DNA: The Early Experiments

Dr Solomon Genet
 Topics to be Covered

Beginnings of the science of genetics

Discovery of DNA and chromosomes
Experiments showing that DNA is the
genetic material
Griffith and Avery
Hershey and Chase
First hints into the structure of DNA
 Beginnings of Genetics
Gregor Mendel in 1865 studied and suggested
Patterns of inheritance in pea plants
 Mendels Peas
Y y
X

Y Y
Y = dominant
X
y = recessive
Worked with pure-breeding plants.
Each pea plant produces both pollen (the male gamete) and
eggs, which allowed Mendel to self-pollinate the plants. He did
this for several generation, until he had plants that, when self-
pollinated, always produced offspring that were identical to the
parent in the traits being studied.
Mendels Peas
Y y
X

Y Y
Y = dominant
X
y = recessive

Y y
and 3 yellow: 1 green
Mendels Peas
YY yy
X

Yy Yy
X

1 : 2 : 1
YY Yy yy
 Discovery of DNA
1865: Gregor Mendel
Patterns of inheritance in pea plants
1869: Johann Friedrich Miescher
Isolation of DNA (nuclein)

Extracted nuclein from white

blood cells from pus-covered
bandages at a local clinic
 Chromosomes
1865: Gregor Mendel
Patterns of inheritance in pea plants
1869: Johann Friedrich Miescher
Isolation of DNA (nuclein)
1882: Walter Fleming
Observes chromosome behavior during cell
division (mitosis)
 Chromosomes in Mitosis

http://scienceclassics.nas.edu/moore/chap02.html
Chromosome Theory of Inheritance
1865 Mendel: patterns of inheritance
1869 Miescher: isolation of DNA (nuclein)
1882 Fleming: chromosome behavior during
mitosis
1902 Sutton, Boveri: chromosome theory of
inheritance

Walter Sutton Theodor Boveri

 Grasshopper Chromosomes
Grasshoppers have 22 chromosomes in each
cell
These chromosomes come in 11 different sizes,
with two chromosomes of each size
During meiosis (cell divisions that produce eggs
and sperm), the number of chromosomes is
reduced to 11, one of each size
Diploid
QuickTime and a
TIFF (LZW) decompressor
Haploid (eggs & sperm) are needed to see this picture.
Mendel Revisited
YY yy
X

Yy Yy
X

1 : 2 : 1
YY Yy yy
 Genes and Chromosomes
1865 Mendel: patterns of inheritance
1869 Miescher: isolation of DNA (nuclein)
1882 Fleming: chromosome behavior during mitosis
1902 Sutton, Boveri: chromosome theory of
inheritance
1911 Thomas Hunt Morgan
studied fruit flies (Drosophila melanogaster)
shows genes are on chromosomes, in a linear array
yellow white miniature
body eyes wings
 Nucleic acid or protein?
1865 Mendel: patterns of inheritance
1869 Miescher: isolation of DNA (nuclein)
1882 Fleming: chromosome behavior during mitosis
1902 Sutton, Boveri: chromosome theory of inheritance
1911 Morgan: linear arrangement of genes on chromosomes

1920-30s Chromosomes = nucleic acid + protein

nucleic acid --> four nucleotides
proteins --> 20 amino acids

Is it DNA or Proteins that serves as genetic material?

Griffith: Pneumococcus virulence

1923--Frederick Griffith identifies two forms of

pneumococcus bacteria
R strain:
rough colonies
non-virulent
S strain:
smooth colonies
virulent
 Griffith: Pneumococcus virulence

R strain does not

kill mice

S strain does
Bacteria can be killed by heating

S strain
 Adding dead S strain
can make R strain virulent!
(S)

(R)
Griffith, 1928
(R plus dead S)
 DNA from S strain
makes R strain virulent
(R)

(S)

Avery, MacLeod,
and McCarty,
1944

(R + DNA
from S) (S)
 DNA is the Genetic Material
1865 Mendel: patterns of inheritance
1869 Miescher: isolation of DNA (nuclein)
1882 Fleming: chromosome behavior during
mitosis
1902 Sutton, Boveri: chromosome theory of
inheritance
1911 Morgan: linear arrangement of genes on
chromosomes
1944 Avery, MacLeod, and McCarty: DNA
carries genetic information
Phage: Viruses that infect bacteria
Protein coat
DNA
 DNA or protein?

