DNA STRUCTURE,

REPLICATION AND
CONSENSUS SEQUENCES

DR. BONUKE ANYONA

 Learning Objectives
• By the end of this Topic, you should be able
to:
i. Define DNA and describe its structure
ii. Describe DNA replication mechanisms
iii. Describe DNA consensus sequences
 Discovery of DNA

• Nucleic Acids were discovered in 1869 by

Friedrich Mieschner as a substance contained
within nuclei
• 1929 Phoebus Levene discovered that DNA
was a polymer of nucleotides
• During the ’30s & 40’s proteins rather than
DNA was thought to hold genetic information
Griffith Discovers Transformation

 1928
 Attempting to develop a vaccine
 Isolated two strains of Streptococcus
pneumoniae
 Rough strain was harmless
 Smooth strain was pathogenic
 Transformation
•1. Mice injected with •2. Mice injected with live •3. Mice injected with •4. Mice injected with live R
live cells of harmless cells of killer strain S. heat-killed S cells. cells plus heat-killed S cells.
strain R. (harmless) (Pathogenic) (Harmless) (Pathogenic)

•Mice live. No live R cells •Mice die. Live S cells in •Mice live. No live S cells •Mice die. Live S cells in
in their blood. their blood. in their blood. their blood.
Transformation
What happened in the fourth experiment?
 The harmless R cells had been
transformed by material from the dead S
cells

 Descendents of the transformed cells were

also pathogenic

 Why?
Transformation
Griffith’s conclusion:
- information specifying virulence passed from
the dead S strain cells into the live R strain cells
- Griffith called the transfer of this information
transformation

7
 The Genetic Material
Avery, MacLeod, & McCarty, 1944
repeated Griffith’s experiment using purified cell
extracts and discovered:
- removal of all protein from the transforming
material did not destroy its ability to transform
R strain cells
- DNA-digesting enzymes destroyed all
transforming ability
- the transforming material is DNA
8
Oswald & Avery
What is the transforming material?
 Cell extracts treated with protein-digesting
enzymes could still transform bacteria

 Cell extracts treated with DNA-digesting

enzymes lost their transforming ability

 Concluded that DNA, not protein,

transforms bacteria
Bacteriophages
 Viruses that infect
bacteria
 Consist of protein and •bacteri
al cell •plasma
DNA wall membran
e
 Inject their hereditary
material into bacteria
•cytoplas
m
Hershey & Chase’s Experiments

• Created labeled bacteriophages

– Radioactive sulfur – Labels Proteins
– Radioactive phosphorus – Labels Nucleic Acids

• Allowed labeled viruses to infect bacteria

• Asked: Where are the radioactive labels after

infection?
Hershey – Chase Experiment
Hershey – Chase Experiment

13
 What is DNA?
• DNA is a Nucleic Acid
• Polymer of Nucleotides
• Each nucleotide consists of
– Deoxyribose (5-carbon sugar)
– Phosphate group
– A nitrogen-containing base
• Four bases
– Adenine, Guanine, Thymine, Cytosine
Nucleotide Structure
Composition of DNA
• Chargaff showed that:
– Amount of adenine relative to guanine differs
among species
– Amount of adenine always equals amount of
thymine and amount of guanine always equals
amount of cytosine
A=T and G=C
 Rosalind Franklin’s Work
• Was an expert in X-ray crystallography
• Used this technique to examine DNA fibers
• Concluded that DNA was some sort of helix
Watson-Crick Model

• DNA consists of two nucleotide strands:

Double Helix
• Strands run in opposite directions - Antiparallel
• Strands are held together by hydrogen bonds between
bases
• A binds with T and C with G
• The sides of the ladder are a sugar-phosphate backbone,
while the “rungs” of the ladder are the bases
DNA is antiparallel
 Base-pairing rule
•The four bases of DNA are:
•Adenine (A) Guanine (G)
Thymine (T) Cytosine (C)
•Adenine always hydrogen bonds
with Thymine (A-T)
•Guanine always hydrogen bonds
with Cytosine (G-C)
•These bonding patterns are called
base pairings (bp)
• The pattern of base pairing is the mechanism by which DNA
holds information.
• Humans have a > 3 billion of these base pairings
• Less than 5% of our DNA actually forms genes
• There are about 30,000 genes encoded in our DNA, nearly half
of these genes either have yet to be discovered or their function
is unknown
DNA is written out like this:
CTCGAGGGGCCTAGACATTGCCCTCCAGAGAGAGCAC
CCAACACCCTCCAGGCTTGACCGGCCAGGGTGTCC
CCTTCCTACCTTGGAGAGAGCAGCCCCAGGGCATC
CTGCAGGGGGTGCTGGGACACCAGCTGGCCTTCAA
GGTCTCTGCCTCCCTCCAGCCACCCCACTACACGCT
GCTGGGATCCTGGA
Genetic Information
Transfer
 Central dogma

replication transcription translation

DNA RNA protein

reverse
transcription
 Central dogma
• Replication: synthesis of daughter DNA
from parental DNA
• Transcription: synthesis of RNA using
DNA as the template
• Translation: protein synthesis using
mRNA molecules as the template
• Reverse transcription: synthesis of DNA
using RNA as the template
DNA
Replication
 Section 1

