CHAPTER 11

NUCLEIC ACID
STRUCTURE AND
DNA REPLICATION

Prepared by
Brenda Leady, University of Toledo

Copyright (c) The McGraw-Hill

Companies, Inc. Permission required
for reproduction or display. 1
 Some properties of life

(b) Evolutionary
adaptation
(a) Order
(c) Response to the
environment

(e) Energy
processing
(d) Regulation

(f) Growth and (g) Reproduction

development

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Reproduction
 Not only at the level of organism but also
at the level of cell
 What is being copied?
 Shape
 Characteristics
 Fate
 Genetic materials – by replication

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Genetic material must be able to:

 Contain the information necessary to

construct an entire organism
 Pass from parent to offspring and from cell
to cell during cell division
 Be accurately copied
 Account for the known variation within and
between species

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 Late 1800s scientists postulated a
biochemical basis
 Researchers became convinced
chromosomes carry genetic information
 1920s to 1940s expected the protein
portion of chromosomes to be the genetic
material

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Griffith’s bacterial transformations
 Late 1920s Frederick Griffith was working with
bacteria named Streptococcus pneumoniae
 S. pneumoniae
 Strains that secrete capsules look smooth (S form)
and can cause fatal infections in mice
 Strains that do not secrete capsules look rough (R
form) and infections are not fatal in mice

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 Rough strains (R) without capsule are
not fatal
 No living bacteria found in blood
 Smooth strains (S) with capsule are
fatal
 Capsule prevents immune system from
killing bacteria
 Living bacteria found in blood
 If mice are injected with heat-killed
type S, they survive
 Mixing live R with heat-killed S kills the
mouse
 Blood contains living S bacteria
 Transformation

 Genetic material from the heat-killed

type S bacteria had been transferred
to the living type R bacteria
 This trait gave them the capsule and
was passed on to their offspring

 Griffith did not know the biochemical

basis of his transforming principle

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Avery, MacLeod, and McCarty used purification
methods to reveal that DNA is the genetic material
 1940s interested in bacterial transformation
 Only purified DNA from type S could transform type R
 Purified DNA might still contain traces of contamination that may be the transforming principle
 Added DNase, RNase and proteases
 RNase and protease had no effect
 With DNase no transformation
 DNA is the genetic material
Hershey and Chase
 1952, studying T2 virus infecting Escherichia coli
 Bacteriophage or phage
 Phage coat made entirely of protein
 DNA found inside capsid

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 Shearing force from a blender will separate the
phage coat from the bacteria
 35S will label proteins only

 32P will label DNA only

 Experiment to find what is injected into bacteria-

DNA or protein?
 Results support DNA as the genetic material

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 The Hershey-Chase Experiment
1 Mix radioactively 2 Agitate in a blender 3 Centrifuge the mixture 4 Measure the
labeled phages with to separate phages so bacteria form a radioactivity in
bacteria. The phages outside the bacteria pellet at the bottom of the pellet and
infect the bacterial from the cells and the test tube. liquid.
cells. their contents.

Radioactive Empty Radioactivity

Phage protein protein shell in liquid
Bacterium Phage
DNA
DNA
Batch 1
Radioactive
protein
Centrifuge

Pellet

Radioactive
Batch 2 DNA
Radioactive
DNA Centrifuge
Radioactivity
Pellet in pellet
Figure 10.1B
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12
Levels of DNA structure
1. Nucleotides are the building
blocks of DNA (and RNA).
2. A strand of DNA (or RNA)
3. Two strands form a double
helix.
4. In living cells, DNA is
associated with an array of
different proteins to form
chromosomes.
5. A genome is the complete
complement of an organism’s
genetic material.

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Nucleotides
3 components
Phosphate group
Pentose sugar
Nitrogenous base

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DNA
 3 components
 Phosphate group
 Pentose sugar
 Deoxyribose
 Nitrogenous base
 Purines
 Adenine (A),
guanine (G)
 Pyrimidines
 Cytosine (C),
thymine (T)

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RNA
 3 components
 Phosphate group
 Pentose sugar
 Ribose
 Nitrogenous base
 Purines
 Adenine (A),
guanine (G)
 Pyrimidines
 Cytosine (C),
uracil (U)

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 Conventional numbering system
 Sugar carbons 1’ to 5’
 Base attached to 1’
 Phosphate attached to 5’