Alfred Hershey
and Martha
Chase, 1952
Radioactively
label either DNA
or protein of
bacteriophage
T2
The Blender Experiment
 Separate Protein from DNA

Protein coats are

removed
DNA remains in cells
Cells are still
infected: produce new
phage
 DNA is the Genetic Material
1865 Mendel: patterns of inheritance
1869 Miescher: isolation of DNA (nuclein)
1882 Fleming: chromosome behavior during
mitosis
1902 Sutton, Boveri: chromosome theory of
inheritance
1911 Morgan: linear arrangement of genes on
chromosomes
DNA carries
1944 Avery, MacLeod, and McCarty genetic
information
1952 Hershey and Chase
 What is DNA, anyway?

Weakly acidic
Rich in phosphorus
Contains the sugar deoxyribose
Contains four different nucleotides (adenine,
guanine, thymine, and cytosine)
Found in the nuclei of cells (in chromosomes)
Transmits genetic information--but how?
 Chargaffs Rules

1950: Erwin Chargaff measures the

amounts of each of the four bases
In a number of different species,
Adenine = Thymine
Guanine = Cytosine
Hints that DNA is more complex
than previously believed
 X-ray crystallography
Make crystals of chemical of interest (so that
molecules line up in a defined pattern)
Put crystal in X-ray beam
Beam bends (diffracts) when it hits an atom
Can calculate structure from diffraction
pattern

Rosalind Maurice
Franklin Wilkins
X-ray diffraction pattern of DNA
 Preview: Structure of DNA

James Watson and Francis Crick, 1953

 Summary
Traits are inherited through pairs of genes,
one from each parent
Genes are carried in a linear array on
chromosomes
Genes are made of DNA
How does DNA encode genetic information?

Practice Question
If thymine makes up 15% of the bases in a
specific DNA molecule, what percentage of the
bases are cytosine?
10 minutes Break
Structure of DNA

Dr Solomon Genet
What was known before the double
helix
DNA is made up nucleotide subunits
Each nucleotide is made of: sugar, phosphate, base
There are 4 bases in DNA:
Adenine and guanine (purines), thymine and
cytosine (pyrimidines)

From Chargaffs work:

No. purines = No. pyrimidines
No. Adenines = No. Thymines
No. Guanines = No. Cytosines

From Franklin and Wilkins X-ray diffraction:

Structure of DNA is helical
 Nucleotides are the building
blocks of DNA

Without
phosphate=
Nucleoside
 The Pentose Sugars

DeoxyriboNucleic Acid: RiboNucleic Acid:

Nucleotide bases

in RNA in DNA
 Bases are linked to sugars via
glycosidic linkages

Sugar Base

(deoxyribose) (adenine)
 Sugar-phosphate backbone
DNA

Sugar
Phosphate

Phosphodiester
linkage
 DNA is a Double Helix:

Watson and Crick used four types of

information to deduce the double helix
structure:
Biophysical data of various kinds..
X-ray diffraction patterns (helical
nature plus dimensions)
The base ratios (Chargaff)
Model building
Watson-Crick model for DNA Structure

10.4
35.4
base
pairs

= 2 nm
Hydrogen Bonds Hold Base Pairs Together

H b o nds
2

bo n ds
3H
 View Down the Helix Axis

Bases

Sugar/phosphate
backbone
DNA: 3-D model

http://en.wikipedia.org/wiki/DNA
 DNA Sequence is written from 5' to 3'

A T G

5' end
3' end
Sugar/phosphate backbone is constant
Sequence written as: 5'-ATG-3'
Anti-parallel strand is: 3'-TAC-5'

(Therefore written: 5'-CAT-3')

The 2 strands are complementary and
antiparallel
 Nucleic Acid Length and
Nomenclature
Nucleic acid length is given as the
number of base pairs (bp)
1000 bp = 1 kb (kilobase)
1 million bp = 1 Mb (megabase)
Short lengths of DNA (less than ~ 50
bp) are called oligonucleotides
Polynucleotides are longer
Structure of DNA could explain
replication and heredity!