General Concepts of
DNA Replication
 DNA Replication
• A reaction in which daughter DNAs are
synthesized using the parental DNAs as the
template.
• Transferring the genetic information to the
descendant generation with a high fidelity

replication

parental DNA
daughter DNA
 Daughter strand synthesis
• Chemical formulation:

• The nature of DNA replication is a series of

3´- 5´phosphodiester bond formation
catalyzed by a group of enzymes.
Phosphodiester bond formation
 DNA replication system
Requirements
Template: double stranded DNA
Substrate: dNTP
Primer: short RNA fragment with a free 3
´-OH end
Enzyme: DNA-dependent DNA polymerase
(DDDP), other enzymes,
protein factor
Characteristics of replication

 Semi-conservative replication
 Bidirectional replication
 Semi-continuous replication
 High fidelity
§1.1 Semi-Conservative Replication
 Semi-conservative replication

Half of the parental DNA molecule is conserved

in each new double helix, paired with a newly
synthesized complementary strand. This is called semi-
conservative replication

Significance
The genetic information is ensured to be transferred from
one generation to the next generation with a high fidelity.
Semiconservative replication
 §1.2 Bidirectional Replication
• Replication starts from unwinding the dsDNA at
a particular point (called origin), followed by the
synthesis on each strand.
• The parental dsDNA and two newly formed
dsDNA form a Y-shape structure called
replication fork. 5'
3'

3'
5'
3'
5'

5'

direction of 3'

replication
 Bidirectional replication
• Once the dsDNA is
opened at the origin,
two replication forks
are formed
spontaneously.
• These two replication
forks move in
opposite directions
as the syntheses
continue.
Replication of Prokaryotes

The replication
process starts
from the origin,
and proceeds in
two opposite
directions.
It is named 
replication.
 Replication of eukaryotes
• Chromosomes of eukaryotes have multiple
origins.
• The space between two adjacent origins is called
the replicon, a functional unit of replication.
§1.3 Semi-continuous Replication

The daughter strands on two template strands

are synthesized differently since the replication
process obeys the principle that DNA is
synthesized from the 5´end to the 3´end.

3'
5' 3' 5'
direction of unwinding
3'
5'
 Leading strand

On the template having the 3´- end, the

daughter strand is synthesized continuously
in the 5’-3’ direction. This strand is referred
to as the leading strand.

3'
5' 3' 5'
direction of unwinding
3'
5'
Semi-continuous replication
3'

5'
replication fork

3'
5'
3'
replication direction 5'

3'

5'
Okazaki fragment
3'

5'
leading strand
 Okazaki fragments

• Many DNA fragments are synthesized

sequentially on the DNA template strand having
the 5´- end.
• These DNA fragments are called Okazaki
fragments. They are 1000 – 2000 nt long for
prokaryotes and 100-150 nt long for eukaryotes.
• The daughter strand consisting of Okazaki
fragments is called the lagging strand.
 Semi-continuous replication

Continuous synthesis of the leading strand

and discontinuous synthesis of the lagging
strand represent a unique feature of DNA
replication. It is referred to as the semi-
continuous replication.
 Section 2

Enzymology
of DNA Replication
 Enzymes and protein factors
protein Mr # function

Dna A protein 50,000 1 recognize origin

Dna B protein 300,000 6 open dsDNA
Dna C protein 29,000 1 assist Dna B binding
DNA pol Elongate the DNA
strands
Dna G protein 60,000 1 synthesize RNA primer
SSB 75,600 4 single-strand binding

DNA topoisomerase 400,000 4 release supercoil

constraint
2.1 DNA Polymerase
DNA-pol of prokaryotes
• The first DNA- dependent
DNA polymerase (short
for DNA-pol I) was
discovered in 1958 by
Arthur Kornberg
– Received Nobel Prize in
physiology and medicine in
1959.
2.1 DNA Polymerase
• Later, DNA-pol II and DNA-pol III were
identified in experiments using mutated
E. coli cell line.
• All of them possess the following
biological activity.
1. 53 polymerizing
2. Exonuclease
DNA-pol of E. coli
 DNA-pol I

