It is divided into 2 stages – G1 phase where initiation of replication takes place. Cdk activity occurs in this phase. S phase is the stage where actual DNA replication occurs
Initiation of replication takes place at specific region called ARS – Autonomously Replicating Sequences which includes the origin of replication of 100bp sequences
The locally denatured segment of DNA at the ARS is called replication bubble and the 2 single strands of DNA at the bubble on which new complementary strands are formed are called template strands
As the DNA unwinds, a Y-shaped structure is formed that remains linked to the replication bubble. It is called as the replication fork. There are many replication forks in a linear eukaryotic chromosome due to the presence of multi-ARS regions. In such cases, bidirectional replication takes place
Initiation of replication is done by an initiator protein called ORC (Origin Recognition Complex) that binds the DNA and starts unwinding it
MCM loader is loaded by CDT1 which unwinds the remaining DNA helix
RPA – Replication protein A binds to the two separated strands of DNA that stabilizes them and prevents them from reforming double stranded DNA. Gyrase releases tension on the DNA strands generated by rapid unwinding of the DNA
The DNA strand along with ORC, RPA, MCM loader and CDT1 forms the pre-replicative complex (pre-RC)
Replication begins in the S phase. DNA pol A/primase synthesizes the RNA primer (a nucleotide sequence of 20-30 nucleotides) that initiates the DNA replication by providing 3’ –OH end to the DNA pol δ. However, the DNA pol α/primase extends the primer as a short stretch of DNA of 200 nucleotides beyond the length of the primer and subsequently dissociates
DNA pol δ catalyzes DNA synthesis. The sliding clamp in this case is called PCNA (proliferating cell nuclear antigen) that is loaded by RFC (Replication factor C). It adds about 75 nucleotides per second
The strand that synthesizes continuously in 5’ to 3’ direction is called the leading strand while the strand synthesized in the opposite direction is called lagging strand. The leading strand requires one primer whereas the lagging strand requires several primers
After the synthesis of the strands is completed, RNase H1 removes the primer while DNA pol ε fills up the gap with DNA. Eventually, DNA ligase 1 joins the nicks by forming a phosphodiester bond except for one last nucleotide of the primer. This nucleotide is removed later by an enzyme called FEN 1 (Flap endonuclease)
Depiction of bidirectional replication from multiple ARSEukaryotic replication fork
End Replication of linear chromosomes
When all the primers are removed after DNA replication, there is always a problem of replicating the ends of the newly synthesized DNA as there are no further primers to extend the DNA and fill up the gaps
This is accomplished by an enzyme called telomerase. It has 2 components –
Protein component that is involved in the telomere synthesis
RNA component that acts as the template for the telomere DNA synthesis
The protein component – TERT stands for Telomerase Reverse Transcriptase as this enzyme telomerase is involved in reverse transcription (ie., synthesis of DNA from RNA)
The telomerase tethers to the 3’ end of the parent DNA strand. It extends this DNA strand by adding new nucleotides to it in the sequence – TTAGGG. This is repeated for a number of times
After the template DNA strand has been sufficiently lengthened, the complementary strand to it is synthesized by the normal replication mechanism. Primer is synthesized subsequently followed by the extension of the primer of DNA pol α/primase, DNA pol δ, DNA pol ε, followed by DNA ligase that joins the nicks in DNA backbone
However, after removing the final primer, the overhang still remains but the total length of the chromosome is restored
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