Watson & Crick noted that

replication could
involve:
1. Separating 2 strands
2. Synthesising new
complementary strands
 DNA is Flexible

Possible rotation about 7 bonds in the sugar-phosphate

backbone
6 freely rotate, limited rotation round bond 4
Purines occur in syn or anti, pyrimidines only in anti due
to steric hindrance
Different DNA Structures Exist: B-Form

Watson and
Crick structure
= B-form

B-form is the
most stable
under
physiological
conditions
It is right-handed
Different DNA Structures Exist: A-Form

A-form DNA forms under

conditions of higher salt
or alcohol:

Still right-handed
11 base-pairs per turn
Shallower minor groove
Deeper major groove
Different DNA Structures Exist: Z-Form

Z-form DNA can form

especially with
alternating Cs & Gs:

Left-handed!
12 base-pairs per turn
Deep minor groove
Short stretches exist in cells
 Grooves allow Access to Proteins

DNA-
binding
protein

DNA of
specific
sequence

Base sequence influences structure important here

 DNA Structure: Summary 1
2 antiparallel strands in a right-handed helix (B
and A)
Complementary base pairing:
A base pairs via two H bonds with T
G base pairs via three H bonds with C
Bases are on the inside, sugar-phosphate
backbone on the outside
10.4 bp per helical turn (B-form)
Structure maintained by H bonds and base
stacking interactions
Sequence written 5' to 3
 Cells have a lot of DNA!

DNA molecules are the largest macromolecules in the

cell and are commonly packaged into chromosomes
Most bacteria and viruses single chromosome
Eukaryotes many chromosomes
Chromosomes are divided into genes (code for proteins)
All the cells genes and the DNA between genes
(intergenic DNA) makes up the genome
DNA from a lysed E. coli cell
E. coli
Cells have a lot of DNA

E. coli = 2 m
E. coli DNA = 1.7 mm
You have ~2 m of DNA in each cell
(1014 cells)
Your total body DNA
2 x 1011 km: further than the sun!

How do cells package all that DNA?

 DNA Supercoiling
The coiling of a coil
The result of
structural strain on
the coil
 DNA supercoiling
Even isolated closed-circular DNA plasmids
are generally supercoiled

relaxed supercoiled
 DNA Supercoiling
Most cellular DNA is
underwound
Fewer helical turns
than expected (>10.4
bp/turn)
Removal of a turn
induces structural
strain
Strain on helix: relieved
by supercoiling
Or strand separation
 Consider a 260-bp
strand of B-DNA
Relaxed DNA, 10.4 bp
per turn
25 turns (260/10.4)
Join ends to produce
relaxed circular DNA
 What if 2 turns are
unwound before
circularising?
2 options
Here unwound
circle with Lk = 23
 DNA Supercoiling
OR this -ve
superhelix
(>10.4 bp/turn)
Lk =23
 Topoisomerase & DNA Supercoiling
Linking number is altered by topoisomerases
Transiently break DNA strand(s)
Pass other DNA strand(s) through the
break
Rejoin the broken ends

topoisomers
differ only by linking number
 Topoisomerases: Facts
Type I topoisomerases cleave 1 DNA strand
Type II topoisomerases cleave both strands
Topoisomers can only be interconverted by cleaving
1 or both of the strands and then rejoining them
Topoisomers can be separated by centrifugation or
electrophoresis
Supercoiled forms are more compact
Topisomerase has strand cutting (nuclease) activity
and strand sealing (ligase) activity
Dont require ATP, they store energy from breaking
phosphodiester bonds and use it to reseal the nick
 Plectonemic and Solenoidal Supercoiling
Plectonemic
coiling more
stable in solution
(isolated DNA)
Solenoidal
coiling
stabilised by
protein binding
Solenoidal interconvertible
coiling is the
more compact
structure

Pletonemic coiling does not give the compaction

required to package DNA into cells
Levels of DNA Organisation
 Chromatin
DNA in cells is found associated with proteins
collectively known as chromatin
Proteins include histones and non-histones
Histones are very tightly associated with DNA
like beads on a string
packaged into ordered nucleosomes
core structure of chromatin
not present in bacteria
small, basic proteins (rich in basic amino acids)
H1, H2A, H2B, H3, H4 (histone proteins)
Nucleosomes are the repeating units of
chromatin
 Nucleosomes
Each nucleosome contains a histone
octamer at its core
2 x H2A
2 x H2B
2 x H3
2 x H4

Histone tails
are highly
regulated!
 Nucleosomes
140 bp core DNA wrapped around each octamer
Nearly 2 turns (1.8)
Nucleosomes
Histone H1 acts
as a linker, but is
not part of the
nucleosome core
Chromatin Assembly
DNA wrapping around
each histone core
induces supercoiling of
linker DNA

