Biology 1010

Chapter 14 Lecture
• In 1953, James Watson &
Francis Crick introduced an
elegant double-helical
model for the structure of
deoxyribonucleic acid
(DNA)

• DNA, the substance of

inheritance, is the most
celebrated molecule of our
time

• Hereditary information is
encoded in DNA and
reproduced in all cells of
the body (DNA replication)
 Griffith’s DNA Experiment
• In 1928, Frederick Griffith performed one of the first experiments suggesting that
bacteria are capable of transferring genetic information

• Griffith worked with two strains of Streptococcus pneumoniae bacteria which infect mice
- a type S (smooth) and type R (rough) strain

• When he mixed heat-killed remains of the pathogenic strain with living cells of the
harmless strain, some living cells became pathogenic

• He called this phenomenon transformation, now defined as a change in genotype and

phenotype due to assimilation of foreign DNA

• Griffith's findings were followed by research in the late 1930s and early 40s that isolated
DNA as the material that communicated this genetic information
Experiment Mixture of ***
Living Living Heat-killed heat-killed
S cells R cells S cells S cells and
(control) (control) (control) living R cells

Results
Mouse dies Mouse healthy Mouse healthy Mouse dies

Living S cells
• Later work by Oswald Avery and others identified the transforming
substance as DNA

• Many biologists remained skeptical, mainly because little was known

about DNA and they thought proteins were better candidates for the
genetic material
 Hershey and Chase

• In 1952, Alfred Hershey and Martha Chase confirmed that DNA is genetic
material, using a virus called T2

• While DNA had been known to biologists since 1869, many scientists still
assumed that proteins carried the information for inheritance because
DNA appeared simpler than proteins

• Hershey and Chase showed that when viruses, which are composed of
DNA and protein, infect bacteria, their DNA enters the host bacterial
cell, but most of their protein does not
Experiment
Batch 1: Radioactive sulfur (35S) in viral protein
***
1 Labeled viruses 2 Agitation frees outside 3 Centrifuged cells
infect cells. viral parts from cells. form a pellet.
Radioactive 4 Radioactivity
protein (viral protein)
found in liquid

Centrifuge

Pellet
Batch 2: Radioactive phosphorus (32P) in viral DNA
Radioactive
DNA

Centrifuge
4 Radioactivity (viral
Pellet DNA) found in pellet
 Chargaff’s Rules
 It was known that DNA is a polymer of nucleotides, each consisting of a
nitrogenous base, a sugar, and a phosphate group

 In 1950, Erwin Chargaff reported that DNA composition varies from one
species to the next
 Two findings became known as Chargaff’s Rules: The base composition of
DNA varies between species
 In any species the number of A and T bases is equal and the number of G and
C bases is equal
 The basis for these rules was not understood until the discovery of the double helix
Phosphate

3 end

Sugar
DNA (deoxyribose)
nucleotide Nitrogenous
base
Sugar– Nitrogenous bases
phosphate 5 end
backbone
Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)
3 end

DNA
nucleotide
 The DNA Helix

• James Watson and Francis Crick were first to determine the structure of
DNA

• Maurice Wilkins and Rosalind Franklin were using a technique called X-

ray crystallography to study molecular structure

• Franklin produced a picture of the DNA molecule using this technique

The pattern in the photo suggested that the DNA molecule was made
up of two strands, forming a double helix

Rosalind Franklin Franklin’s X-ray diffraction

photograph of DNA
 5 end
C G
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
 Watson & Crick
• Watson and Crick built models of a double helix to conform to the X-ray
measurements and the chemistry of DNA
• Franklin had concluded that there were two outer sugar-phosphate backbones, with
the nitrogenous bases paired in the molecule’s interior

• Watson built a model in which the backbones were antiparallel (their

subunits run in opposite directions)

• At first, Watson and Crick thought the bases paired like with like (A with A,
and so on), but such pairings did not result in a uniform width
• Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent
with the X-ray data
Purine  purine: too wide

Pyrimidine  pyrimidine: too narrow

Purine  pyrimidine: width

consistent with X-ray data
 Watson & Crick
• 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
Sugar
Sugar
Adenine (A) Thymine (T)

Sugar
Sugar

Guanine (G) Cytosine (C)

 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) Parental (b) Separation of parental (c) Formation of new

molecule strands into templates strands complementary
to template strands
 DNA Replication is Semiconservative
• 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

• 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)
Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model

