DNA Replication in Eukaryotes

The replication in prokaryotic cell is much simpler than that of the eukaryotic cell. The
eukaryotic DNA is much larger, more condensed and linear as compared to prokaryotic DNA.
Also the eukaryotic replication occurs in a separate phase of the cell cycle known
as S (synthesis) phase and has to be well regulated unlike prokaryotic cell where the DNA
replication can take place continuously during growth. However, as in prokaryotes, the DNA
replication process in eukaryotes, can be divided into three
stages: Initiation, Elongation and Termination.

Initiation of DNA replication:

Origin:

As seen in the last post, the replication begins at a particular site in the genome known
as origin. The replication origin have conserved DNA sequences. As the eukaryotic
genome is much larger than the prokaryotic genome, there are hundreds of origin in
eukaryotic genome. Hence the replication can begin simultaneously and complete the
replication faster. Origin has the sequence to which the ORC binds.

The origin recognition complex (ORC):

ORC or the origin recognition complex is a complex of six subunits arranged in a C-

shaped structure encircling the DNA at origin (fig 1A). This complex binds and recruits
other factors to establish a pre-replicative complex (pre-RC).

ORC/Cdc6/DNA complex:

Cell division cycle 6 protein or Cdc6 binds the ORC/origin DNA complex, during late
M phase of the cell cycle (fig 1B). ATP binding is required for ORC/Cdc6/DNA
complex formation.
OCCM Complex:

Cdt1/MCM2–7 heptamer then binds the ORC/Cdc6 complex and loads MCM2–
7 onto the dsDNA. MCM2–7, the minichromosome maintenance 2–7 consists of six
spirally arranged subunits which forms the core of the replicative DNA helicase.
Cdt1 or the chromatin licensing and DNA replication factor 1 stabilises a single
Mcm2-7 hexamer and loads it onto the chromosome.

The binding of Cdt1/MCM2–7 to ORC/Cdc6 complex leads to the formation

of OCCM Complex (fig 1C).

OM intermediate:

The formation of this complex is accompanied with ATP hydrolysis and the release
of Cdc6 and Cdt1. The release of these two proteins leads to the formation of
the ORC/MCM2–7 (OM) intermediate.

OCM complex:

To the OM intermediate another Cdc6 molecule binds to form an ORC/Cdc6/MCM2–

7 (OCM) complex (fig 1D), which can now bind another MCM2–7 hexamer.

Double hexamer:

As another molecule of MCM2–7 hexamer is brought to the OCM complex, the two
hexamers are positioned to form a head-to-head MCM2–7 double hexamer (DH)
and Cdc6, ORC and Cdt1 are released as still in G1 phase (fig 1E).

Pre-replication complex:

The DH encircles the ds DNA and results in the formation of the pre-
replication complex (pre-RC) formation. The process of formation of the pre-RC is
also called as DNA licensing. The pre-RC DH is inactive and unable to carry out the
unwinding activities.

Pre-initiation complex (pre-IC) formation:

During S phase, the kinase called DDK phosphorylates and activates the MCM2–7
DHs. The complex of the proteins with activated MCM2-7 is called as preinitiation
complex (pre-IC) formation (fig 1F).

Fig 1: Eukaryotic initiation of DNA replication (Riera et al., 2017)

CMG complex:
The phosphorylation of DH exposes a Sld3-binding site on the Mcm2–7 complex. This
favours the binding of the Sld3/Sld7 complex along with Cdc45 to replication origins.

Fig 2: Unwinding of DNA. (Watase et al., 2012)

S-phase-specific cyclin-dependent kinase (CDK) phosphorylates Sld2 and Sld3. This
allows the protein DNA replication regulator Dpb11 to get recruited at the origin, which
interacts with both the phosphoproteins, Sld2 and Sld3.

These three proteins Sld 2, Sld3 and Dpb11 form a platform for the binding of the
accessory factors Cdc45 and GINS with Mcm2–7. The association of Cdc45 and GINS
results in the formation of the Cdc45/MCM2–7/GINS (CMG) complex and Sld2,
Sld3, and Dpb11 get disassociated.
Fig 1: Structures of the budding yeast OCCM, DH, and CMG (CMG bound to
a replication fork) (Riera et al., 2017).
Hence through a highly regulated process, the MCM2-7 double hexamer, which
encircled the duplex DNA, is converted into two CMG particles, each encircling single-
stranded DNA.

