DNA Replication in Eukaryotes

Learning objectives: DNA Replication in Eukaryotes

1. Explain the process of replication.

2. Explain the main features of eukaryotic replication.
3. State 3 mechanisms used by cells as cell cycle check-points in DNA replication.
4. Explain the assembly of the replication initiation complex (RIC).
5. Explain why this RIC is needed.
6. Describe at what point does an origin of replication become licensed to undergo replication.
7. Explain the function of CDKs and DDKs in DNA replication.
8. Describe the assemblies of the Pre-RC and the RIC as they occur in different phases of the cell cycle.
9. Assess the relationship between replication and the cell cycle.
10. Explain the importance of multiple origins to DNA replication.
11. Explain DNA replication initiation, elongation and termination.
12. Describe FIVE (5) phosphorylation events that serve to regulate the functions of named replication proteins and
the forward direction of DNA synthesis.
13. Describe how DNA replication is positively and negatively controlled.
14. Prepare a table detailing all the proteins and their respective functions involved in DNA replication and control.
3 Main Concepts of DNA Replication in Eukaryotes

 
Concept 1: Assembly of a replication initiation complex
 
Concept 2: The mechanics of replication
 
Concept 3: Regulation / Control of replication
 
Concept 1: Initiation of replication requires assembly of pre-RC
followed by a replication initiation complex

1. Origins of replication

• DNA replication is initiated from multiple sites on chromosomes, called origins.

• origins – aka autonomously replicating sequences (ARS) - exist as short, well-defined
sequences of about 150 bp.
• origins operate asynchronously during S-phase in order to ensure efficient replication
of the chromosomes within the time-frame of the cell cycle.
• Origins may be 30-300kb apart according to species and tissue type
• Origins are activated in clusters (~20-80 origins) called replication units.
 This Orc??
2. Assembly of the pre-replicative complex
The ORC
• all eukaryotes utilize similar proteins and a
conserved mechanism for initiating and
This is the
regulating initiation of DNA replication. correct ORC
• DNA synthesis is initiated by the binding of
initiator proteins to the origins of
replication.
• Many eukaryotes have a multi-subunit
protein called the origin recognition
complex (ORC) that binds specifically to ARS
sequences.
• The ORC complex (made up of 6 proteins)
forms the core of the origin complex to
which other components are loaded in a
step-wise manner.
Assembly of pre-replication initiation complex at the origin
• The ORC recruits the pre-replicative complex
(cell division cycle 6 (Cdc6), Cdc10-dependent
transcript 1 (Cdt1) (also known as licensing
factors) and the minichromosome maintenance
(MCM, 2-7 of them) proteins to replication
origins during the G1 phase.
• Loading of MCMs at the origin is called DNA
licensing and completes the assembly of the
pre-RIC complex. This complex is activated to
give the Replication Inititation Complex (RIC) by
cyclin-dependent kinases (Cdks) and the cell
division cycle 7–dumb bell former 4 (Cdc7–Dbf4)
complex in S phase to promote the initiation of
DNA replication.
• Cdc6 and Cdt1 leave the complex.
• The helicase activity of the MCM’s partially
unwinds the DNA, which in turn attracts Fig. Assembly of pre-replication
replication protein A (RPA, 3 subunits RPA1,
RPA2 and RPA3).
initiation complex at the origin
• RPA stabilizes the ssDNA.
Assembly of the replicative complex

Topoisomerase enzymes also act to stabilize ssDNA and reduce torsional stress.
Topoisomerase I and II remove supercoils in DNA but although type II is more efficient, it requires energy
from ATP hydrolysis.
Mechanics of DNA Replication in Eukaryotes
In eukaryotes, there are 5 forms of DNA Polymerase alpha, beta, delta, epsilon,
and gamma but only alpha and delta are used in DNA synthesis, gamma is used in DNA synthesis of mitochondrial DNA,
beta and epsilon are involved in DNA repair. DNA Pol alpha has primase activity which allows production of a short RNA strand
(~10 bases long). The distance between 2 primers varies according to species but usually 100-200 bases separate 2 primers.