Topoisomerase can relax

linker DNA by inducing
negative supercoil
Nucleosomes are only the first stage in DNA
condensation
Length
200 bp linear DNA 680 (68 nm)

200 bp in nucleosome 100 (10 nm)

Packing ratio ~ 7

7.8 x 109 bp in human 200 m

metaphase Packing ratio ~ 102-103!
chromosomes
Higher order chromatin structure: the
Solenoid
 DNA Structure: Summary 2
Many levels of organisation besides the
double helix
Cellular DNA has supercoiling properties
Topoisomerases modify linking number
DNA exists as chromatin in cells
Nucleosomes: repeating units of chromatin
 Chromosomal Elements
Cellular DNA made of genes and intergenic
DNA
Gene region of DNA coding for a protein
(related to phenotype)
Intergenic DNA is not junk:
regulatory sequences provide signals
mark beginning or end of genes
influence process of converting genetic
information to protein
Eukaryotic Chromosome:Repetitive DNA
Bacteria usually 1 chromosome/cell and each
chromosome has 1 copy of each gene
Eukaryotes have many stretches of DNA
sequences repeated several times in the total DNA
of a cell.
1. Highly repetitive: About 10-15% of mammalian
DNA. This class includes tandem repeats (repeats
placed next to each other)
2. Moderately repetitive: Roughly 25-40% of
mammalian DNA. This class includes interspersed
repeats (randomly interspersed)
3. Single copy (or very low copy number): This class
accounts for 50-60% of mammalian DNA.
Some repeated DNA may just be junk
Eukaryotic Chromosomes:
Centromere and Telomere
centromere

Telomere
(end)

Chromosome
(2 chromatids)
 Heterochromatin and Euchromatin

Heterochromatin:
Compacted chromatin
Transcriptionally silent (not present where
most genes are)
Concentrated at centromeres and telomeres
Highly repetitive sequences
Euchromatin:
Less compact chromatin
Transcriptionally active (where most genes
are located genes)
 Centromeres and Telomeres
Centromeres
-functions during cell division
-Rich in A=T pairs
-Many tandem repeats
-Heterochromatic

Telomeres
-TTAGGG repeats (100s-1000s)
-Heterochromatic
-Have specialised replication mechanism
 Introns and Exons
Many eukaryotic genes contain intervening sections of
DNA that do not code for proteins (introns)
Coding regions are exons
Few prokaryotic genes contain introns
 Pseudogenes
Some DNA sequences look like genes, but are not
e.g. GLUT6
Lost the ability to produce proteins
Evolutionary relics: human genome is continually
undergoing change!
Once a pseudogene has become non-functional it
will degrade through accumulation of more
mutations and eventually will no longer be
recognizable as a gene relic.
Gene Structure

Intragenic DNA
Genome organisation

Only 1.5 % of the

genome codes for
proteins
 Organelles of Eukaryotic Cells also
Contain DNA
Mitochondria and chloroplasts have DNA
-remnant of ancient bacteria?
Mitochondrial DNA molecules smaller than nuclear
chromosomes
Mitochondrial DNA codes for mitochondrial RNA
and a few mitochondrial proteins
Over 95% mitochondrial proteins actually encoded
by nuclear DNA
Mutation rate is higher (used in study of ancestry)
Maternally inherited
 Chromosomes
Human chromosomes in metaphase of mitosis:
46 per cell:
Maternal and
paternal copies of
Ch. 1-22
(autosomes)
+ XX or XY (sex
chromosomes)
Different shapes
and sizes
Karyotype
Normal Male
Karyotype:
46, XY
Maternal and
paternal copies
of chromosomes
1-22
(autosomes)
+ XY (sex
chromosomes)
Aneuploidy

Karyotype from a

female with Down

Syndrome

(47, XX, +21)

 DNA Structure: Summary 3
Cellular DNA is made up of genes and
intergenic DNA
Genes are made up of exons (coding) and
introns (non-coding)
Heterochromatin is found in regions of DNA
not containing genes, and particularly at
centromeres and telomeres
Euchromatin is located at areas of active
genes
Some organelles contain DNA
 Central dogma of molecular
genetics
Function
Functional and physical
unit of heredity passed
from parent to offspring
Protein
translation
RNA

