DNA: The Genetic Material

Chapter 14

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Review of Nucleotide Structure
Different Nitrogenous Bases
 How is DNA Synthesized?
where a free P is attached
to the 5’ C of one terminal
sugar

• Phosphodiester bond forms

between the 5’ phosphate
group of one sugar and 3’
Sugar-phosphate
hydroxyl group of the backbone
adjacent sugar

• Linear strands of DNA have a

free 5’ phosphate group and
a free 3’ hydroxyl group
where a free OH is
attached to the 3’ C of
another terminal sugar
 3-D Structure of DNA
• Rosalind Franklin and Maurice Wilkins
– X-ray diffraction studies suggested
DNA had helical shape with a
diameter of about 2 nanometers (nm)

• James Watson and Francis Crick

– figure out that DNA is a double helix
• two strands of nucleotides
• bases point inward and form base-
pairs
• purines pairing with pyrimidines
– strands are antiparallel
• one strand runs 5’ to 3’
• other strand runs 3’ to 5’
 Base-Pairing

• Pyrimidines always
pair with purines

• T forms 2 hydrogen
bonds with A

• C forms 3 hydrogen
bonds with G
DNA Replication is Semiconservative

• The parent DNA molecule unzips

• Each parent strand serves as a template

for a new strand in daughter DNA
molecule

- confirmed by Meselson-Stahl
experiment
 DNA Replication Process
• Replication of DNA begins at several specific sites along
the DNA molecule called origins of replication or
replication origins

• DNA polymerase III works together with other enzymes

to generate DNA strands

Replication
forks
 Action of DNA Polymerase III

• DNA polymerase III moves along the template strand in a 3'-

5‘ direction and it adds free nucleotides only to a free 3’ OH
of the daughter strand

• Daughter strand is formed (synthesized) in a 5'-3’ direction

 DNA Replication Continues
• DNA gyrase stops DNA from twisting
• DNA helicase opens up the helix
• DNA polymerase III requires a primer to which it can
add the first nucleotide
– DNA primase (an RNA polymerase) constructs an RNA
primer
• DNA polymerase III adds nucleotides to 3’ end
– leading strand replicates toward replication fork.
• synthesized continuously
– lagging strand elongates from replication fork.
• synthesized discontinuously
– production of Okazaki fragments
• DNA polymerase I removes RNA primers and fills gaps
• DNA ligase attaches Okazaki fragments to lagging strand
 DNA Replication Fork
Prevent parent strands
from reforming a double helix
Stops DNA from twisting
by releasing the tension
in double helix

Breaks H bonds between

parent strands, allowing
them to separate apart
 DNA Replication Fork
DNA primase = RNA polymerase DNA polymerase III extends
that makes RNA primer RNA primer
5’ 5’ 3’

3’ 5’ 3’ 5’
TCGAGCA T C G A G CA

RNA primer is a short, single-stranded

sequence of 6-15 ribonucleotides long
 DNA Synthesis

• Only one primer is required for the initiation and propagation

of the leading daughter strand synthesis

• The leading daughter strand is synthesized continuously in

the 5’ to 3’ direction
 DNA Synthesis

• The lagging daughter strand is synthesized discontinuously in

small segments, called Okazaki fragments

• Multiple RNA primers are needed to synthesize the lagging

daughter strand
 DNA Synthesis

• When DNA polymerase III encounters the preceding primer, it

lifts its lower arm up

• DNA polymerase I removes RNA primer and fills the gap

between Okazaki fragments with deoxynucleotides
 DNA Synthesis

• DNA ligase joins Okazaki fragments together by making the

phosphodiester bonds

• DNA polymerase III puts its lower arm into a new location
 DNA Synthesis

• DNA polymerase III then synthesizes a new Okazaki fragment

until it reaches the previous RNA primer
 5’ A T C G A A C C C T 3’
Parent DNA
molecule

DNA polymerase III moves from left to right

 Why do Chromosomes Become Shorter?

• The 3’ end of the lagging

strand cannot be replicated
– so over time, the
telomeres become
shorter in dividing cells
 Telomerase: Keeping Telomeres Long Enough

3’

• Telomerase is a reverse transcriptase, an enzyme that reverses

transcription
- transcription = when DNA is used to make RNA
- reverse transcription = when RNA is used to make DNA
How does Telomerase Work?
How can DNA be Damaged?
Photorepair
Excision Repair
The Meselson-Stahl Experiment
• E. coli were grown in a heavy isotope of
nitrogen, 15N

• All the DNA incorporated 15N

• Cells were then switched to media containing

lighter isotope of nitrogen, 14N

• DNA was extracted from the cells at various

time intervals
The Meselson-Stahl Experiment
 Is Genetic Information in Protein or DNA?
• Avery Experiment
- worked with bacteria that transferred genetic material
between each other (transformation)
- removed almost all proteins from bacteria, and found no
reduction in transforming activity

• Hershey-Chase
- used different radioactive isotopes to label DNA and
protein in bacteriophages
- new bacteriophage passed genetic information into host
cell, which was then used to produce new viruses
- found material used to specify new generations of
viruses were made of DNA
 The Hershey-Chase Experiment
Bacteriophage protein was
labeled with radioactive sulfur
(35S)

Bacteriophage DNA was

labeled with radioactive
phosphorus (32P)