually, the leading strand of one replication bubble reaches the lagging strand of another bubble, and the lagging strand will reach the 5’ end of the previous Okazaki Fragments in the same bubble.
DNA polymerase halts when it reaches a section of DNA template that has already been replicated. However, DNA polymerase cannot catalyze formation of a phosphodiester bond between the two segments of the new DNA strand, and it draws drops off. These unattached sections of the sugar-phosphate backbone in an otherwise full-replicated DNA strand are called nicks.
Once all the template nucleotide has been replicated, the replication process is not yet over. RNA primer needs to be replaced with DNA, and nicks in the sugar-phosphate backbone need to be connected.
Cell cycle regulation for Eukaryotic cells
DNA replication is a tightly orchestrated process that is controlled within the context of the cell cycle. Progress through the cell cycle and in turn DNA replication is tightly regulated by the formation and the activation of Pre-Replicative Complex (pre-RC) which is achieved through the activation and inactivation of cyclin-dependent kinases (Cdks). Specifically, it is the interactions of cyclins and cyclin-dependent kinases that are responsible for the transition from G1 into S phase.
During the G1 phase of the cell cycle there are no levels of cdk activity. This low level of cdk activity allows for the formation of new pre-RC complexes but it is not sufficient for DNA replication to be initiated by the newly formed pre-RCs. During the remaining phases of the cell cycle there are elevated levels of cdk activity. This high level of cdk activity is responsible for initiating DNA replication as well as inhibiting new pre-RC complex formation. Once DNA replication has been initiated the pre-RC complex is broken down. Due to the fact that cdk levels remain high during the S phase, G2, and M phases of the cell cycle no jet pre-RC complexes can be formed. This all helps to ensure that no initiation can occur until the cell division is complete.