First Second
(a) Conservative Parent cell
replication replication
model

(b) Semiconservative
model

(c) Dispersive
model
Experiment
1 Bacteria 2 Bacteria ***
cultured in transferred
medium to medium
with 15N with 14N
(heavy (lighter
isotope) isotope)

Results Less
3 DNA sample 4 DNA sample
centrifuged centrifuged dense
after first after second
replication replication More
dense
Conclusion
Predictions: First replication Second replication

Conservative
model

Semiconservative
model

Dispersive
model
 DNA Replication

• The copying of DNA is remarkable in its speed and accuracy

• More than a dozen enzymes and other proteins participate in DNA

replication

• Much more is known about how this “replication machine” works in

bacteria than in eukaryotes

• Most of the process is similar between prokaryotes and eukaryotes

 Getting Started
For eukaryotes…
• Replication begins at particular sites called origins of replication, where the two
DNA strands are separated, opening up a replication “bubble”

• At each end of a replication bubble is a replication fork, a Y-shaped region

where the parental strands of DNA are being unwound

• Multiple replication bubbles form and eventually fuse to speed up DNA replication

For prokaryotes…
• Prokaryotes have ONE origin of replication for their ONE circular chromosome

• Replication happens very quickly and without many mistakes

 Origin of replication in an E. coli cell Origins of replication in a eukaryotic cell ***
Parental
Origin of (template) strand Origin of Double-stranded
replication replication DNA molecule
Daughter
(new) strand
Parental (template)Daughter (new)
Replication strandstrand
Double- fork
stranded
DNA Replication
Bubble Replication fork
molecule bubble
Two
daughter DNA
molecules

Two daughter DNA molecules

0.25 m
0.5 m
 Getting Started
• Helicases are enzymes that untwist the double helix at the replication
forks

• Single-strand binding proteins (SSBs) bind to and stabilize single-

stranded DNA
• Without these, the two strands of DNA will want to bind together again

• Topoisomerase relieves the strain caused by tight twisting ahead of the

replication fork by breaking, swiveling, and rejoining DNA strands
 Primase

Topoisomerase
3

5 RNA
3 primer
5
Replication
3 fork

Helicase
5

Single-strand binding
proteins
 Synthesizing DNA
• Multiple replication bubbles form and eventually fuse to speed up DNA replication

• DNA polymerases can’t initiate synthesis of a polynucleotide, they only add

nucleotides to an already existing chain base-paired with the template

• The initial nucleotide strand is a short RNA primer

• The enzyme, primase, starts an RNA chain from a single RNA nucleotide and
adds RNA nucleotides one at a time using the parental DNA as a template

• The primer is short (5 -10 nucleotides long)

• The new DNA strand will start from the 3 end of the RNA primer
 Synthesizing DNA
• Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork

• Prokaryotes have 3 DNA polymerases: DNA pol I, DNA pol II, DNA pol III
• DNA pol III is required for DNA synthesis
• DNA pol I and DNA pol II are required for repair

• In Eukaryotes, the leading strand is synthesized by the enzyme pol δ, the lagging strand is
synthesized by pol ε

• Most DNA polymerases require a primer and a DNA template strand

• The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in
human cells
 Synthesizing DNA

• Each nucleotide that is added to a growing DNA consists of a sugar

attached to a base and three phosphate groups

• dATP is used to make DNA and is similar to the ATP of energy metabolism

• The difference is in the sugars: dATP has deoxyribose, while ATP has
ribose

• As each monomer nucleotide joins the DNA strand, it loses two phosphate
groups as a molecule of pyrophosphate
 New strand Template strand
5 3 5 3

Sugar A T A T
Phosphate Base

C G C G

G C DNA G C
poly-
merase
3 A T A
T
P P Pi
P 3
P C C
Pyro-
phosphate
Nucleotide
5 5
2 Pi
 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
 Overview
Leading
strand Origin of replication Lagging
strand

Primer

Leading
Lagging strand Overall strand
directions
of replication
Origin of replication
3
5

5 RNA primer
3
3 Sliding clamp

Parental DNA DNA pol III

5
3
5

5 Continuous elongation
3 in the 5 to 3 direction
3

5
 Antiparallel Elongation

• 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

• After formation of Okazaki fragments, DNA polymerase I removes the

RNA primers and replaces the nucleotides with DNA

• The remaining gaps are joined together by DNA ligase

 Overview
Lagging Origin of replication
Leading strand Lagging
strand strand

Leading RNA primer

strand for fragment 2
Overall directions Okazaki
of replication 5
3 fragment 2 4 DNA pol III
1 Primase makes
makes Okazaki
3 fragment 2.
RNA primer.
5 3
3
Template 5
strand 5 5
5 DNA pol I
3
replaces RNA
RNA primer 2 DNA pol III with DNA.
3 for fragment 1 makes Okazaki
fragment 1.
5 3
3 5
6 DNA ligase forms
5
5 bonds between
3 DNA fragments.
3 DNA pol III
3
detaches.
Okazaki
5
fragment 1 3
5
3
5 Overall direction of replication
 Overview
Origin of replication
Leading strand Leading strand Lagging
template strand

