DNA replication in eukaryotes involves three main stages: initiation, elongation, and termination. Initiation begins at origins of replication, where the pre-replication complex forms. During elongation, DNA polymerase adds nucleotides to grow new DNA strands by copying existing template strands. Elongation of the leading strand is continuous while the lagging strand occurs in fragments called Okazaki fragments. Termination occurs when the replication forks meet, and the DNA strands are fully replicated. Telomeres protect chromosome ends during replication to prevent shortening with each cell division.

1. Prepared by
Faisal Ghazi Lazim
DNA Replication
in Eukaryotes

2. Definition of DNA Replication
The process of making an identical copy of a duplex (double-stranded)
DNA, using existing DNA as a template for the synthesis of new DNA
strands.
The replication process does not begin at any random spot along the
DNA molecule. It begins at a certain sequence of nucleotides, the origin
of replication. Each eukaryote chromosome contains a multiple origin of
replication.
The hydrogen bonds between the two complementary DNA strands
break more easily at the origin so that the double helix can be opened in
both directions starting from this point.
The opening of the double helix creates two replication forks, which
form a replication bubble .Replication proceeds in both directions away
from the origin of replication, expanding the replication bubble.
Eventually, the two replication forks meet at the DNA replication
terminus opposite the origin of replication, and the result is two separate
and complete circular chromosomes.

3. Replication Fork Formation
The replication fork(s) are at one or both ends of a distinct
replicative structure called a replication eye or bubble,
which can be visualized experimentally
The replication bubble can result from either bidirectional or
unidirectional replication
In bidirectional replication, two replication forks move in
opposite directions from the origin, and hence each end of the
bubble is a replication fork.
 In unidirectional replication, one replication fork moves in
one direction from the origin. In this case, one end of a
replication bubble is a replication fork and the other end is the
origin of replication.

4. Replication bubble

5. Modes of Replication
A. Semiconservative Replication
The double-stranded DNA contains one
parental and one daughter strand following
Replication.
B. Conservative Replication
Both parental strands stay together after
DNA replication.
C. Dispersive Replication
Parental and daughter DNA are interspersed
in both strands following replication.

6. DNA Polymerase
All the DNA polymerases add nucleotides to a growing DNA
chain exclusively in a 5' to 3‘ direction.
So if no 3’ to 5’ synthesizing activity can be found, how is
the new strand oriented 3’ to 5’ in the direction of the
replication fork synthesized?
The solution to this problem is discontinuous synthesis of
that strand, whereas the other strand is synthesized
continuously.

7. The enzyme DNA polymerase adds
nucleotides to the 3′ end of each
strand of DNA. Newly added
nucleotides have bases that are
complementary to those on the
template strand.
nucleotides are added to the 3′ end
of the growing daughter strand —
the end at which the DNA strand
has a free hydroxyl (–OH) group on
the 3′ carbon of the sugar

8. Type of the DNA Polymerase
DNA polymerase α (alpha):
DNA polymerase α associated with enzyme Primase, forms RNA primer
which are 8-10 nucleotide long.
Later DNA polymerase α elongates this RNA primer by more 20 nucleotides
and then leaves the place.
DNA polymerase ε (epsilon):
DNA polymerase ε synthesis nucleotides on the leading strand.
It will continuously add nucleotides leading to continuous process of
replication.
Thus it will require only one RNA primer at the beginning.

9. Type of the DNA Polymerase
DNA polymerase δ (delta):
DNA polymerase δ helps the synthesis of DNA on lagging strand.
On the lagging strand multiple RNA primers are required.
On the lagging strand, DNA polymerase δ synthesize small
fragments of DNA called Okazaki fragments.
At the end of each Okazaki fragment, DNA polymerase δ runs to
previous Okazaki fragment and replaces the RNA primer
nucleotides with DNA nucleotides.
this leads to flap formation which is removed and the nick
between is replaced by enzyme DNA ligases.

10. Eukaryotic genome origins
In eukaryotes, the budding yeast Saccharomyces cerevisiae were first
identified by their ability to support the replication of mini-chromosomes
or plasmids, giving rise to the name Autonomously replicating sequences or
ARS elements. Each budding yeast origin consists of a short (~11 bp)
essential DNA sequence (called the ARS consensus sequence or ACS) that
recruits replication proteins.
In other eukaryotes, including humans, the base pair sequences at the
replication origins vary. Despite this sequence variation, all the origins
form a base for assembly of a group of proteins known collectively as the
pre-replication complex (pre-RC).
First, the origin DNA is bound by the origin recognition complex (ORC)
which, with help from two further protein factors (Cdc6 and Cdt1), load the
mini chromosome maintenance (or MCM) protein complex . Once
assembled, this complex of proteins indicates that the replication origin is
ready for activation. Once the replication origin is activated, the cell's DNA
will be replicated.