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Strands
 Nucleotides covalently
bonded
 Phosphodiester bond –
phosphate group links 2
sugars
 Phosphates and sugars
from backbone
 Bases project from
backbone
 Directionality- 5’ to 3’
 5’ – TACG – 3’
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Solving DNA structure
 1953, James Watson and Francis Crick, with Maurice
Wilkins, proposed the structure of the DNA double helix
 Watson and Crick used Linus Pauling’s method of
working out protein structures using simple ball-and-stick
models
 Rosalind Franklin’s X-ray diffraction results provided
crucial information that DNA was as composed of two
antiparallel sugar-phosphate backbones, with the
nitrogenous bases paired in the molecule’s interior.
 Erwin Chargoff analyzed base composition of DNA that
also provided important information

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 Watson and Crick put
together these pieces
of information
 Found ball-and-stick
model consistent with
data
 Watson and Crick
awarded Nobel Prize
in 1962
 Rosalind Franklin had
died (1958) and the
Nobel is not awarded
posthumously 20
21
One-paged article had opened new era of life science

22
Controversies of King’s College London in
1953
 Watson and Crick then indirectly obtained a
prepublication version of Franklin's DNA X-ray diffraction
data possibly without her knowledge, and a
prepublication manuscript by Pauling and Corey, giving
them critical insights into the DNA structure.
 Franklin eventually left King's College London in March
1953 to move to Birkbeck College.
 Franklin was diagnosed with ovarian cancer in 1956 at
age 37, and died two years later, without an award, but
not without recognition -- eventually.

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 DNA is
 Double stranded
 Helical
 Sugar-phosphate
backbone
 Bases on the inside
 Stabilized by
hydrogen bonding
 Base pairs with
specific pairing

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 AT/GC or Chargoff’s rule
 A pairs with T
 G pairs with C
 Keeps with consistent
 10 base pairs per turn
 2 DNA strands are
complementary
 5’ – GCGGATTT – 3’
 3’ – CGCCTAAA – 5’
 2 strands are antiparallel
 One strand 5’ to 3’
 Other stand 3’ to 5’

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 Space-filling model shows grooves
 Major groove
 Where proteins bind
 Minor groove

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NUCLEIC ACID STRUCTURE – Summary

Nucleic acids are genetic materials of living organism on earth

Griffith’s experiment suggested that the existence of genetic
material in the cell
Avery’s experiment showed that DNA is the genetic material in
bacteria

Nucleotide
Composed of three major part: phosphate backbone, sugar ring,
base

Structure
Anti-parallel double helical structure
One turn per every 10 nucleotides

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Replication
 3 different models for
DNA replication
proposed in late 1950s
 Semiconservative (2
old-new pair)
 Conservative (1 old pair
+ 1 new pair)
 Dispersive (2 old+new
mixed chromosmes)
 Newly made strands are
daughter strands
 Original strands are
parental strands

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 In 1958, Matthew Meselson
and Franklin Stahl devised
experiment to differentiate
among 3 proposed
mechanisms
 Nitrogen comes in a common
light form (14N) and a rare
heavy form (15N)
 Grew E.coli in medium with
only 15N
 Then switched to medium with
only 14N
 Collected sample after each
generation
 Original parental strands
would be 15N while newly
made strands would be 14N
 Results consistent with
semiconservative mechanism

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 During replication 2
parental strands
separate and serve
as template strands
 New nucleotides must
obey the AT/GC rule
 End result 2 new
double helices with
same base sequence
as original

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 Origin of replication
 Site of start point for
replication
 Bidirectional replication
 Replication proceeds
outward in opposite
directions
 Bacteria have a single
origin
 Eukaryotes require
multiple origins

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Replication of E. coli DNA

 Replication forks: represent the region of active DNA synthesis

 At each fork, the parental strand of DNA separated and two new
daughter strand were synthesized

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 Origin of replication provides an
opening called a replication bubble
that forms two replication forks
 DNA replication occurs near the fork
 Synthesis begins with a primer
 Proceeds 5’ to 3’
 Leading strand made in direction
fork is moving
 Synthesized as one long
continuous molecule
 Lagging strand made as Okazaki
fragments that have to be connected
later

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 DNA helicase
 Binds to DNA and travels 5’ to
3’ using ATP to separate
strand and move fork forward
 DNA topoisomerase
 Relives additional coiling
ahead of replication fork
 DNA molecules can coil and bend in space,
leading to changes in topology such as
formation of supercoils
 Top I; cut one strand and ligation, Top II; cut ds
DNA and ligation.

