DNA replication is precisely controlled to occur once per cell cycle. It is linked to the cell cycle through two principles: 1) Initiation of DNA replication commits the cell to further division as replication must complete before division. 2) Signals ensure each replicon is activated only once per cell cycle. Replication initiates at origins through the assembly of pre-replication complexes containing the MCM helicase, which is activated by CDK and DDK to begin DNA unwinding and replication. The cell cycle is halted in response to DNA damage to allow for repair before replication and division resume.

1. -BY
SHREYA AHUJA
ROLL NO. 11

2. REPLICATION AND CELL CYCLE
Whether a cell has only one chromosome (as in prokaryotes) or many chromosomes (as in eukaryotes), the entire
genome must be replicated precisely once for every cell division.
How is this act of replication linked to cell cycle?
Two general principles are used to compare the state of replication with the condition of the cell cycle:
• Initiation of DNA replication commits the cell (prokaryote or eukaryote) to a further division. Replication is controlled
at the stage of initiation. Once replication has started, it continues until the entire genome has been duplicated.
• If replication proceeds, the consequent division cannot be permitted to occur until the replication event has been
completed. Indeed, the completion of replication may provide a trigger for cell division. The duplicate genomes are
then segregated one to each daughter cell.
Trigger for cell division

3. • In prokaryotes, the initiation of replication is a single event involving a unique site on the bacterial chromosome, and
the process of division is accomplished by the development of a septum that grows from the cell wall and divides the
cell into two.
• In eukaryotic cells, initiation of replication is identified by the start of S-Phase, , a protracted period during which
DNA synthesis occurs, and which involves many individual initiation events. The act of division is accomplished by the
reorganization of the cell at mitosis.
• The unit of DNA in which an individual act of replication occurs is called the replicon. Each replicon “fires” once and
only once in each cell cycle. It has an origin at which replication is initiated and a terminus at which replication stops.
• Each eukaryotic chromosome contains a large number of replicons; thus the unit of segregation includes many units
of replication. This adds another dimension to the problem of control: All of the replicons must be fired during one
cell cycle. They are not, however, active simultaneously. But each replicon must be activated no more than once in
each cell cycle.
• Hence, some signals must distinguish the replicated from the non-replicated replicons to ensure that replicons do
not fire a second time. And since, many replicons are activated independently, there must be another signal to
indicate when the entire process of replicating all replicons is completed.
• Additionally, there are multiple copies of each organelle DNA per cell, and the control of organelle DNA replication
must be related to cell cycle.
FACTS ABOUT INITIATION OF REPLICATION

4. AUTONOMOUSLY REPLICATING SEQUENCE IN EUKARYOTES
• An autonomously replicating sequence (ARS) contains the origin of replication
in the yeast genome. It contains four regions (A, B1, B2, and B3), named in
order of their effect on plasmid stability; when these regions are mutated,
replication does not initiate.
• As seen above the ARS are considerably A-T rich which makes it easy for
replicative proteins to disrupt the H-bonding in that area. ORC protein complex
(Origin Recognition Complex) is bound at the ARS throughout the cell cycle,
allowing replicative proteins access to the ARS.
• Mutational analysis for the yeast ARS elements have shown that any mutation
in the B1, B2 and B3 regions result in a reduction of function of the ARS
element. A mutation in the A region results in a complete loss of function.
• Melting of DNA occurs within domain B2, induced by attachment of ARS Binding
factor 1 to B3. A1 and B1 domain binds with Origin Recognition Complex.
• 5'- T/A T T T A Y R T T T T/A -3

5. • The process of duplicating DNA is called DNA replication,
and it takes place by first unwinding the duplex DNA
molecule, starting at many locations called DNA replication
origins, followed by an unzipping process that unwinds the
DNA as it is being copied.
• However, replication does not start at all the different
origins at once. Rather, there is a defined temporal order in
which these origins fire. Frequently a few adjacent origins
open up to duplicate a segment of a chromosome, followed
some time later by another group of origins opening up in
an adjacent segment.
• Replication does not necessarily start at exactly the same
origin sites every time, but the segments appear to replicate
in the same temporal sequence regardless of exactly where
the segments are.

