prepared by:

Ellen Mercado, MS
☂ By the 1940s, it became clear that deoxyribonucleic acids (DNA)
carry the hereditary information.

☂ Other work in the 1940s demonstrated that a gene controls the

manufacture of one protein.

☂ Thus the expression of a gene in terms of an enzyme protein led to

the study of protein synthesis and its control.

☂ The discovery of the double-helical nature of DNA by Watson &

Crick explained how genetic information could be duplicated and
passed on to succeeding generations.
Information
Transfer
In
Cells
 Genes, , and
☂ Gene: a segment of DNA that ☂ Chromosome 1: largest human chromosome,
carries sequence of nitrogen with ~249 million nucleotide base pairs &
representing approximately 8% of the entire
bases to direct the synthesis of a DNA content of a human cell
certainprotein, tRNA, or mRNA. ☂ Research into chromosome 1 has shown that it
☂ After the Human Genome Project, contains ~4,220 genes & was the last to have
scientists found that there were its DNA sequencing completed by The Human
around 20,000 genes within the Genome Project, over 30 years ago.
genome. Remarkably, these genes ☂ bacterial gene: continuous
comprise only about 1-2% of the 3 human gene: discontinuous
billion bp of DNA. ☂ Exon: a section of DNA, when transcribed,
☂ Chromosome 1 likely contains codes for a protein or RNA
2,000 to 2,100 genes that provide ☂ Intron: a section of DNA or mRNA that does
not code for a protein.
instructions for making proteins.
 DNA Replication ● DNA is synthesized from its
5’ -> 3’ end (from the 3’ -> 5’
direction of the template).
● involves separation of the 2 original strands ● The leading strand is synthesized
and synthesis of 2 new daughter strands continuously in the 5’ -> 3’
using the original strands as templates direction toward the replication
● DNA double helix unwinds at a specific fork.
point called an origin of replication. ● The lagging strand is synthesized
semidiscontinuously as a series
● The 2 polynucleotide chains are synthesized of Okazaki fragments, also in the
in opposite directions from the origin of
5’ -> 3’ direction, but away from
replication; thus DNA replication is the replication fork.
bidirectional.
● Okazaki fragments of the lagging
● At each origin of replication, there are 2 strand are joined by the DNA
replication forks, points at which new ligase.
polynucleotide strands are formed.
 DNA Replication DNA REPLICATION or DNA SYNTHESIS
● DNA makes duplicate copies of itself
● The 2 strands of DNA molecule separate
● Each strand then serves as a pattern/template in
making a new DNA molecule
● occurs only once in each cell cycle (in S phase)
old DNA
● occurs before a cell divides so that each daughter cell
new DNA strand
will have a copy
strand ● Results in 2 identical double-stranded DNA molecules

DNA is double-stranded. In replication process, the

strands are separated creating a replication fork.

Because each of these double-stranded molecules of DNA

consists of a single strand of old DNA (the template) and a
single strand of newly replicated DNA (the complement),
this is called Semi-Conservative Replication.
 Parent First Second
cell replication replication
Suggested Models
(a) Conservative
of DNA Replication model

(b) Semiconservative
model

(c) Dispersive model

The Recognized Model: Semi-conservative Mode
of DNA Replication
DNA Replication
DNA Replication requires:
öA strand of DNA to serve as a template
öSubstrates/Free nucleotides from the 4 types of deoxyribonucleoside
triphosphates (dNTPs): (1) dATP (2) dGTP (3) dCTP (4) dTTP
öDNA polymerase III - an enzyme that brings and links the free
nucleotides to the replicating DNA strand
öA source of chemical energy provided by any of the dNTPs: the bond
between 1st & 2nd phosphates of dNTP is broken to give off energy
required to drive the enzyme-catalyzed reaction

dNTPs: Deoxyadenosine triphosphate, Deoxyguanosine triphosphate,

Deoxythymidine triphosphate, and Deoxycytidine triphosphate
Energy Source for DNA Replication
☂Energy is obtained when DNA
polymerase breaks the bond
between the 1st and 2nd phosphates
of the incoming dNTP.
☂Pyrophosphate (PPi) is removed,
leaving the nucleotide which is
joined to free 3ʹ hydroxyl (3’ OH) of
the growing DNA strand.
☂Nucleotides have a hydroxyl (-OH)
group attached to the 3ʹ carbon of
the deoxyribose sugar.