DNA Pol- δ has 5’-3’ proof-

reading ability which allows it
to correct errors during
synthesis.
Mechanics of DNA Replication in Eukaryotes

After DNA polymerase (Pol-α)-dependent synthesis of

an RNA/DNA primer on the ssDNA, Replication
Factor C (RFC) induces a DNA polymerase switch,
from Pol-α to Pol-δ. To accomplish this, RFC binds to the
RNA primer and loads proliferating cell nuclear antigen
(PCNA) onto the DNA, which, in turn, recruits Pol- δ.
Mechanics of DNA Replication in Eukaryotes
Initiation of Extension from the 3’ end of the
RNA primer RNA primer RNA primer to produce new
Template DNA synthesis synthesis DNA strand

RNA primer is
required for DNA
polymerase to build
new copy of DNA.

DNA Pol alpha has

primase activity to
enable generation of
RNA primers.

DNA Pol delta builds

the new copy of DNA
from the 3’ end of
the RNA primer using
the template DNA
and
Mechanics of DNA Replication in Eukaryotes
DNA Pol δ holoenzyme (comprising PCNA, RFC and DNA Pol-δ) carries out processive DNA
synthesis continuously on the leading strand, it performs discontinuous assembly on the
lagging strand. The latter process involves first extending the RNA/DNA primers from the
3’ end that are produced by Pol-δ to create Okazaki fragments.
Mechanics of DNA Replication in Eukaryotes
Extension occurs from the 3’end of the primers because DNA Pol–δ can only polymerize
in a 5’ to 3’ direction. Hence, synthesis of the 3’-5’ DNA strand is continuous (leading
strand) and uninterrupted whereas on the 5’-3’ DNA strand, synthesis is discontinuous
(lagging strand).

When Pol-δ encounters the previous RNA/DNA primer during the elongation step of the
lagging strand, it performs strand displacement to release the primer.
 Mechanics of DNA Replication in Eukaryotes

Next, PCNA binds flap endonuclease 1

(FEN1), which then cuts the displaced
RNA/DNA, possibly in conjunction with the
endonuclease Dna2. Finally, PCNA recruits
DNA ligase I (LigI) to seal the DNA strand.
Overview of Mechanics of DNA Replication in Eukaryotes
Yes, that’s correct! DNA
replication must be coupled
with nucleosome
disassembly and re-assembly
Concept 3: Regulation of DNA Replication: Positive and negative
control; phosphorylation events

DNA replication must be achieved according to the following:-

 
1. must be accurate to maintain genome integrity.
2. must be duplicated precisely before cell division occurs.
3. chromosome replication must occur only once per cell cycle.
4. chromosomes must not be replicated twice
Regulation of DNA Replication

The following regulation check points exist:-

1. DNA replication is coordinately controlled at each origin to prevent replication of already

replicated DNA – coordination is asynchronous to prevent duplication.
2. After completion of the S phase, cells in G2 cannot initiate replication unless they
undergo mitosis.
3. there must be DNA replication licensing at the G2-S-transition of the cell cycle. This
means that each origin must have a “license” (aka pre-RC) to begin replication.
4. Origins must be “de-licensed” once they are finished with replication and this is
accomplished by the MCM complex and CDK. CDKs are the master regulators of the cell cycle
wrt DNA replication and its segregation at mitosis.
5. Geminin is another protein that must be degraded at the end of mitosis in order for
another round of licensing to occur in higher eukaryotes.
Control of Replication
With their multiple origins, how does the eukaryotic
cell know which origins have been already replicated
and which still await replication?

IMPORTANT: When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA
of the G2 nucleus does not begin replicating again even though replication is proceeding
normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized
DNA be replicated again.