6. • PRIMING OF DNA REPLICATION: DNA double helix is
unwound and an initial priming event by DNA polymerase α
occurs on the leading strand. Priming of the DNA helix
consists of synthesis of an RNA primer to allow DNA
synthesis by DNA polymerase α. Priming occurs once at the
origin on the leading strand and at the start of each Okazaki
fragment on the lagging strand.
• DNA replication is initiated from specific sequences called
origins of replication, and eukaryotic cells have multiple
replication origins. To initiate DNA replication, multiple
replicative proteins assemble on and dissociate from these
replicative origins. The individual factors described below
work together to direct the formation of the pre-replication
complex (pre-RC), a key intermediate in the replication
initiation process.
PRE-REPLICATIVE COMPLEX

7. REGULATION OF DNA REPLICATION

9. ACTIVATION OF CDK
Y - Tyrosine residues
T – Threonine residues
CAK – CDK activating kinase

10. o CDK is the key regulator of pre-RC assembly
o CDK acts by inhibiting the individual components of the pre-RC. CDK phosphorylates Cdc6 to mark it for degradation by the SCF in late
G1 and early S phase
o CDK also phosphorylates ORC proteins. It has been suggested that phosphorylation affects the ability of the ORC to bind other
components of the pre-RC.
o In metazoans, studies have shown that Geminin prevents pre-RC assembly by binding to cdt1 and preventing its association with the
pre-RC. Since geminin is degraded by the APC/C, pre-RC assembly can proceed only when APC/C activity is high, which occurs in G1.

13.  Origin licensing: The hexameric replicative helicase, Mcm2-7, is loaded in an
inactive form into pre-replicative complexes (pre-RCs) at potential origin sites.
This helicase loading step requires the ATP-dependent activity of the Origin
Recognition Complex (ORC), which marks potential origin sites on the
chromosome, Cdc6, which like several of the ORC subunits is a member of the
AAA+ family of ATPases.
 ORC and Cdc6 cooperatively load two heptamers of the Cdt1·Mcm2-7 complex
into a head-to-head Mcm2-7 double hexamer onto DNA
 Chromosomal sites containing functional pre-RCs serve as binding sites for
additional replication factors including Mcm10, Sld3, and Cdc45. Function
unclear.
 DDK and CDK promote the activation of a subset of “licensed” origins by
activating the MCM2-7 helicase and by promoting the assembly of further
initiation factors around pre-RCs to form pre-initiation complexes or replisomes.
 CDK phosphorylates two replication proteins, Sld3 and Sld2, both of which bind
to Dpb11 when phosphorylated. Dpb11 has two pairs of tandem BRCT domains,
known as a phosphopeptide-binding domain. The N-terminal pair of the BRCT
domains binds to phosphorylated Sld3, and the C-terminal pair binds to
phosphorylated Sld2. These phosphorylation-dependent interactions are
essential, and represent the minimal requirement for CDK-dependent activation
of DNA replication in budding. DDK phosphorylates Mcm, and this
phosphorylation is thought to enhance the interaction between Mcm and other
replication proteins by alleviating an inhibitory activity in Mcm4

14. CELL CYCLE IS HALTED IN RESPONSE TO DNA DAMAGE
Maintenance of the G1/S DNA
damage checkpoint in the
presence and absence of Cdk2.
In response to DNA damage,
activation of p53-p21 pathway is
not altered in the absence of
Cdk2, the primary target of p21
at the G1/S checkpoint. In the
presence of Cdk2, the induced
p21 inhibits Cdk2/cyclin E
complexes in response to DNA
damage. In the absence of Cdk2,
Cdk1/cyclin E complexes are
responsible for promotion of the
G1/S transition and as a result
become the target for p21
inhibition in response to DNA
damage. Nevertheless, Cdk1 is
not fully capable to rescue the
functions of Cdk2 in DNA damage
repair.