11. Pre-replication complex
 (pre-RC) is a protein complex that forms at the origin of
replication during the initiation step of DNA replication. Formation
of the pre-RC is required for DNA replication to occur. Complete
and faithful replication of the genome ensures that each daughter
cell will carry the same genetic information as the parent cell.
Accordingly, formation of the pre-RC is a very important part of the
cell cycle.
The eukaryotic pre-RC is the most complex and highly regulated pre-
RC. In most eukaryotes it is composed of six ORC proteins (ORC1-6),
Cdc6, Cdt1, and a heterohexamer of the six MCM proteins (MCM2-7).
The MCM heterohexamer arguably arose via MCM gene duplication
events and subsequent divergent evolution.

13. Steps of the Pre-replication complex
 First - association of Origin recognizing complex (ORC) with
replication origin.
 Second - binding of Cdc6 protein to ORC.
 Third - binding of Cdt1 and minichromosome maintenance protein.
 This replicative complex assembly occurs during G1 phase prior to S
phase.
*During the transition between G1 phase to S phase, CDK proteins and
DDK proteins get attached to the Pre-replication complex.
It transforms the Pre-replication complex into active replication fork.

15. Proliferating cell nucleus antigen (PCNA)

16. Replication Factor C (RFC)

18. The Origin Number Paradox
*Genome 20 Mb (yeast) up to 6,000 Mb (human)
*Fork rate 10 bp / sec (frog) - 50 bp / sec (mammal)
*Amount replicated by 2 forks in 8 hr (human cells) = 2 x 50 x
28,800 = 2,880,000 (~ 3 Mb, a 2,000-fold deficit)
*46 chromosomes (human cells) - with one origin per
chromosome, at least 92 replication forks gives approx. 140 Mb
replicated in 8 hours (still a 40-fold deficit

19. Why so many origins?

20. Restoration of chromatin after replication
The principle chromatin
assembly reactions during DNA
replication. Reaction
(a): parental nucleosomes are
partially disrupted during DNA
replication and the histones are
directly transferred to the
replicated DNA, reassembling
into nucleosomes. Reaction
(b): the assembly of new
nucleosomes from newly
synthesized and soluble
histones is mediated by a
chromatin assembly factor

21. DNA Replication Process
*There are three Stages of DNA replication:
1. Initiation
2. Elongation
3. Termination

22. Initiation
In eukaryotes, clusters (tandem arrays) of about 20–50 replicons initiate
simultaneously at defined times throughout S-phase.
1. Those which replicate early in S-phase comprise predominantly euchromatin
(which includes transcriptionally active DNA)
2. while those activated late in S-phase are mainly within heterochromatin (see
Topic D3).
3. Centromeric and telomeric DNA replicates last.
In yeast replication origins are termed autonomously replicating sequences
(ARSs).
The minimum length of DNA that will support replication is only 11 bp and has the
consensus sequence [A/T]TTTAT[A/G]TTT[A/T], though additional copies of this
sequence are required for optimal efficiency.
This sequence is bound by the origin recognition complex (ORC) which, when
activated by CDKs, permits opening of the DNA for copying.
In contrast to prokaryotes, eukaryotic replicons can only initiate once per cell cycle.
A protein (licensing factor) which is absolutely required for initiation and inactivated
after use can only gain access to the nucleus when the nuclear envelope dissolves
at mitosis, thus preventing premature re-initiation.

23. Elongation
During elongation, 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.
DNA polymerase contains a groove that allows it to bind to a single-stranded template
DNA and travel one nucleotide at time. For example, when DNA polymerase meets an
adenosine nucleotide on the template strand, it adds a thymidine to the 3′ end of the
newly synthesized strand, and then moves to the next nucleotide on the template strand.
This process will continue until the DNA polymerase reaches the end of the template
strand.

24. Elongation
DNA polymerase cannot initiate new strand synthesis; it only adds new nucleotides at
the 3′ end of an existing strand. All newly synthesized polynucleotide strands must be
initiated by a specialized RNA polymerase called primase. Primase initiates
polynucleotide synthesis and by creating a short RNA polynucleotide strand
complementary to template DNA strand. This short stretch of RNA nucleotides is called
the primer. Once RNA primer has been synthesized at the template DNA, primase exits,
and DNA polymerase extends the new strand with nucleotides complementary to the
template DNA.
Eventually, the RNA nucleotides in the primer are removed and replaced with DNA
nucleotides. Once DNA replication is finished, the daughter molecules are made entirely
of continuous DNA nucleotides, with no RNA portions.