The completely assembled CMG complex bound with another protein Mcm10 is
highly active in ATP hydrolysis-driven 3′–5′ DNA unwinding i.e. the helicase activity,
and is the main component at the eukaryotic DNA replication fork (Fig 1G).

As the origin is unwound, the heterotrimeric single‐stranded DNA‐binding

protein RPA or Replication protein A (RPA) stabilises the ss DNA. RPA can be defined
as eukaryotic single-stranded DNA binding (SSB) protein.

Eventually Pol ε interacts directly with the CMG through GINS and Pol α is loaded
with help of the protein ctf4 on to the helicase. Pol α is associated with DNA primase to
form a four subunit, the DNA Polymerase α-Primase complex, which
can synthesize RNA primer and further DNA extensions.

DNA Polymerase α-Primase Complex

After the initial DNA synthesis by pol α- primase complex, the leading and lagging
strand DNA synthesis is then taken over by the polymerase ε and polymerase δ,
respectively.

Eukaryotic Elongation and Termination

Eukaryotic replication bubble

During Initiation, a repertoire of proteins bind and unwind the DNA at the origin. To
this protein complex the polymerases are loaded. The polymerases work together with
other proteins for the elongation of the daughter strands.

Elongation

The first polymerase to initiate the DNA synthesis is the DNA polymerase α, which
exists in the form of DNA polymerase α-primase complex. The primase subunit
synthesizes the RNA primers (around 7-12 nucleotides) which are then transferred to
the polymerase domain and extended with DNA bases (around 20-25
nucleotides). Replication factor C (RFC) initiates a reaction called polymerase
switching. The DNA strands are then extended by other polymerases namely; the DNA
polymerase δ and DNA polymerase ε.

Leading strand synthesis:

Fig 1: Synthesis of leading or continuous strand
As DNA pol α completes synthesizing RNA primer and adding DNA bases,
the RFC causes dissociation of DNA pol α and assembles proliferating cell nuclear
antigen (PCNA) in the region of the primer terminus. PCNA is a DNA clamp for DNA
polymerases.

Pol ε has been reported as the main leading strand synthesis polymerase
(in Saccharomyces cerevisiae). In the leading strand as the replication is continuous and
the primer is synthesized only once and the extension is carried out (fig 1).
Lagging strand synthesis:

Fig 2: Synthesis of lagging or discontinuous strand with series of Okazaki

fragments.
Polymerases δ is the major polymerase in lagging-strand synthesis. The replication in
the lagging strands is discontinuous and takes place with formation of
several Okazaki fragments (fig 2).

Okazaki fragments (fig 3) generated in eukaryotes during lagging-strand synthesis are

around 200 bases (prokaryotes, around 2000 bases) long. As several Okazaki fragments
are made, Polymerase switching during synthesis of Okazaki fragments is of higher
importance.

Hence an Okazaki fragment is made up of RNA nucleotides (7-10 nucleotides), then

around 10-20 nucleotides of DNA bases are added by the DNA pol α. Following which,
the RFC causes polymerase switching and the deoxyribonucleotides are added by DNA
pol δ held by the PCNA, the sliding clamp (see the figure below). DNA pol δ
dissociates after the synthesis of the entire DNA stretch, as it approaches the previous
RNA primer.

The proteins involved in the replication, especially PCNA (Boehm et al., 2016)
RNA primers are removed by RNase H1, such that one ribonucleotide remains still
attached to the DNA (3′ end) part of the Okazaki fragment. This left out ribonucleotide
is then removed by flap endonuclease 1 (FEN 1). Polymerase δ then fills the gaps
formed between Okazaki fragments following the primer removal. The nick formed
between the Okazaki fragment and the lagging strand are filled in by DNA ligase I,
hence forming single lagging strand.
Fig 4: Removal of RNA primer and linking of individual Okazaki fragments.
Nucleosome Assembly:

During elongation, there is continuous disassembly as reassembly of the nucleosome

packaging along the DNA, too. As we know, one of the feature of the eukaryotic DNA
is that it is packaged in form of highly compact structures called the Chromosomes. It
involves winding of the negatively charged DNA around the basic proteins
called histones to form structures called nucleosomes (fig below). Each nucleosome is
composed of 8 histone proteins, two each of H2A, H2B, H3 and H4. Histone H1 forms
the linker between two nucleosomes.

