Eukaryotic DNA replication is a highly regulated and complex process that ensures the accurate duplication of the entire genome.

1. Process of Replication in Eukaryotes
Eukaryotic DNA replication is a highly regulated and complex process that
ensures the accurate duplication of the entire genome. The replication
occurs during the S phase of the cell cycle and involves several key steps:
1. Initiation
 Origin Recognition: DNA replication begins at specific sequences
called origins of replication. Eukaryotic chromosomes contain multiple
origins to allow rapid replication of large genomes.
 Pre-replication Complex Formation (Pre-RC): During the G1 phase,
the Origin Recognition Complex (ORC) binds to the origin, recruiting
other proteins such as Cdc6, Cdt1, and the Mini-Chromosome
Maintenance (MCM) complex (a helicase). This forms the pre-
replication complex.
 Activation of Replication: In the S phase, cyclin-dependent kinases
(CDKs) and Dbf4-dependent kinase (DDK) activate the MCM helicase,
allowing it to unwind DNA, creating replication forks. This marks the
transition from the pre-replicative to the replicative state.
2. Unwinding of DNA
 Helicase Activity: The MCM helicase unwinds the double-stranded
DNA into two single strands, creating a replication fork.
 Single-Strand Binding Proteins (RPA): Replication Protein A (RPA)
binds to the single-stranded DNA to prevent it from reannealing or
forming secondary structures.
3. Primer Synthesis
 Primase: A primase, a component of the DNA polymerase α complex,
synthesizes a short RNA primer complementary to the single-stranded
DNA. This primer provides a 3’ hydroxyl group for the addition of
nucleotides by DNA polymerases.
4. Elongation
 Leading Strand Synthesis: On the leading strand, DNA polymerase ε
synthesizes DNA continuously in the 5’ to 3’ direction, following the
replication fork.

2.  Lagging Strand Synthesis: On the lagging strand, DNA polymerase δ
synthesizes DNA in short fragments called Okazaki fragments. Each
fragment is initiated by an RNA primer synthesized by DNA
polymerase α, and the DNA is synthesized in the 5’ to 3’ direction away
from the replication fork.
5. Removal of RNA Primers and Ligation
 RNase H and FEN1: RNA primers are removed by RNase H and Flap
Endonuclease 1 (FEN1).
 DNA Polymerase δ: After the removal of RNA primers, DNA
polymerase δ fills the gaps with DNA nucleotides.
 DNA Ligase: DNA ligase seals the nicks between the Okazaki
fragments, forming a continuous DNA strand.
6. Termination
 Replication continues until two replication forks meet, or the replication
machinery reaches the end of the chromosome (telomeres).
 Telomere Replication: The enzyme telomerase adds repetitive
sequences to the ends of chromosomes (telomeres) to prevent loss of
important genetic information due to the end-replication problem.
Telomerase contains an RNA template and acts as a reverse
transcriptase.
7. Proofreading and Error Correction
 Proofreading: DNA polymerases have proofreading activity (3’ to 5’
exonuclease activity) that allows them to remove incorrectly paired
nucleotides immediately after they are added.
 Mismatch Repair: After replication, mismatch repair enzymes correct
any errors that escaped the proofreading mechanism.
8. Regulation of Replication
 Cell Cycle Control: The initiation of replication is tightly regulated by
the cell cycle. CDKs and other cell cycle regulators ensure that
replication occurs only once per cell cycle.
 Checkpoints: DNA damage checkpoints, such as the G1/S and G2/M
checkpoints, monitor the integrity of the DNA before and after

3. replication. If DNA damage is detected, the replication process can be
paused to allow repair.
Key Enzymes and Proteins
 Origin Recognition Complex (ORC)
 Cdc6 and Cdt1
 MCM Helicase
 DNA Polymerases α, δ, and ε
 Primase
 RPA (Replication Protein A)
 RNase H
 FEN1 (Flap Endonuclease 1)
 DNA Ligase
 Telomerase
Important Features of Eukaryotic DNA Replication
 Multiple Origins of Replication: Eukaryotic chromosomes have
multiple origins of replication to ensure that the entire genome is
replicated efficiently.
 Linear Chromosomes: Eukaryotic chromosomes are linear, unlike the
circular chromosomes of prokaryotes, leading to specific challenges at
the chromosome ends (telomeres).
 Replication Licensing: Eukaryotic cells ensure that replication occurs
only once per cell cycle by tightly regulating the assembly of the pre-
replication complex.
This process ensures that eukaryotic cells accurately duplicate their DNA in
preparation for cell division.