☂the 3’ OH of a growing chain of

nucleotides attacks the α-phosphate
on the next dNTP to be incorporated
(blue at top right), resulting in a
phosphodiester linkage and the
release of pyrophosphate (PPi).
 DNA polymerase III
DNA polymerase Ü Catalyzes the synthesis of new DNA
molecule
Ü key enzymes in replication
Ü Requires 3’-OH end as primer for
Ü Once the 2 strands have separated at the the addition of the next nucleotide
replication fork, the nucleotides must be lined
to the growing chain of nucleotides
up in proper order for DNA synthesis.
Ü Nucleophilic attack from the 3’-OH
Ü In the absence of DNA polymerase, alignment is
group of DNA primer results in
slow.
covalent attachment of the new
Ü provides the speed and specificity of alignment
nucleotide
Ü Along the lagging strand (3’ -> 5’), the Ü Catalyzes ONLY in 5’ to 3’ direction
polymerase can synthesize only short fragments
(Okazaki) because these enzymes only work
from 5’ -> 3’. DNA Polymerase I
Ü Joining the Okazaki fragments and any Ü has 3’ to 5’ (proofreading)
remaining nicks is catalyzed by DNA ligase. exonucleases activity; cleaves RNA
primer from the elongating DNA
strand
DNA Replication & Enzymes Ä Primers: short oligonucleotides
that are 4 to 15 nucleotides long.
ÄSingle-strand binding protein (SSBP): prevents They are required to start the
the separated parental strands from reannealing; synthesis of both daughter
binds to a DNA strand at replication fork. strands.
ÄDNA Topoisomerase/DNA Gyrase: relaxes the Ä DNA Ligase: catalyzes the
tension of supercoiled twists created in formation of a covalent
unwinding the parental without rotation; breaks phosphodiester bond between
phosphodiester bond in one parental strand adjacent nucleotides that have
ahead of replication fork, creating swivel point been separated by a nick
on opposite strand (absence of a covalent bond
ÄDNA Primase: synthesizes the RNA primer between a 3’-OH end and a 5’-
strands. Primases are placed at about every 50 PO4-3 end).
nucleotides in the lagging strand synthesis.
♻ Each nucleotide that is
added to a growing DNA
strand is a nucleoside
triphosphate.
♻ dATP supplies adenine to
DNA and is similar to the
ATP of energy metabolism.
♻ The difference is in their
sugars: dATP has
deoxyribose while ATP has
ribose.
♻ As each monomer of dATP
joins the DNA strand, it
loses two phosphate
groups as a
pyrophosphate molecule.
Adenosine 5’-triphosphate (ATP) serves as a common currency
into which energy gained from food is converted and stored.
ester N H2
ATP anhydride
is N
N
O O O
a -
O- P-O- P-O- P-O-CH 2 O N N
Nucleotide! O- O- O- H H
H H
HO OH
AMP
ADP
Adenosine 5'-triphosphate
(ATP)
 Primase adds short
Single-strand binding primer to template
Helicase unwinds the
proteins stabilize strand
parental double helix
separate strands