Single-strand Lagging strand Leading strand

binding proteins Overall directions
Leading strand of replication
Helicase

5 DNA pol III

3 Primer
3 5 Primase
3
Parental DNA DNA pol III Lagging strand
5
Lagging strand 3 DNA pol I DNA ligase
5
template 3

5
 Overview
Origin of replication
Leading strand Lagging
strand

Lagging strand Leading strand

Overall directions
of replication
 DNA pol III Lagging strand
5
3 DNA pol I DNA ligase
5
3

5
https://www.youtube.com/watch?v=0Ha9nppnwOc
Important!
 Proofreading and Repairing DNA

• DNA polymerases proofread newly made DNA, replacing any incorrect

nucleotides

• In mismatch repair of DNA, other enzymes correct errors in base pairing

• A hereditary defect in one such enzyme is associated with a form of colon

cancer

• This defect allows cancer-causing errors to accumulate in DNA faster than

normal
 Altered Nucleotides ***
• DNA replication is very accurate, but mistakes can occasionally occur, such as a DNA
polymerase inserting a wrong base
• Uncorrected mistakes may sometimes lead to serious consequences, such as cancer

• Repair mechanisms correct the mistakes

• In rare cases, mistakes are not corrected, leading to mutations
• In other cases, repair enzymes are themselves mutated or defective

• DNA can also be damaged by exposure to harmful chemical or physical agents such as
cigarette smoke and X-rays, or spontaneous changes

• In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA
• Error rate after proofreading repair is low but not zero

• Sequence changes may become permanent and can be passed on to the next generation
• These changes (mutations) are the source of the genetic variation upon which natural selection operates
 5 3

3 5

Nuclease

5 3

3 5
DNA
polymerase

5 3

3 5

DNA ligase

5 3

3 5
 Xeroderma Pigmentosa
• A well-studied example of DNA mutations not being corrected is seen in people
suffering from xeroderma pigmentosa
• Affected individuals have skin that is highly sensitive to UV rays from the sun

• When individuals are exposed to UV, thymine dimers are formed

• People with xeroderma pigmentosa are not able to repair the damage

• These dimers are not repaired because of a defect in the nucleotide excision repair
enzymes
• The thymine dimers distort the structure of the DNA double helix and this may cause problems
during DNA replication

• People with xeroderma pigmentosa may have a higher risk of contracting skin cancer
than those who do not have the condition
 Mutations
• Errors during DNA replication are not the only reason why mutations arise in DNA

• Mutations are variations in the nucleotide sequence of a genome

• There are two main types: induced or spontaneous

• Induced mutations are those that result from an exposure to chemicals, UV rays,

x-rays, or some other environmental agent

• Spontaneous mutations occur without any exposure to any environmental agent;

they are a result of natural reactions taking place within the body
 Mutations
• Mutations may have a wide range of effects

• Some mutations are not expressed; these are known as silent mutations

• Point mutations are those mutations that affect a single base pair

• The most common of these are substitutions, in which one base is replaced by another base

• Mutations can also be the result of the addition of a base, known as an insertion

• Or they can be caused by the removal of a base, also known as deletion

• Sometimes a piece of DNA from one chromosome may get translocated to another
chromosome or to another region of the same chromosome
• This is also known as translocation
 End Replication Problem in Eukaryotes

• PROBLEM - In linear DNA, when the replication fork reaches the end of the helix,
there is no place to produce the RNA primer needed to start the final Okazaki
fragment on the lagging strand

• This would lead to the shortening of the chromosome after each replication cycle
and eventually lead to the loss of coding sequences
 Telomeres

• Eukaryotic chromosomal DNA molecules have special nucleotide

sequences at their ends called telomeres
• Telomeres do not prevent the shortening of DNA molecules, but they do postpone it
• In humans and other vertebrate animals, a six base pair sequence of TTAGGG is
repeated 100 - 1000 times