• Mainly
responsible for
proofreading
and filling the
gaps, repairing
DNA damage
 DNA-pol II

• Temporary functional when DNA-pol I and

DNA-pol III are not functional
• Still capable for doing synthesis on the
damaged template
• Participating in DNA repairing
 DNA-pol III
• A heterodimer enzyme composed of ten
different subunits
• Having the highest polymerization
activity (105 nt/min)
• The true enzyme responsible for the
elongation process
 Structure of DNA-pol III

α ： has 5´→ 3´
polymerizing activity
ε ： has 3´→ 5´
exonuclease activity and
plays a key role to ensure
the replication fidelity.
θ: maintain heterodimer
structure
• Structure of DNA-pol III
 DNA-pol of eukaryotes
DNA-pol : initiate replication and DnaG,
synthesize primers primase
DNA-pol : replication with low repairing
fidelity

DNA-pol : polymerization in
mitochondria
DNA-pol : elongation DNA-pol III
DNA-pol : proofreading and DNA-pol I
filling gap
§2.2 Primase

• Also called DnaG

• Primase is able to synthesize primers using
free NTPs as the substrate and the ssDNA
as the template.
• Primers are short RNA fragments of several
(8-30) nucleotides long.
Role of Primase Enzyme
Role of Primase Enzyme

• Primers provide free 3´-OH groups to react

with the -P atom of dNTP to form
phosphoester bonds.
• Primase, DnaB, DnaC and a replication
origin form a Primosome Complex at the
initiation phase.
 §2.3 Helicase

• Also referred to as DnaB.

• It opens the double strand DNA with
consuming ATP.
• The opening process with the assistance of
DnaA and DnaC
 §2.4 SSB protein
• Stand for single strand DNA binding protein
• SSB protein maintains the DNA template in the
single strand form in order to
• prevent the dsDNA formation;
• protect the vulnerable ssDNA from
nucleases.
§2.5 Topoisomerase
• Opening the dsDNA will create supercoil
ahead of replication forks.
• The supercoil constraint needs to be released
by topoisomerases.
§2.5 Topoisomerase
• The interconversion of topoisomers of dsDNA
is catalyzed by a topoisomerase in a three-step
process:
• Cleavage of one or both strands of DNA
• Passage of a segment of DNA through this
break
• Resealing of the DNA break
 Topoisomerase I (topo I)

• Also called -protein in prokaryotes.

• It cuts a phosphoester bond on one DNA
strand, rotates the broken DNA freely around
the other strand to relax the constraint, and
reseals the cut.
 Topoisomerase II (topo II)
• It is named gyrase in prokaryotes.
• It cuts phosphoester bonds on both strands of
dsDNA, releases the supercoil constraint, and
reforms the phosphoester bonds.
• It can change dsDNA into the negative supercoil
state with consumption of ATP.
§2.6 DNA Ligase
• Connect two adjacent ssDNA strands by
joining the 3´-OH of one DNA strand to the 5
´-P of another DNA strand.
• Sealing the nick in the process of replication,
repairing, recombination and splicing.
§2.6 DNA Ligase

3' 5'
5' 3'
RNAase
3' 5'
5' OH P 3'

dNTP DNA polymerase

3' 5'
5' P 3'
ATP DNA ligase
3' 5'
5' 3'
§2.7 Replication Fidelity
• Replication based on the principle of base
pairing is crucial to the high accuracy of the
genetic information transfer.
• Enzymes use two mechanisms to ensure the
replication fidelity.
– Proofreading and real-time correction
– Base selection
 Proofreading and correction
• DNA-pol I has the function to correct the
mismatched nucleotides.
• It identifies the mismatched nucleotide,
removes it using the 3´- 5´ exonuclease
activity, add a correct base, and continues
the replication.
 Exonuclease functions
5´→3´ exonuclease 3´→5´ exonuclease
activity activity
cut primer or excise excise mismatched
mutated segment nuleotides

5' 3'
C T T C A G G A

G A A G T C C G G C G
3' 5'
 Section 3

DNA Replication
Process
 Synthesis Phase (S phase)
• S phase during interphase of the cell cycle
• Nucleus of eukaryotesti

S
phase
DNA replication
takes
place in the S phase. G1 interphase G2

Mitosis
-prophase
-metaphase
-anaphase
-telophase
Sequential actions

• Initiation: recognize the starting point,

separate dsDNA, primer synthesis, …
• Elongation: add dNTPs to the existing
strand, form phosphoester bonds, correct
the mismatch bases, extending the DNA
strand, …
• Termination: stop the replication
3.1 Replication of
Prokaryotes
§3.1 Replication of Prokaryotes
a. Initiation