Enzymes in DNA Replication

Ligase joins Okazaki

DNA polymerase III fragments and seals
binds nucleotides DNA polymerase I other nicks in sugar-
to form new strands (Exonuclease) removes phosphate backbone
RNA primer and inserts
the correct bases
DNA Replication Enzymes: Leading Strand
1. Helicase opens the DNA helix by breaking H bonds between the nitrogenous
bases. Unwinding can occur at either end or in the middle.
2. Topoisomerase II or DNA gyrase relaxes supercoiled chromosome to make DNA
more accessible for the initiation of replication; helps relieve the stress on DNA
when unwinding.
3. Single-strand binding proteins (SSBP) bind to single-stranded DNA to prevent
reannealing of hydrogen bonds between DNA strands.
4. DNA primase creates a single RNA primer to start the replication. Primase
protein makes a short segment of RNA primer complementary to the DNA.
5. Sliding clamp (usually not shown in illustrations) helps hold DNA pol III in place
when nucleotides are being added to the growing DNA strand.
6. DNA polymerase III slides along the leading strand in the 3’ to 5’ direction
synthesizing the matching strand in the 5’ to 3’ direction. This means that DNA
polymerase III adds nucleotides to the three prime (3')- end of a DNA strand,
one nucleotide at a time.
DNA Replication Enzymes: L a g g i n g Strand
1. Helicase opens the DNA helix by breaking 5. DNA polymerase adds a dNTP onto
hydrogen bonds between nitrogenous bases. the 3" end of the primer & initiates
2. Gyrase (or Topoisomerase II) relaxes lagging strand synthesis.
supercoiled DNA to make DNA more (Pyrophosphate is removed from dNTP).
accessible for initiation of replication; helps 6. The polymerase extends the primer for
relieve the stress on DNA when unwinding. about several nucleotides until it comes
3. Single-stranded binding proteins (SSBP) in contact with the 5' end of the
bind to each single-stranded DNA to prevent preceding primer.
reannealing (re-attachment) of hydrogen 7. Each RNA primer is degraded &
bonds between the separated DNA strands. replaced with required nucleotide by
4. DNA primase creates RNA primer to start DNA pol I.
the replication and before every synthesis of 8. DNA ligase seals the nicks between the
Okazaki fragment (1000-2000 bases in Okazaki fragments on the lagging strand
prokaryotes; 100-200 bases in eukaryotes) to create one continuous DNA strand.
 Lys-CH2 CH2 CH2 CH2 NH3 + acetylation
DNA Replication Lysine side chain deacetylation
(has a positive charge) O
Lys-CH2 CH2 CH2 CH2 NH3 + acetylation Lys-CH2 CH2 CH2 CH2 NH-CCH 3
Lysine side chain deacetylation Acetylated lysine side chain
(has a positive charge) O no charge)
(has
Lys-CH2 CH2 CH2 CH2 NH-CCH 3
Opening Up the Superstructure Acetylated lysine side chain
• During replication, the condensed superstructure of chromosomes is
(has no charge)
opened by a signal transduction mechanism.
• One step of this mechanism involves acetylation and deacetylation of key
lysine residues.
• Acetylation removes a positive charge and thus weakens the DNA-histone
interactions.
DNA Replication or DNA Synthesis Two Oddities of DNA
• The copying of DNA is remarkable in its speed Polymerase III
and accuracy.
• Replication begins at particular sites called ´ cannot begin DNA synthesis
origins of replication, where the 2 DNA by linking together the 2
strands are separated, opening up a individual nucleotides…
replication “bubble”. Rather, it can only elongate
• Eukaryotic chromosome may have a strand starting with either
hundreds or even thousands of origins of an RNA primer or 3’ OH end
replication. (hydroxyl terminus) of an
• Replication proceeds in both directions from existing DNA strand.
each origin, until the entire molecule is copied.
• Nucleotides are always added to the growing ´ Directionality of strand
strand at the 3' end (with free -OH group). synthesis - DNA pol III can
• The rate of elongation is ~ 500 nucleotides per only attach nucleotides in
second in bacteria and 50 nucleotides/second the 5’ to 3’ direction.
in human cells.
Ü Initiation of replication occurs at
DNA Replication: Initiation
specific nucleotide sequence
called the Origin of Replication,
where various proteins bind to
begin the replication.
Ü E. coli has a single origin of
replication ( oriC ) as most
prokaryotes do.
Ü Eukaryotes have hundreds origin
of replication.
Ü As the DNA opens up, Y-shaped
structures called replication forks
are formed.
DNA Replication: Initiation
2 replication forks are
formed at the origin of
replication, allowing for
bidirectional replication
and formation of a
structure that looks like a
bubble when viewed
through a transmission
electron microscope; as a
result, this structure is
called a replication
bubble.
In E. coli, the entire genome
is replicated in just 40
minutes,
at a pace of approximately
1,000 nucleotides per
second.