1 gene = the DNA transcription

sequence that encodes
1 specific function DNA (1 gene)
 Practice Questions
1. Which of the following statements regarding the Watson-Crick
model for DNA structure is NOT TRUE:
a) The two strands of the double-helix are antiparallel
b) Nucleotides are joined together by 3-5 phosphodiester bonds
c) There are approximately 10 base pairs per helical turn
d) Adenine forms three hydrogen bonds with Thymine
e) It is the most common structure of DNA in cells

2. If the sequence of one strand of DNA is 5-

ATGCTCTACGTG-3, the sequence of the complementary strand
is:
a) 5-TACGAGATGCAC-3
b) 5-CACGTAGAGCAT-3
c) 5-ATGCTCTACGTG-3
d) 5-CGATGTGCTAGA-3
e) 3-GTGCATCTCGTA-5
 Practice Questions
3) Topoisomerases promote DNA supercoilling by:
a) Adding and removing bases from the double helix
b) Modifying the linking number of the double helix
c) Promoting the conversion of B-DNA to Z-DNA
d) Looping DNA around histones
e) Allowing other proteins to bind inside the grooves of the
double helix

4. In double-stranded DNA:
a) only a right-handed helix is possible.
b) sequences rich in AT base pairs are denatured less
readily than those rich in GC pairs.
c) the sequence of bases has no effect on the overall
structure.
d) the two strands are parallel.
e) the two strands have complementary sequences
 Practice Questions
5) DNA in a closed-circular, double-stranded molecule with no net
bending of the DNA axis on itself is:
a) a left-handed helix.
b) a mixed right- and left-handed helix.
c) relaxed.
d) supercoiled.
e) underwound.

6. The linking number (Lk) of a closed-circular, double-stranded DNA

molecule is changed by:
a) breaking a strand, then rejoining it.
b) breaking a strand, unwinding or rewinding the DNA, then
rejoining it.
c) supercoiling without the breaking of any phosphodiester bonds.
d) breaking all hydrogen bonds in the DNA.
e) underwinding without the breaking of any phosphodiester
bonds.
 Practice Questions
7) The chromosomal region that is the point of attachment of the
mitotic spindle is the:
a) centromere
b) endomere
c) intron
d) exon
e) telomere

8. Introns:
a) are frequently present in prokaryotic genes but are rare in
eukaryotic genes.
b) Code for large proteins
c) Are always shorter than exons
d) Are often found in eukaryotic genes
e) Code for molecules other than proteins
Physical and Chemical
Properties of DNA

Dr Solomon Genet
Denaturing and Annealing of DNA
Annealing of DNA
Hypo- and Hyper- Chromic Effect
Increasing UV light absorption;
Hyper-chromic effect

C
T

G
A
Double-
stranded Single-
DNA stranded Free
DNA nucleotides

Decreasing UV light absorption;

Hypo-chromic effect
ssDNA absorbs more UV light than
ds DNA
DNA Melting Points
Melting Point:
Temperature
at which half
of a sample is
ssDNA and
half is dsDNA
Depends on pH
and ionic
strength and
on the size
and base
composition
of the DNA
DNA Melting Points
 DNA Melting Points
Regions that are rich in A=T base pairs will specifically
denature while most of the DNA remains double-stranded
 Practice Questions
1. Which is true about UV light absorption by
DNA?
a) dsDNA absorbs more UV light than free nucleotides
b) ssDNA absorbs more light than free nucleotides
c) Pairing 2 strands of DNA will cause the hyper-
chromic effect
d) Denaturing dsDNA to ssDNA will cause the
hypochromic effect
e) Denaturing dsDNA to ssDNA causes the
hyperchromic effect
2. Explain why DNA with a high GC content has a
higher melting point
 DNA Replication
Dr Solomon Genet
MUHAS

http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471
661791/animations/replication/replication.swf
http://www.biostudio.com/d_%20DNA%20Replication
%20Nucleotide%20Polymerization.htm
http://biology-animations.blogspot.com/2007/10/molecular-
biology-of-dna-replication.html
 Objectives
Draw out the possible outcomes from the
Meselson-Stahl experiment and explain the
conclusions drawn from the study
List the steps involved in DNA Replication and
the proteins that mediate each step
Compare the mechanism of DNA replication on
the leading versus lagging strand
Identify the 5 and 3 ends of the leading and
lagging DNA template strands, the RNA primer,
and the nascent DNA molecule.
Draw the nucleophilic attack that results in the
next entering nucleotide becoming covalently
attached to the most recently added nucleotide
List the functions of telomerase
 DNA Replication
If laid out end to end, DNA is 1.8 meters long
(packed into a 0.0001 cm wide nucleus)
Each cell must accurately replicate its DNA once
and only once before dividing
Animal cells replicate DNA at approximately
1000 nucleotides/hour, so this is a relatively fast
process
Mistakes in DNA Replication (i.e. replicating the
DNA more than or less than one time/mutations)
could kill a cellHow is this process controlled?
Base Pairing Enables DNA
Replication
Each DNA strand
acts as a
template for a
complimentary
strand
A pairs to T, C
with G etc.
How does DNA serve as a template
for its own replication?
Parental
DNA