 Single-strand binding
proteins
 Keep parental strands open to
act as templates
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 DNA polymerase
 Covalently links nucleotides
 Deoxynuceloside triphosphates

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 Deoxynuceloside triphosphates
 Free nucleotides with 3 phosphate groups
 Breaking covalent bond to release
pyrophosphate (2 phosphate groups) provides
energy to connect adjacent nucleotides

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 DNA polymerase has 2 enzymatic features to explain leading and
lagging strands
1. DNA polymerase unable to begin DNA synthesis on a bare
template strand
 DNA primase must make a short RNA primer
 RNA primer will be removed and replaced with DNA
later
2. DNA polymerase can only work 5’ to 3’

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 In the leading strand
 DNA primase makes one RNA
primer
 DNA polymerase attaches
nucleotides in a 5’ to 3’ direction
as it slides forward
 In the lagging strand
 DNA synthesized 5’ to 3’ but in a
direction away from the fork
 Okazaki fragments made as a
short RNA primer made by DNA
primase at the 5’ end and then
DNA laid down by DNA
polymerase
 RNA primers will be removed by
DNA polymerase and filled in
with DNA
 DNA ligase will join adjacent
DNA fragments
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Model of the E. coli Replication Fork

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DNA replication is very accurate
 3 reasons
1. Hydrogen bonding between A and T or G
and C more stable than mismatches
2. Active site of DNA polymerase unlikely to
form bonds if pairs mismatched
3. DNA polymerase removes mismatched pairs
 Proofreading results in DNA polymerase backing
up and digesting linkages
 Other DNA repair enzymes

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The family of DNA polymerases
 3 important issues for DNA polymerase are
speed, fidelity, and completeness
 Nearly all living species have more than 1
type of DNA polymerase
 Genomes of most species have several DNA
polymerase genes due to gene duplication
 Independent genetic changes produce
enzymes with specialized functions suited to
the organism
 E. coli has 5 DNA polymerases
 DNA polymerase III with multiple subunits
responsible for majority of replication
 DNA polymerase I has a single subunit whose
job is to rapidly remove RNA primers and fill in
DNA
 DNA polymerases II, IV and V are involved in
DNA repair and replicating damaged DNA
 DNA polymerases I and III stall at DNA damage
 DNA polymerases II, IV and V don’t stall but go slower
and make sure replication is complete
 Humans have 12 or more DNA polymerases
 Designated with Greek letters
 DNA polymerase α has its own built in primase
subunit
 DNA polymerase δ and ε extend DNA at a faster
rate
 DNA polymerase γ replicates mitochondrial DNA
 When DNA polymerases α, δ or ε encounter
abnormalities they may be unable to replicate
 Lesion-replicating polymerases may be able to
synthesize complementary strands to the
damaged area
 DNA polymerase
cannot copy the tip
of the DNA strand
with a 3’ end
 No place for
upstream primer to
be made
 If this replication
problem were not
solved, linear
chromosomes would
become
progressively
shorter

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 Telomere: end shorting problem

 Specialized form of DNA replication only in

eukaryotes in the telomeres
 Telomeres are a series of repeat
sequences within DNA and special
proteins
 Telomere at 3’ does not have a
complementary strand and is called a 3’
overhang

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47
Structure of a Telomere

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 Telomerase

 Telomerase prevents
chromosome
shortening
 Attaches many
copies of repeated
DNA sequences to
the ends of the
chromosomes
 Provides upstream
site for RNA primer

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Telomeres and aging
 Body cells have a predetermined life span
 Skin sample grown in a dish will double a
finite number of times
 Infants,
about 80 times
 Older person, 10 to 20 times

 Senescent cells have lost the capacity to

divide

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 Progressive shortening of telomeres
correlated with cellular senescence
 Telomerase present in germ-line cells and
in rapidly dividing somatic cells
 Telomerase function reduces with age
 Inserting a highly active telomerase gene
into cells in the lab causes them to
continue to divide
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Telomeres and cancer
 When cells become cancerous they divide
uncontrollably
 In 90% of all types of human cancers,
telomerase is found at high levels
 Prevents telomere shortening and may
play a role in continued growth of cancer
cells
 Mechanism unknown
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Eukaryotic chromosome structure

 Typical eukaryotic chromosome may be

hundreds of millions of base pairs long
 Length would be 1 meter
 Must fit in cell 10-100µm

 Chromosome
 Discrete unit of genetic material
 Chromosomes composed of chromatin
 DNA-protein complex
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3 levels of DNA compaction
1. DNA wrapping
 DNA wrapped around histones to form
nucleosome
 Shortens length of DNA molecule 7-fold

2. 30-nm fiber
 Current model suggests asymmetric, 3D
zigzag of nucleosomes
 Shortens length another 7-fold

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3. Radial loop domains
 Interaction between
30-nm fibers and
nuclear matrix
 Each chromosome
located in discrete
territory
 Level of compaction
of chromosomes not
uniform
 Heterochromatin
 Euchromatin

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Cell division
 When cells prepare to divide,
chromosomes become even more
compacted
 Euchromatin not as compact
 Hetrochromatin much more compact
 Metaphase chromosomes highly compacted

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