In eukaryotes, the pace is

much slower: about 40
nucleotides per second.
The coordination of the
protein complexes required
for the steps of replication
and the speed at which
replication must occur are
impressive, especially
considering that enzymes
are also proofreading,
which leaves very few errors
behind.
DNA Replication Enzymes
The Lagging Strand
(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 µm
(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 µm
• In order to begin making a Step 1: Initiation (Priming the Strand)
new strand, a helper strand
called Primer or RNA Primer
(typically 10-12 nucleotides
long) is needed to prime the
process.
• Primer is a short, single
strand of RNA (ribonucleic
acid) and is complementary
to the DNA template strand.
• Primers are formed by
enzyme called DNA Primase.
• DNA Polymerase III enzyme
can then add nucleotides to
the 3' end of the RNA
primer.
 Step 2 - Strand Elongation

Leading Strand – replicated continuously in 5’ to 3’ direction

Lagging Strand – synthesized in segments (Okazaki fragments) in 5’ to 3’ direction
Step 3: Termination of Replication
Eukaryotes initiate DNA replication at
Telomeres are regions of repetitive DNA close to the
multiple points in the chromosome, so
ends and help prevent loss of genes due to this
replication forks meet and terminate
shortening. Shortening of the telomeres is a normal
at many points in the chromosome.
process in somatic cells. This shortens the telomeres
of the daughter DNA chromosome. As a result, cells
In eukaryotes, a dedicated replisome
can only divide a certain number of times before the
removal pathway was recently
DNA loss prevents further division. This phenomenon
identified, which operates late during
is called the Hayflick limit. (max 60 times for human cell)
termination, after the DNA is fully
replicated.
Within the germ cell line, which passes DNA to the
next generation, telomerase extends the repetitive
Eukaryotes have linear chromosomes,
sequences of the telomere region to prevent
DNA replication is unable to reach the
degradation. Telomerase can become mistakenly
very end of the chromosomes. Due to
active in somatic cells, sometimes leading
this problem, DNA is lost in each
to cancer formation. Increased telomerase activity is
replication cycle from the end of the
one of the hallmarks of cancer.
chromosome.
Step 3: Termination of Replication
Termination requires that the progress of
the DNA replication fork must stop or be
blocked. Termination at a specific locus or
gene location involves the interaction
between two components:
(1) a termination site sequence in the
DNA, and
Bacteria have circular chromosomes,
(2) a protein which binds to this sequence
to physically stop DNA replication. termination of replication occurs
when the two replication forks meet
In various bacterial species, this is named each other on the opposite end of
the DNA replication terminus site-binding the parental chromosome.
protein, or Ter protein.
 In eukaryotes, a dedicated replisome
Termination of DNA replication occurs when removal pathway has recently been
● 2 replication forks meet on the same stretch of DNA identified, which operates late during
● forks converge until all intervening DNA is unwound termination, after the DNA is fully
● any remaining gaps are filled and ligated replicated.
● catenanes are removed
● replication proteins are unloaded It is unclear whether any comparable
pathway exists in bacteria.
Termination Once the primers are removed, a
Eukaryotic chromosomes have multiple origins of replication, free-floating DNA polymerase lands
which initiate replication almost simultaneously. Each origin of at the 3ʹ end of the preceding DNA
replication forms a bubble of duplicated DNA on either side of fragment and extends the DNA
the origin of replication. Eventually, the leading strand of one
over the gap. However, this creates
replication bubble reaches the lagging strand of another bubble,
and the lagging strand will reach the 5ʹ end of the previous
new nicks (unconnected sugar-
Okazaki fragment in the same bubble. phosphate backbone).
DNA polymerase III halts when it reaches a section of DNA In the final stage of DNA
template that has already been replicated. However, DNA replication, the enzyme ligase joins
polymerase III cannot catalyze the formation of a the sugar-phosphate backbones at
phosphodiester bond between the 2 segments of the new DNA each nick site. After ligase has
strand, and it drops off. connected all nicks, the new strand
These unattached sections of the sugar-phosphate backbone in is one long continuous DNA strand,
an otherwise full-replicated DNA strand are called nicks.
and the daughter DNA molecule is
Once all the template nucleotides have been replicated, the
replication process is not yet over. RNA primers need to be
complete.
replaced with DNA, and nicks in the sugar-phosphate backbone
need to be connected.
 Intercalating Agents: are compounds with
Chemical Inhibitors of DNA Replication fused aromatic ring systems that can wedge
(intercalate) between the stacked base pairs
Some types of drugs function by inhibiting of DNA. This disrupts the structure of the
DNA replication. DNA so that the replicative enzymes have
difficulty in synthesizing DNA past the
Substrate Analogs: analogs of dNTP's which function as "intercalated" sites.
chain terminators can be incorporated into DNA. These
analogs are usually either missing the 3' hydroxyl group Anthracycline glycosides and Actinomycin D
or have a chemical group, other than hydroxyl, in the 3' are intercalators used to treat a variety of
position. cancers.