Semi-
Conservative Dispersive
Conservative

+ + +
 Meselson-Stahl DNA Replication
Experiment 1957
To track the pairing of template DNA strands with
newly synthesised DNA strands, the following
experiment was performed:
1. E. coli grown in 15N Nitrogen (heavy isotope)
(parental DNA labeled)
2. E. coli media switched to 14N nitrogen (light
isotope)
3. After 1,2, or 3 generations, DNA harvested from
E. coli
4. DNA was mixed with cesium chloride to
separate heavy and light DNA - centrifuge
 Meselson-Stahl DNA Replication
Experiment

In the Meselson-Stahl DNA replication

experiment, if the cells were first grown for
many generations in 15N containing media,
and then switched to 14N containing media,
what percent of the DNA had 1 light strand
and 1 heavy strand after 2 generations of
growth in 14N growth media?
Meselson-Stahl DNA Replication
Experiment Possible outcomes
Meselson-Stahl DNA Replication
Experiment -- Results
DNA Replication is Semi-Conservative
Parental
DNA

Semi-
Conservative Dispersive
Conservative

+ + +
 Steps Involved in DNA Replication
1. Identification of origins of replication
2. Unwinding (denaturation) of dsDNA to provide
a ssDNA template
3. Formation of the RNA primer
4. DNA Polymerase binds
5. Initiation of DNA synthesis and elongation
6. The torsional strain caused by unwinding of
DNA is relieved (throughout this process)
7. Removal of the RNA primer
8. Ligation of the newly synthesised DNA
segments in the lagging strand
DNA Helix is Opened at Replication Origins
This process can occur at
normal body temp
because:

1. Replication origin A-T rich

sequences attract Initiator
proteins

2. Only a small segment of

the DNA is opened at one
time (100 bp)

3. Replication origins are

A-T rich (2 H-bonds/pair
instead of 3 H-bonds in G-
C pairs)
Is Replication Bidirectional or
Unidirectional?
Replication can be Bi-directional from
Multiple Origins in Eukaryotes
DNA is Synthesised in 5 to 3 Direction

Enzyme: DNA polymerase

 The DNA Replication Fork is
Asymmetrical

How are both strands synthesised

simultaneously?
DNA Replication Forks are Asymmetrical
 Proteins Involved in DNA Replication
Protein Function
Initiator Proteins Bind to origin, facilitate binding of other
proteins
DNA Polymerases Deoxynucleotide polymerisation,
proofreading
Helicases Unwinding of DNA
Topoisomerases/ Relieve torsional strain that results from
gyrase helicase-induced unwinding
DNA primase Initiates synthesis of RNA primers
Single-strand Prevent premature reannealing of dsDNA
binding proteins
DNA ligase Seals the single strand nick between the
nascent chain and Okazaki fragments on
lagging strand
Telomerase Allows completion of DNA synthesis at ends
of chromosomes
 Helicases

Disrupt double helix in energy requiring reaction

Depends on hydrolysis of nucleoside 5-triphosphates (ATP)
Helicase is dimeric
Interacts with dsDNA and ssDNA
Subunits alternate binding to dsDNA release ssDNA,
rebind to dsDNA
ATP hydrolysis drives unwinding of DNA base pairs
Werners syndrome, Blooms syndrome: defective helicase
Single-Stranded Binding Protein (SSB)
 The Tangle Problem
"Since the two chains in our
model are intertwined, it is
essential for them to
untwist if they are to
separate. ...... Although it is
difficult at the moment to
see how these processes
occur without everything
getting tangled, we do not
feel that this objection
would be insuperable."
-J. D. Watson and F. H. C.
Crick, 1953
The Tangle Problem
Type I Topoisomerase
Type I Topoisomerase
Type I Topoisomerase
Type I Topoisomerase
 Type II Topoisomerase
Another part of same
DNA molecule binds to
N-gate
DNA binds to C- Gate
gate closes
trapping
DNA

