CELL CYCLE

D R . I S R A T H R I S T Y, M . B I O M E D

B I O C H E M I S T RY D E PA R T M E N T
U N I V E R S I T Y O F M U H A M M A D I YA H S U M AT E R A U TA R A
 DNA REPLICATION

Process of duplication of the entire genome prior to cell

division

Biological significance
• extreme accuracy of DNA replication is necessary in
order to preserve the integrity of the genome in
successive generations
• In eukaryotes , replication only occurs during the S
phase of the cell cycle.
• Replication rate in eukaryotes is slower resulting in a
higher fidelity/accuracy of replication in eukaryotes
• Watson and Crick reasoned that the pairing was more
specific, dictated by the base structures
• They determined that adenine (A) paired only with
thymine (T), and guanine (G) paired only with cytosine
(C)
• The Watson-Crick model explains Chargaff’s rules: in
any organism the amount of A = T, and the amount of G
=C
FIGURE 16.7

G
5 end
C
C G Hydrogen bond
3 end
G C
G C T A

3.4 nm
T A

G C G C
C G

A T

1 nm C G
T A
C G
G C
C G A T

A T 3 end
A T
0.34 nm
T A 5 end

(a) Key features of (b) Partial chemical structure (c) Space-filling

DNA structure model
THE BASIC PRINCIPLE: BASE
PAIRING TO A TEMPLATE STRAND
• Since the two strands of DNA are complementary, each
strand acts as a template for building a new strand in
replication
• In DNA replication, the parent molecule unwinds, and
two new daughter strands are built based on base-
pairing rules
FIGURE 16.9-3

A T A T A T A T
C G C G C G C G
T A T A T A T A
A T A T A T A T
G C G C G C G C

(a) Parent molecule (b) Separation of (c) “Daughter” DNA molecules,

strands each consisting of one
parental strand and one
new strand
FIGURE 16.10
Parent First Second
cell replication replication

(a) Conservative
model

(b) Semiconservative
model

(c) Dispersive model

• Watson and Crick’s semiconservative model of replication
predicts that when a double helix replicates, each daughter
molecule will have one old strand (derived or “conserved” from
the parent molecule) and one newly made strand
• Competing models were the conservative model (the two parent
strands rejoin) and the dispersive model (each strand is a mix of
old and new)
FIGURE 16.12A

(a) Origin of replication in an E. coli cell

Origin of
replication Parental (template) strand

Daughter (new) strand

Double-
stranded Replication fork
DNA molecule Replication
bubble

Two
daughter
DNA molecules

0.5 m
FIGURE 16.12B

(b) Origins of replication in a eukaryotic cell

Double-stranded
Origin of replication DNA molecule

Parental (template) Daughter (new)

strand strand

Bubble Replication fork

Two daughter DNA molecules

0.25 m
DNA REPLICATION STEPS

1:Initiation and Unwinding

2:Primer Synthesis
3:Elongation
DNA REPLICATION
1: INITIATION AND UNWINDING
Initiation and Unwinding
•DNA replication occurs when the complementary
strands of DNA break apart and unwind.
•This is accomplished with the help of enzymes called
helicases.
•Each half will then be the template for a new,
complementary strand.
•Because the newly unwound single strands have a
tendency to rejoin, another group of proteins, the
single-strand binding proteins, keep the single
strands stable until elongation begins.
• At the end of each replication bubble is a replication fork, a Y-
shaped region where new DNA strands are elongating
• Helicases are enzymes that untwist the double helix at the
replication forks
• Single-strand binding proteins bind to and stabilize single-
stranded DNA
• Topoisomerase corrects “overwinding” ahead of replication
forks by breaking, swiveling, and rejoining DNA strands
DNA REPLICATION
•A third family of proteins, the topoisomerases, change DNA
supercoiling by inserting or removing superhelical twists.
•The point at which the double helix separates is called the
replication fork, because of the shape of the molecule.
DNA REPLICATION
2:PRIMER SYNTHESIS
Primer Synthesis
• Primase enzyme starts the actual
synthesis of the new DNA molecule.
• Primase synthesize primers (A short
segment of nucleotides - about 10 to 12
bases - used to initiate DNA synthesis in
the polymerase chain reaction).
• Primer synthesis marks the beginning of
the actual synthesis of the new DNA
molecule.
DNA REPLICATION
• Primase are required because DNA polymerases, the enzymes
responsible for the actual addition of new nucleotides to the new
DNA strand, can only add deoxyribonucleotides to the 3'-OH group
of an existing chain and cannot begin synthesis de novo.
• DNA replication can proceed only in the 5'-to-3' direction.
FIGURE 16.13

Primase

3
Topoisomerase
5 RNA
3 primer
5
3

Helicase

5
Single-strand binding
proteins
DNA REPLICATION
3: ELONGATION
Elongation
• At this point enzymes called DNA polymerases move along each of
the separated DNA strands, adding nucleotides to the exposed
bases according to the base pairing rules.
DNA REPLICATION
• DNA is always synthesized in the
5'-to-3' direction, meaning that
nucleotides are added only to the 3'
end of the growing strand.
• As shown in Figure, the 5'-phosphate
group of the new nucleotide binds to
the 3'-OH group of the last nucleotide
of the growing strand.
• Consequently, synthesis proceeds
immediately only along the so-called
leading strand. This immediate
replication is known as continuous
replication.
ANTIPARALLEL ELONGATION
• The antiparallel structure of the double helix affects replication
• DNA polymerases add nucleotides only to the free 3end of a growing strand;
therefore, a new DNA strand can elongate only in the 5to3direction
• Along one template strand of DNA, the DNA polymerase synthesizes a
leading strand continuously, moving toward the replication fork
• To elongate the other new strand, called the lagging strand, DNA
polymerase must work in the direction away from the replication fork
• The lagging strand is synthesized as a series of segments called Okazaki
fragments, which are joined together by DNA ligase
E) SEMI-DISCONTINUOUS
REPLICATION