• The replication starts at a particular

point called origin.
• The origin of E. coli, ori C, is at the
location 82.
• The structure of the origin is 248 bp long
and AT-rich.
Genome of E. coli
 Structure of ori C
• Three 13 bp consensus sequences
• Two pairs of anti-consensus repeats
 Formation of replication fork
• DnaA recognizes ori C.
• DnaB and DnaC join the DNA-DnaA
complex, open the local AT-rich region,
and move on the template downstream
further to separate enough space.
• DnaA is replaced gradually.
• SSB protein binds the complex to stabilize
ssDNA.
 Primer synthesis

• Primase joins and forms a complex called

primosome.
• Primase starts the synthesis of primers on
the ssDNA template using NTP as the
substrates in the 5´- 3´ direction at the
expense of ATP.
• The short RNA fragments provide free 3´-
OH groups for DNA elongation.
 Releasing supercoil constraint
• The supercoil constraints are generated
ahead of the replication forks.
• Topoisomerase binds to the dsDNA region
just before the replication forks to release
the supercoil constraint.
• The negatively supercoiled DNA serves as
a better template than the positively
supercoiled DNA.
 Primosome complex

Dna B Dna C
Dna A primase 3'

5'
3'

5'
DNA topomerase
b. Elongation

• dNTPs are continuously connected to the

primer or the nascent DNA chain by
DNA-pol III.
• The core enzymes ( 、  、 and  )
catalyze the synthesis of leading and
lagging strands, respectively.
• The nature of the chain elongation is the
series formation of the phosphodiester
bonds.
• The synthesis direction
of the leading strand is
the same as that of the
replication fork.
• The synthesis direction
of the latest Okazaki
fragment is also the
same as that of the
replication fork.
 Lagging strand synthesis
• Primers on Okazaki fragments are
digested by RNase.
• The gaps are filled by DNA-pol I in the 5
´→3´direction.
• The nick between the 5´end of one
fragment and the 3´end of the next
fragment is sealed by ligase.
3' 5'
5' 3'

RNAase
3' 5'
5' OH P 3'

dNTP DNA polymerase

3' 5'
5' P 3'
ATP DNA ligase
3' 5'
5' 3'
c. Termination

• The replication of E. coli is bidirectional

from one origin, and the two replication
forks must meet at one point called ter at
bp position 32.
• All the primers will be removed, and all
the fragments will be connected by DNA-
pol I and ligase.
3.2 Replication of
Eukaryotes
§3.2 Replication of Eukaryotes
• DNA replication is closely related with
cell cycle.
• Multiple origins on one chromosome, and
replications are activated in a sequential
order rather than simultaneously.
Cell cycle
 Initiation

• The eukaryotic origins are shorter than

that of E. coli.
• Requires DNA-pol  (primase activity)
and DNA-pol  (polymerase activity and
helicase activity).
• Needs topoisomerase and replication
factors (RF) to assist.
b. Elongation

• DNA replication and nucleosome

assembling occur simultaneously.
• Overall replication speed is compatible
with that of prokaryotes.
c. Termination
3' 5'
5' 3'
3' 5'
5' 3'

connection of discontinuous
segment
3' 5'
5' 3'
3' 5'
5' 3'
 Telomere
• The terminal structure of eukaryotic DNA
of chromosomes is called telomere.
• Telomere is composed of terminal DNA
sequence and protein.
• The sequence of typical telomeres is rich
in T and G.
• The telomere structure is crucial to keep
the termini of chromosomes in the cell
from becoming entangled and sticking to
each other.
 Telomerase
• The eukaryotic cells use telomerase to
maintain the integrity of DNA telomere.
• The telomerase is composed of
telomerase RNA
telomerase association protein
telomerase reverse transcriptase

• It is able to synthesize DNA using RNA as

the template.
 Significance of Telomerase
• Telomerase may play important roles is
cancer cell biology and in cell aging.
 Section 4

Other Replication Modes

§4.1 Reverse Transcription
• The genetic information carrier of some
biological systems is ssRNA instead of dsDNA
(such as ssRNA viruses).
• The information flow is from RNA to DNA,
opposite to the normal process.
• This special replication mode is called
reverse transcription.
Viral infection of RNA virus

Reverse
transcription

Reverse transcription is a process in which

ssRNA is used as the template to synthesize
Process of Reverse transcription

• Synthesis of ssDNA complementary to

ssRNA, forming a RNA-DNA hybrid.
• Hydrolysis of ssRNA in the RNA-DNA
hybrid by RNase activity of reverse
transcriptase, leaving ssDNA.
• Synthesis of the second ssDNA using the
left ssDNA as the template, forming a
DNA-DNA duplex.
 Reverse transcriptase Enzyme