DNA Damaging Agents: a variety of

compounds such as Cisplatin, cause chemical
damage to DNA and are used in the
treatment of cancers.

Topoisomerase Inhibitors: Nalidixic acid and

Fluoroquinolones are antibiotics used to
inhibit bacterial topoisomerases.
Replication
Overall direction 3’
of replication
3’ 5’
5’

3’

5’ 3’
5’

DNA polymerase enzyme adds DNA nucleotides

to the RNA primer.
Replication
Overall direction
3’
of replication
3’ 5’

5’
3’

5’ 3’
5’

DNA polymerase enzyme adds DNA nucleotides

to the RNA primer.
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.
Replication
Overall direction
3’
of replication
3’ 5’

5’
3’

5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Replication
Overall direction
3’
of replication
3’ 5’

5’
Okazaki fragment
3’

5’ 3’ 5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication
Overall direction
3’
of replication
3’ 5’

5’
Okazaki fragment
3’

5’ 3’ 5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication

3’
3’ 5’

5’
3’
5’ 3’ 5’ 3’5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication

3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
Replication

3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Exonuclease activity of DNA polymerase I

removes RNA primers.
Replication

3’
3’

5’
3’
5’ 3’5’ 3’
5’

Polymerase activity of DNA polymerase I fills the gaps.

Ligase forms bonds between sugar-phosphate backbone.
 3¢
5¢ 3¢
Template
strand 5¢
 3¢
5¢ 3¢
Template
strand 5¢
3¢ RNA primer
for fragment 1
5¢
1 3¢
5¢
 3¢
5¢ 3¢
Template
strand 5¢
3¢ RNA primer
for fragment 1
5¢
1 3¢
5¢

3¢ Okazaki
fragment 1
5¢
1 3¢
5¢
 3¢
5¢ 3¢
Template
strand 5¢
3¢ RNA primer
for fragment 1
5¢
1 3¢
5¢

3¢ Okazaki
fragment 1
5¢
1 3¢
RNA primer
for fragment 2 5¢
5¢
3¢
2
Okazaki
fragment 2 1 3¢
5¢
 3¢
5¢ 3¢
Template
strand 5¢
3¢ RNA primer
for fragment 1
5¢
1 3¢
5¢

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

2
1 3¢
5¢ 5¢
3¢
 3¢
5¢ 3¢
Template
strand 5¢
3¢ RNA primer
for fragment 1
5¢
1 3¢
5¢

3¢ Okazaki
fragment 1
5¢
1 3¢
RNA primer
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
 3¢
5¢ 3¢
Template
strand 5¢
3¢ RNA primer
for fragment 1
5¢
1 3¢
5¢

3¢ Okazaki
fragment 1
5¢
1 3¢
RNA primer
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
 l m us cles !
h os e m en t a
Flex t 1. DNA Replication. Show the 2 synthesized DNA
strands after DNA synthesis or DNA Replication
Legend:
Blue – original DNA strand Nucleotide Bases Two Newly Replicated DNA
Green – Newly replicated/ of Original DNA After the DNA Replication
Synthesized strand
A-T A-T A-T
T-A T-A T-A
T-A T-A T-A
C-G C-G C-G
C-G C-G C-G
A-T A-T A-T
G-C G-C G-C
C-G C-G C-G
A-T A-T A-T
A-T A-T A-T