25. Leading strand synthesis
The leading strand is the template strand that is being
replicated in the same direction as the movement of the
replication fork.
Nucleotides are added by the DNA Polymerase ε.
DNA polymerase requires the RNA primer produce by
Primase.
Elongation take place in 5’ to 3’ direction.
Finally the primer are removed by RNAse H and the gap is
sealed by the DNA Ligase.

26. Lagging strand synthesis
DNA replication on lagging strand is discontinuous and
elongation opposite direction to replication fork.
Nucleotide are added by the DNA Polymerase δ.
Lagging strand used more RNA Primer for loading
nucleotide.
DNA polymerase will synthesize short fragments of DNA
called Okazaki fragments which are added to the 3' end of
the primer. These fragments can be anywhere between
100-400 nucleotides long in eukaryotes.

27. Termination
DNA polymerase halts when it reaches a section of DNA template that has already been
replicated.
RNA primers need to be replaced with DNA, and nicks in the sugar-phosphate
backbone need to be connected.
The group of cellular enzymes that remove RNA primers include the proteins FEN1
(flap endonulcease 1) and RNase H. The enzymes FEN1 and RNase H remove RNA
primers at the start of each leading strand and at the start of each Okazaki fragment,
leaving gaps of unreplicated template DNA. Once the primers are removed, a free-
floating DNA polymerase lands at the 3′ end of the preceding DNA fragment and
extends the DNA over the gap. However, this creates new nicks (unconnected sugar-
phosphate backbone)
In the final stage of DNA replication, the enzyme ligase joins the sugar-phosphate
backbones at each nick site. After ligase has connected all nicks, the new strand is one
long continuous DNA strand, and the daughter DNA molecule is complete.

29. Eukaryotic DNA replication fork

30. Summary of DNA Replication

31. The End Problem of Linear DNA Replication
Linear chromosomes have an end problem. After DNA replication, each newly synthesized
DNA strand is shorter at its 5′ end than at the parental DNA strand’s 5′ end. This produces a
3′ overhang at one end (and one end only) of each daughter DNA strand, such that the two
daughter DNAs have their 3′ overhangs at opposite ends.
Every RNA primer synthesized during replication can be removed and replaced with DNA
strands except the RNA primer at the 5′ end of the newly synthesized strand. This small
section of RNA can only be removed, not replaced with DNA. Enzymes RNase H and FEN1
remove RNA primers, but DNA Polymerase will add new DNA only if the DNA Polymerase
has an existing strand 5′ to it (“behind” it) to extend. However, there is no more DNA in the 5′
direction after the final RNA primer, so DNA polymerse cannot replace the RNA with DNA.
Therefore, both daughter DNA strands have an incomplete 5′ strand with 3′ overhang.
In the absence of additional cellular processes, nucleases would digest these single-stranded 3′
overhangs. Each daughter DNA would become shorter than the parental DNA, and eventually
entire DNA would be lost. To prevent this shortening, the ends of linear eukaryotic
chromosomes have special structures called telomeres.

32. Telomere Replication
The ends of the linear chromosomes are known as telomeres: repetitive sequences that
code for no particular gene. These telomeres protect the important genes from being
deleted as cells divide and as DNA strands shorten during replication.
In humans, a six base pair sequence, TTAGGG, is repeated 100 to 1000 times. After
each round of DNA replication, some telomeric sequences are lost at the 5′ end of the
newly synthesized strand on each daughter DNA, but because these are noncoding
sequences, their loss does not adversely affect the cell. However, even these sequences
are not unlimited. After sufficient rounds of replication, all the telomeric repeats are
lost, and the DNA risks losing coding sequences with subsequent rounds.
The discovery of the enzyme telomerase helped in the understanding of how
chromosome ends are maintained. The telomerase enzyme attaches to the end of a
chromosome and contains a catalytic part and a built-in RNA template. Telomerase
adds complementary RNA bases to the 3′ end of the DNA strand. Once the 3′ end of
the lagging strand template is sufficiently elongated, DNA polymerase adds the
complementary nucleotides to the ends of the chromosomes; thus, the ends of the
chromosomes are replicated.

33. Steps of telomerase action
.

34. Proofreading
DNA polymerases are the enzymes that build DNA in cells.
During DNA replication (copying), most DNA polymerases can
“check their work” with each base that they add.
This process is called proofreading.
If the polymerase detects that a
wrong (incorrectly paired) nucleotide
has been added, it will remove and
replace the nucleotide right away,
before continuing with DNA synthesis

35. Thanks