Two control mechanisms have been identified — one positive and one negative. This
redundancy probably reflects the crucial importance of precise replication to the
integrity of the genome.
Control of Replication

MCM Licensing and helicase activity: positive control of replication

In order to be replicated, each origin of replication must be bound by:

• an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout
the process.
• Accessory proteins called licensing factors. These accumulate in the nucleus during G1
of the cell cycle. They include:
• Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with
• MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be
replicated. MCMs have helicase activity for partial unwinding of dsDNA to produce the
replication bubble which is needed to access the bases by DNA pol-d.
• Once replication begins in S phase, Cdc-6 and Cdt-1 leave the ORCs (the latter by
ubiquination and destruction in proteasomes).
Control of Replication

Geminin: negative control of replication

G2 nuclei also contain at least one protein — called geminin — that

prevents assembly of MCM proteins on freshly-synthesized DNA
(probably by blocking the actions of Cdt1).
As the cell completes mitosis, geminin is degraded so the DNA of the two
daughter cells will be able to respond to licensing factors and be able to
replicate their DNA at the next S phase. Geminin prevents re-initiation of
replication for the same DNA templates and ensure replication occurs
only once per cell cycle.
DNA Replication in Eukaryotes and the Importance of
Phosphorylation Events
Objective of these phosphorylation events:
to activate or de-activate replication such that
replication occurs
(i) ONLY ONCE PER CELL CYCLE and
(ii) to ensure (ii) PROGRESSION OF THE CELL
CYCLE FROM ONE PHASE TO THE NEXT.
Phosphorylation events that regulate DNA replication include the
following:-
Phosphorylation by Cdks enable activation of Pre-RIC and recruitment of RIC proteins.
DNA Pol-δ is phosphorylated at the p125kDa subunit to allow loading of PCNA.
RPA, has 3 subunits 1,2&3, and is phosphorylated 2X in the cell cycle:
• 1st time: during the S phase-RPA2 is phosphorylated by Cdk2 kinase while bound to ssDNA
• 2nd time: at the end of the S phase-RPA2 is phosphorylated again by Cdk1-cyclin A and Cdk-1 cyclin B
which causes dissociation of RPA protein.
DNA Pol-α is phosphorylated multiple times at its p180kDa subunit:-
• 1st time: Before the S phase, the p180kDa subunit of DNA Pol-α is phosphorylated by Cdc7-Dbf4 enzyme
complex
• 2nd time: At the end of the G1-S phase, the same p180kDa subunit of DNA Pol-α is phosphorylated by
Cdk2-cyclin E which also stimulates the initiation of replication
• 3rd time: At the end of the S phase, p180kDa subunit is phosphorylated again by Cdk2-cyclin A and Cdk1-
cyclin A to prevent re-initiation of replication.
RFC is phosphorylated by calmodulin-dependent protein kinase II (CaMKII) which blocks interaction with
PCNA and prevents loading of PCNA and hence, re-initiation of replication.
FEN1 may require phosphorylation by Cdk1-cyclin A and Cdk2-cyclin A which appears to reduce its
endonuclease activity and prevents PCNA binding; this prevents recruitment of DNA ligase I.
Cell cycle and DNA replication in eukaryotes
1. Assembly of the pre-RIC occurs once in G1 phase of the cell cycle.
2. Synthesis of DNA occurs once in the S-phase. In this way, eukaryotic cells produce one and only one copy of
their genome each time they divide.

The cell cycle is controlled by a large families of related proteins, with different members of these families
controlling progression through distinct phases of the cell cycle.These multiple members of the Cdk family
associate with specific cyclins to drive progression through the different stages of the cell cycle (see Figure). For
example, progression from G1 to S is regulated principally by Cdk2 and Cdk4 (and in some cells Cdk6) in
association with cyclins D and E. Complexes of Cdk4 and Cdk6 with the D-type cyclins (cyclin D1, D2, and D3) play a
critical role in progression through the restriction point in G1. Cyclin E is expressed later in G1, and Cdk2/cyclin E
complexes are required for the G1 to S transition and initiation of DNA synthesis. Complexes of Cdk2 with cyclin A
function in the progression of cells through S phase.

Cell cycle and replication important relationships:

1. Replication only occurs once per cell cycle
2. Re-initiation of the assembly of the RIC is not allowed
3. Replication occurs within the time-frame of the G1 and S phases
4. Progression of the cell cycle is in the forward direction