The nucleosomes.
During replication, two nucleosomes in front of the replication fork, that is unreplicated
DNA, becomes destabilized. Hence the replication fork movement causes DNA
packaging to disorganise to allow the replication proteins to interact with the DNA.

In the replicated portion, the leading and lagging strands were nucleosome-free till
about 225 bp and 285 bp, respectively. Following this region, the DNA was packaged
with histone octomer but some lacked histone H1. After around 450 bp to 650bp from
the replication fork, complete nucleosomes with H1 were detected in the daughter
strands. Hence there is stepwise assembly of daughter strands into complete
nucleosome.

The parental nucleosomes disassociate and randomly bind the daughter DNA strands,
such that each strands has half of the parental nucleosomes. The remaining
nucleosome components required for packaging the two daughter strands, are
synthesized de novo and assembled onto the daughter strands.

Termination

Following the successful elongation, the replication has to be terminated to give two
separate copies of the DNA.
• Involving two adjacent replication forks:

As the eukaryotic cell have a large number of origins, the termination involves merging
of two adjacent replication forks. This includes four different steps:

– Dissolution:

The DNA stretch between the two adjacent forks (fig 5.a) is unwound (fig 5.b) and the
approaching CMGs pass each other (fig 5.c). The last Okazaki fragment is processed
by DNA pol δ and FEN1 (fig 5.d).

– Ligation:

The gaps in the daughter strands (as in normal Okazaki fragments) are filled in and two
oppositely approaching synthesized strands are ligated.

– Dissociation of replisome:

The replisome complex dismantles after the convergence of the two replication fork.
This process involves termination-specific polyubiquitylation of Mcm7 and the
p97/VCP/Cdc48 segregase (fig 5.e).

– Decatenation:

If there are any intertwinings in the daughter DNA strands or catenanes, they are
removed utilising topoisomerase II segregating the two strands.
Fig 5: Termination involves merging of the two neighbouring forks (Dewar & Walter,
2017).
• Involving the ends of the Chromosomes:

As is known the DNA in eukaryotic chromosomes is a linear molecule, the termination

in eukaryotic DNA also involve completing replication at the ends of chromosomes
known as Telomeres (fig 6).

Fig 6: Telomeres form protective end

of eukaryotic linear DNA (Aulinas, 2013).
During the synthesis of Okazaki fragments, RNA primer provide 3′-OH group for 5′
to 3′ replication. On the removal of RNA primer, from the lagging strand at
the chromosome end, the end remains unreplicated and the newly synthesized strand
is shortened (fig 7).
 Fig 7: Shortening of the chromosome ends.
This shortening of the chromosome is prevented by the presence of special repeats of
sequences called telomeres (and telomere-associated proteins) at the ends of DNA in
chromosomes contain. For e.g. human chromosomes are protected by telomeres having
repeated sequences of (TTAGGG)n of about 15–20 kb at birth. These structures protect
the ends of chromosomes from being mistakenly considered as DNA double strand
breaks (DSB).

In normal somatic cells, the telomeric region of eukaryotic chromosomes

are shortened with each round of DNA replication. After certain number of DNA
replications, and hence cell divisions, the telomeres are shortened to an extent that it
leads to replicative cell senescence or apoptosis.

However, in germline and cancer cells, an enzyme called telomerase, extends the ends
of the chromosomes, especially the 5′ end of the lagging strands. The ability to maintain
the length of the telomeres, make these cells immortal.

Telomerase is a reverse transcriptase which has a RNA template, known as template-

encoding RNA molecule (TER) for extension of the telomeric DNA (fig 8). The
basic protein component of telomerase is known as TERT (Telomerase Reverse
Transcriptase).

In humans, the RNA template has the sequence AUCCCAAUC. The newly
synthesized telomeric DNA repeats are added to the overhanging single-stranded 3′
end of the DNA (fig 8).

Fig 8: Synthesis of telomeric DNA repeats by Telomerase (Verhoeven et al, 2014).