Anti parallel strands replicated simultaneously

Leading strand synthesis continuously in 5’– 3’
Lagging strand synthesis in fragments in 5’-3’
FIGURE 16.15
Overview
Leading
Origin of replication Lagging
strand
strand

Primer

Lagging Leading
strand strand Origin of
Overall directions replication
of replication

3
5

5 RNA primer
3
3 Sliding clamp

DNA pol III

Parental DNA 5
3
5

5
3
3

5
FIGURE 16.16B-6
3
5 3
Template
strand 5
3 RNA primer
for fragment 1
5
1 3
5

3 Okazaki
fragment 1
5
1
RNA primer 3
for fragment 2 5
5
3
2
Okazaki
fragment 2 1 3
5
5
3

2
1 3
5 5
3
2
1 3
5
Overall direction of replication
FIGURE 16.17

Overview
Leading Origin of
replication Lagging
strand strand

Leading
Lagging strand
strand Overall directions
Leading strand of replication

5 DNA pol III

3 Primer
Primase
3 5
3
Parental DNA pol III Lagging strand
DNA 5
4 DNA pol I DNA ligase
35
3 2 1 3

5
FIGURE 16.18

DNA pol III

Parental DNA Leading strand
5
5 3 3

3
3 5 5

Connecting Helicase
protein

3 5 Lagging
DNA strand
Lagging strand template
pol III 5 3
 PROOFREADING AND REPAIRING
DNA
• DNA polymerases proofread newly made DNA, replacing any incorrect
nucleotides
• In mismatch repair of DNA, repair enzymes correct errors in base pairing
• DNA can be damaged by exposure to harmful chemical or physical agents such as
cigarette smoke and X-rays; it can also undergo spontaneous changes
• In nucleotide excision repair, a nuclease cuts out and replaces damaged
stretches of DNA
FIGURE 16.19
5 3

3 5
Nuclease

5 3

3 5

DNA
polymerase

5 3

3 5
DNA
ligase

5 3

3 5
FIGURE 16.20
5
Ends of parental Leading strand
DNA strands Lagging strand
3

Last fragment Next-to-last fragment

Lagging strand RNA primer

5
3
Parental strand
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication

5
New leading strand 3
New lagging strand 5
3
Further rounds
of replication

Shorter and shorter daughter molecules

FIGURE 16.UN03

DNA pol III synthesizes

leading strand continuously 3
5
Parental
DNA DNA pol III starts DNA
synthesis at 3 end of primer, Origin of
5 continues in 5  3 direction replication
3
5 Lagging strand synthesized
in short Okazaki fragments,
Helicase later joined by DNA ligase

Primase synthesizes 3
a short RNA primer 5
DNA pol I replaces the RNA
primer with DNA nucleotides
BASIC RULES OF
REPLICATION
A. Semi-conservative
B. Starts at the ‘origin’
C. Synthesis always in the 5-3’ direction
D. Can be uni or bidirectional
E. Semi-discontinuous
F. RNA primers required
 THE MECHANISM OF DNA
REPLICATION

Arthur Kornberg, a Nobel prize winner and other

biochemists deduced steps of replication
– Initiation
• Proteins bind to DNA and open up double helix
• Prepare DNA for complementary base pairing
– Elongation
• Proteins connect the correct sequences of
nucleotides into a continuous new strand of DNA
– Termination
• Proteins release the replication complex
CORE PROTEINS AT THE
REPLICATION FORK
Topoisomerases - Prevents torsion by DNA breaks
Helicases - separates 2 strands
Primase - RNA primer synthesis
Single strand - prevent reannealing
of single strands
binding proteins
- synthesis of new strand
DNA polymerase
- stabilises polymerase
Tethering protein
- seals nick via phosphodiester
DNA ligase linkage
WHAT KIND OF ENZYME
SYNTHESIZES THE NEW DNA
STRAND?

1) RNA polymerase

2) DNA Polymerase

3) Primase

4) Helicase

5) Topoisomerase
EUKARYOTIC CHROMOSOMES HAVE
MULTIPLE ORIGINS OF REPLICATION

1. True
2. False
IN WHAT DIRECTION IS THE NEWLY
SYNTHESIZED DNA PRODUCED?

1. 5'-3'

2. 3'-5'

3. In the direction of the major groove

4. Both 5'-3' and 3'-5' depending on which

strand is being replicated
WHAT IS THE SEQUENCE (1 TO 6) IN
WHICH THESE PROTEINS FUNCTION
DURING DNA REPLICATION

• ____ RNA primase

• ____ DNA ligase
• ____ DNA polymerase
• ____ Topoisomerase
• ____ DNA helicase
• ____ tethering proteins