Both
strands of
first DNA
cleaved

First DNA religated, Second DNA

second released passes through
through C-gate break
Type II Topoisomerase
DNA Polymerase
Proofreads as it
Synthesises
1 error in 108 bp
Error rate lower than
to be expected
simply by accuracy
of base pairing
Incorrect pairing
mutations
DNA polymerase
corrects its own
mistakes if wrong
nt added, cleaved
and replaced
Structure of E. coli DNA Polymerase

P: 53 polymerase activity
E: 35 exonuclease activity
Proofreading Explains 5 to 3 Synthesis Direction
On Lagging strand, DNA is Synthesised
in Fragments
 RNA primer
Polymerase can join
nucleotide to
Lagging
strand nucleotide but it cant
template start a strand
RNA primers made
by DNA primase
Primers removed by
nucleases that
recognise the
RNA/DNA helix
These gaps are filled
by DNA polymerase
DNA ligase joins
chain by joining the
3-OH to 5P
Synthesis of Okazaki Fragments
Primer extended by DNA polymerase

DNA synthesis continues until fragment extends as far

as the primer of previous fragment. New primer
synthesised near replication fork to begin process
Subunits of DNA Polymerase

polymerase
activity

exonuclease
proofreading
activity
clamp
encircles DNA
preventing
dissociation
Putting it all Together at the Replication
Fork

Both strands are produced by a single DNA polymerase III

Accomplished by the looping of the lagging strand
 One more problem
We know that DNA replication requires the
presence of some DNA ahead of the
sequence which is to be copied to serve
as the template for an RNA primer
However, at the extreme end of a linear
molecule, there can never be such a
template
How do cells ensure that part of the genome
is not lost in every round of replication?
Telomeres Allow the Completion of DNA
Synthesis at the Ends of Chromosomes
Only lagging
strand synthesis
shown here
 Summary
DNA Replication is a Semi-conservative process
DNA Replication is:
initiated at origins and moves bi-directionally
asymmetrical (leading v lagging strands)
moves only in the 5 to 3 direction
Practice Questions: DNA Replication
1. What is true about replication origins?
a) Prokaryotes tend to have more origins of replication
the eukaryotes
b) GC rich sequences are common here
c) Only short fragments of about 100 bp are opened up
initially
d) Initiator proteins recognise a nick in the DNA at the
origin
e) Single stranded binding proteins helps to identify the
origin
2. Explain the Meselson-Stahl experiment to show
if DNA replication is conservative or semi-
conservative
Practice Questions : DNA Replication
3. Which is NOT true about DNA
replication?
a) It proceeds in the 53 direction
b) It is normally bi-directional
c) On the lagging strand, replication occurs in
Okazaki fragments
d) It requires helicase to repair errors
e) It requires RNA primers
4. How is the tangle problem overcome
when opening up the DNA helix?
Practice Questions : DNA Replication
5. DNA polymerase, which is true?
a) Separate DNA polymerases act on the leading and
lagging strands
b) Has only 1 enzyme activity which is polymerase
activity
c) Has 53 exonuclease proof-reading activity
d) Has a -subunit which clamps the enzyme to the
DNA preventing it from dissociating
e) Has primase activity
6. Explain how Okazaki fragments are joined
together to make a continuous DNA strand.
Practice Questions : DNA Replication
7. What is NOT a function of DNA polymerase?
a) It recognises and replaces mis-matched base pairs
b) It catalyses the formation of phosphodiester bonds
c) It uses energy from phosphoandydride bonds
d) It is responsible for the high fidelity of DNA
synthesis
e) It rewinds the double helix just ahead of where it
adds new nucleotides to the DNA strand
8. Explain how telomerase enzymes help to
complete the DNA replication process
DNA Repair
Dr Nancy Carmichael
January 2008
 DNA Damage
Despite proofreading in DNA synthesis,
mismatches still occur
Incorrect base pairing
Insertion of one or more nucleotides
Also, DNA can be damaged by environmental
agents
Chemicals e.g. nitrous acid
Radiation e.g. UV light which can fuse 2 adjacent
pyriumidines
High energy radiation can cause double strand breaks
Bases lost or altered at rate of 1000s per day
If damage not repaired - mutation
 DNA Repair
Most DNA repair enzymes are involved in
recognising the lesion, cutting out the damaged
DNA and (using other strand as template) filling
in the gap