Reverse transcriptase is the enzyme for the

reverse transcription. It has activity of three
kinds of enzymes:
• RNA-dependent DNA polymerase
• RNase
• DNA-dependent DNA polymerase
 Significance of RT
• An important discovery in life science and
molecular biology
• RNA plays a key role just like DNA in the
genetic information transfer and gene
expression process.
• RNA could be the molecule developed earlier
than DNA in evolution.
• RT is the supplementary to the central
dogma.
 Significance of RT

• This discovery enriches the understanding

about the cancer-causing theory of viruses.
(cancer genes in RT viruses, and HIV
having RT function)
• Reverse transcriptase has become a
extremely important tool in molecular
biology to select the target genes.
§4.2 Rolling Circle Replication
3' 5'

3'

5'

3'

5'
§4.3 D-loop Replication
 Section 5

DNA Damage and Repair

§5.1 Mutation

Mutation is a change of nucleic acids in

genomic DNA of an organism. The
mutation could occur in the replication
process as well as in other steps of life
process.
 Consequences of mutation

• To create a diversity of the biological world; a

natural evolution of biological systems
• To lead to the functional alternation of
biomolecules, death of cells or tissues, and
some diseases as well
• Changes of genotype, but no effect on
phenotype
§5.2 Causes of Mutation

Chemical
UV radiation modification
Physical carcinogens
factors DNA
damage

infection spontaneous
mutation
T

G
viruses evolution
 Physical damage
O O
N N
R N O R N O
UV CH3
P O P
CH3
N CH3
R N O R N O
N
O
CH3

)
(TT)
 Mutation caused by chemicals

• Carcinogens can cause mutation.

• Carcinogens include:
• Food additives and food preservatives; spoiled
food
• Pollutants: automobile emission; chemical wastes
• Chemicals: pesticides; alkyl derivatives; -NH 2OH
containing materials
§5.3 Types of Mutation
a. Point mutation (mismatch)
Point mutation is referred to as the single
nucleotide alternation.
• Transition: the base alternation from
purine to purine, or from pyrimidine to
pyrimidine.
• Transversion: the base alternation
between purine and pyrimidine, and vise
versa.
Transition mutation
 Hb mutation causing anemia
Single base mutation leads to one AA change,
causing disease.

HbS HbA

 chains CAC CTC

 mRNA GUG GAG

AA residue 6 in  chain Val Glu

b. Deletion and insertion

• Deletion: one or more nucleotides are

deleted from the DNA sequence.
• Insertion: one or more nucleotides are
inserted into the DNA sequence.

Deletion and insertion can cause a reading

frame shift.
Frame-shift mutation

Normal
5´… …GCA GUA CAU GUC … …
Ala Val His Val
Deletion C
5´… …GAG UAC AUG UC … …
Glu Tyr Met Ser
c. Rearrangement
It is an exchange of large DNA fragments.
It can be either reverse the direction or
recombination between chromosomes.
i. Site-specific recombination
ii. Homologous genetic recombination
iii. DNA transposition
§5.4 DNA Repairing

• DNA repairing is a kind response made by

cells after DNA damage occurs, which may
restore their natural structures and normal
biological functions.
• DNA repairing is a supplementary to the
proofreading-correction mechanism in DNA
replication.
 Light repairing

O O
N N
R N O R N O
UV CH3
P O P
CH3
N CH3
R N O R N O
N
O
CH3

)
(TT)
 Excision repairing
• One of the most important and effective
repairing approach.
• UvrA and UvrB: recognize and bind the
damaged region of DNA.
• UvrC: excise the damaged segment.
• DNA-pol Ⅰ: synthesize the DNA segment
to fill the gap.
• DNA ligase: seal the nick.
 Xeroderma pigmentosis (XP)

• XP is an autosomal recessive genetic disease.

• Patients suffer from hyper-sensitivity to UV

which results in multiple skin cancers.

• The cause is due to the low enzymatic

activity for the nucleotide excision-repairing
process, particular thymine dimer.
Excision repairing
 Recombination repairing
• It is used for repairing when a large segment
of DNA is damaged.
• Recombination protein RecA, RecB and
RecC participate in this repairing.
 SOS repairing
• It is responsible for the situation where DNA is
severely damaged and the replication is hard to
continue.
• If workable, the cell could be revived, but may
leave many errors.
• In E. coli, uvr gene and rec gene as well as Lex
A protein constitute a regulatory network.