• It has been proposed that the shortening of telomeres is connected to

aging
• If chromosomes of germ cells became shorter in every cell cycle, essential genes
would eventually be missing from the gametes they produce
 Telomerase
• An enzyme called telomerase catalyzes the lengthening of telomeres by
adding DNA sequence repeats to the 3’ ends of chromosomes
• Telomerase is only found in germ cells (sperm and egg cells) and adult stem cells
• The other cells that undergo cell division continue to have their telomeres shortened
because they lack telomerase
• This means that telomere shortening is associated with aging

• This can resolve the end replication problem because extra nucleotides are
continually added to the ends (telomeres), preventing gene loss with many
cell divisions

• Telomerase can show inappropriately high activity in some cancer cells

• Telomerase is currently under study as a target for cancer therapies
***
A chromosome consists of a DNA molecule together with proteins

• The bacterial chromosome is a double-stranded, circular DNA

molecule associated with a small amount of protein in a region of the cell
called the nucleoid

• Eukaryotic chromosomes have linear DNA molecules associated with a

large amount of protein
• Chromatin, a complex of DNA and protein, is found in the nucleus of eukaryotic
cells
 Chromatid
(700 nm)
Nucleosome
DNA (10 nm in diameter) 30-nm
double helix fiber
(2 nm in diameter)
Loops Scaffold
H1 300-nm
Histone tail fiber
Histones

Replicated chromosome
(1,400 nm)
 Chromatin
• At interphase, most of the chromatin is compacted into a 30-nm fiber, which is folded further
in some areas by looping

• Eukaryotic DNA is wrapped around proteins known as histones to form structures called
nucleosomes
• Histones are evolutionarily conserved proteins that are rich in basic amino acids and form a positively charged
octamer
• DNA is negatively charged because of the phosphate groups and wrapped tightly around the histone core
• This nucleosome is linked to the next one with the help of a linker DNA called the “beads on a string” structure

• In interphase, eukaryotic chromosomes have two distinct regions, a tightly packaged region called
heterochromatin and the less dense region called euchromatin
• Heterochromatin usually contains genes that are not expressed, and is found in the regions of the centromere and
telomeres
• Euchromatin usually contains genes that code for important proteins, with DNA packaged around nucleosomes
but not further compacted
 The Polymerase Chain Reaction (PCR)
• The polymerase chain reaction, PCR, can produce many copies of a
specific target segment of DNA

• A three-step cycle brings about a chain reaction that produces an

exponentially growing population of identical DNA molecules

• The key to PCR is an unusual, heat-stable DNA polymerase called Taq

polymerase
Technique 5 3

Target sequence
Genomic DNA 3 5

1 Denaturation 5 3

3 5
2 Annealing

Cycle 1
yields 2 molecules Primers

3 Extension
New
nucleotides

Cycle 2
yields 4 molecules

Cycle 3
yields 8 molecules;
2 molecules
(in white boxes)
match target sequence
 DNA Sequencing
• The most popular method is the Sanger dideoxy chain termination method
• Based on the use of chain terminators called dideoxynucleotides (ddNTPs)

• The dideoxynucleotides, or ddNTPSs, differ from the deoxynucleotides by the lack

of a free 3' OH group on the five-carbon sugar

• If a ddNTP is added to a growing a DNA strand, the chain is not extended any
further because the free 3' OH group needed to add another nucleotide is not
available
• By using a predetermined ratio of deoxyribonucleotides to dideoxynucleotides, it is possible
to generate DNA fragments of different sizes

• See the video assignment

• Also watch this: https://www.youtube.com/watch?v=ONGdehkB8jU
 Gel Electrophoresis
• To figure out the size of DNA fragments, researchers use a technique
called gel electrophoresis https://www.youtube.com/watch?v=ZDZUAleWX78
• This technique separates a mixture of nucleic acid fragments based on length

• Gel electrophoresis is a process which enables the sorting of molecules based on size
• Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar
or polyacrylamide
• The electric field consists of a negative charge (cathode) at one end which pushes the molecules
through the gel and a positive charge (anode) at the other end that pulls the molecules through the
gel
• The molecules being sorted are dispensed into a well in the gel material
• The gel is placed in an electrophoresis chamber, which is then connected to a power source
• When the electric current is applied, the larger molecules move more slowly through the gel while
the smaller molecules move faster
• The different sized molecules form distinct bands on the gel
Mixture of Power
DNA mol- source
ecules of Cathode Anode
different
sizes
Wells

Gel

Negatively charged DNA molecules will move toward

the positive electrode.

Restriction fragments

Shorter molecules are impeded less than longer

ones, so they move faster through the gel.