1. Strand directed mismatch repair

2. Repair of damage caused by UV light
3. Correction of base alterations (base excision
repair)
4. Repair of double strand breaks
 1. Mismatch repair (MMR)
Mismatched bases and small insertions and
deletions due to errors in DNA replication

ATGCTAGCATGCATATA
TACGATTGTACGTAT AT

Mismatch repair system must:

Recognise mismatch
Identify newly-replicated strand
Replace incorrect base
 How do they Distinguish Template
Strand and New Strand?
Tagging the template DNA with methyl
groups
Tag on GATC sequence which occurs
every 1000 nucleotides
Palindrome (GATC on complementary
strand)
Methylation does not take place
immediately after synthesis
Hemimethylated (template strand is, new
one is not)
DNA Methylation in E. coli

Methylation on N6
of adenines in the
GATC sequence
 Methylation of
Newly Replicated
DNA
For a brief time,
it is possible to
distinguish
between
template and
new DNA
 Mismatch DNA Repair
when strand with mismatch is identified,
endonuclease nicks mismatched strand
and removes the base(s)
gap left is filled using the template strand,
by a 53 DNA polymerase
3-OH end of the new DNA is sealed to the
rest by DNA ligase

Hereditatry nonpolyposis colon cancer

(HNPCC) has a defect in mismatch repair
in humans this is one of the most
common inherited forms of cancer
 2. Repair of Damage by UV
Nucleotide excision repair
Exposure of cell to UV light can result in
covalent joining of 2 adjacent pyrimidines
(usually thymines) producing a dimer
Thymine dimers prevent DNA polymerase from
replicating the DNA strand beyond the dimer
This method also used to remove bulky
chemical adducts formed by benzo(a)pyrene
from cigarette smoke
 Nucleotide Excision Repair
UV-specific
Human: 29-nt endonuclease
fragment removed
(uvrABC
excinuclease)
recognises dimer
and cleaves it out
Gap filled using
DNA polymerase
and ligase
 Xeroderma Pigmentosum
Cells cant repair
the damaged DNA
Accumulation of
mutations
Skin cancers
Most commonly
caused by absence
of UV-specific
excinuclease
 Photoreactivation
Alternate method of repairing pyrimidine dimers
Found in many organisms, but not humans
DNA photolyase is activated by UV light to cleave
dimer back to original two bases
 3. Base Excision Repair (BER)
Correction of base alterations, spontaneous
or by action of chemicals
Cytosine which can be deaminated to uracil
Nitrous acid deaminates cytosine, adenine
and guanine
About 10,000 purine bases are lost this way
every day
Lesions are corrected by base excision
repair
Defect in base excision repair --> MUTYH-
associated polyposis (a type of colon cancer)
 Base Excision Repair
Removal of abnormal bases
Uracil-N-glycosylase recognises uracil
Cleave base from backbone
Leaves an abasic site: apyrimidinic or apurinic
(AP site)
AP site must be recognised and repaired
Specific AP endonucleases recognise it and
makes cut to 5-side of AP site
Deoxyribose-phosphate lyase removes the
empty sugar phosphate residue
DNA polymerase and ligase then fill in the gap
BER: Damaged base is removed
BER: Strand with Abasic Site
is Nicked
BER: Fill Gap and Ligate
 Repair of Double Strand Breaks
Double strand breaks can be caused by
high energy radiation or oxidative free radicals
occur naturally during gene rearrangements
These cant be repaired by methods described
previously
2 systems
homologous recombination repair
nonhomologous end-joining repair
Non-homologous end-joining is error prone and
mutagenic, defects linked with predisposition to
cancer and immuno-deficiency syndromes
Homologous recombination repair uses enzymes
used in genetic recombination between homologous
chromosomes during meiosis
 Practice Question
There has been an outbreak of a severe respiratory
infection in your hospital. Some patients respond well to
treatment with antibiotics, but others do not. You have
isolated two strains of closely-related pathogenic bacteria
from these patients. After extensive analysis in the
laboratory, you find an interesting difference in how the
two strains repair mismatched bases: in the strain that
responds well to antibiotics, mismatches are repaired
efficiently, as you expected. In the other strain, however,
mismatches are repaired correctly only half of the time;
the other half of the time, the base on the template strand
is replaced to complement the base on the newly-
replicated, mutant strand. What might explain these
differences?