Chapter 6
DNA replication, repair and recombination
This is essential to understand
•cancer
•genetic change
•evolution
•key techniques used for molecular biology/ biotechnology
•current antiviral therapeutics
Rgomer@[Link]
ILSB 2121 2nd floor middle of this building on side by
stadium
Objectives
• Be able to explain how genetic information is
replicated and the role of the proteins involved
• Understand the general mechanisms of DNA alteration
and repair
1
Be able to:
• Explain the first experiment that showed that DNA replication is semi-
conservative
• Draw a piece of DNA, labeling the 5 and 3 ends of each strand
• Draw the DNA opened up for replication, with the RNA primers
attached, and label the 5 and 3 ends of the RNA primers
• Draw the new leading strand
• Draw the next RNA primers, labeling the 5 and 3 ends
• Draw the DNA being made on the lagging strand
• Describe how the RNA primer is removed, and explain how ligase seals
the gap
• Name, and describe the function of, the key enzymes in replication
• Describe what telomerase does
• Sketch two mechanisms of DNA repair
• Outline what recombination does
• Describe what a transposon is, and how it can copy itself
• Sketch the molecular mechanism used by a retrovirus
• Describe how acyclovir and AZT work
DNA Replication
06_02_DNA [Link]
Each DNA strand can serve as a template for the
synthesis of a new complementary strand
P- -OH
HO- -P
A-T and G-C complementary bases
2
How is DNA replicated?
Meselson-Stahl Experiment
DNA replication is semiconservative
3
Semiconservative replication
To begin replication, DNA strands must be pried apart
Are replication origins more likely to be A-T rich or G-C rich?
4
Visualization of Replication
DNA synthesis is catalyzed
by DNA polymerase
Nucleotides are added only
to the 3 end of a DNA
strand
DNA synthesis is 5 to 3
5
If DNA is synthesized only in the 5 to 3 direction
how are both strands replicated?
No free 3 end to extend
Discontinuous replication allows backstitching
6
Discontinuous replication
DNA polymerase can add a
nucleotide only to an existing
3 base-pair
To start a new strand we
need a primer
The primer is a short (10 bp)
RNA and is synthesized by
DNA primase
Extension of the RNA primer
by DNA polymerase
generates the Okazaki
fragment (~200 bp)
DNA polymerase will
extend the Okazaki
fragment until it runs into
the next RNA primer
The RNA primer will then
be degraded by a
ribonuclease and a DNA
polymerase called Repair
polymerase replaces the
RNA with DNA
DNA Ligase joins the
fragments (5 P to 3 OH)
7
Ligase is a key enzyme
used in molecular biology
E. coli DNA Human DNA
Add ligase, ATP
Make hybrid genes, proteins, or organisms…..
Multiple proteins act together at the replication fork
Coordinated
replication
8
Proteins involved in replication: study guide
• Initiator proteins: helix opening at replication origin
• DNA Polymerase: polymerization (5 -3 ) proofreading (3 -5 )
• DNA Primase: synthesis of short RNA primer
• Ribonuclease: degradation of RNA primer
• Repair polymerase: replace RNA with DNA
• Ligase: joining of DNA fragments
• DNA Helicase: unzipping DNA helix prior to replication
• Single-stranded DNA-binding protein: prevent reanealing
• Sliding clamp: keeps DNA polymerase attached to template
and on lagging strand, releases when Okazaki fragment is
completed
Telomerase is needed to replicate chromosome ends
Telomeres also help the cell distinguish true chromosome ends
9
DNA Polymerase also has
an error-correcting activity
called Proofreading
Catalyzed by a 3 to 5
exonuclease activity
Error rate is about 1 error
in 107 base pairs
Despite the
proofreading ability of
DNA polymerase,
errors still occur
Permanent changes in
DNA sequence are
mutations
Mutations can be
deleterious, neutral or
even beneficial (basis
for evolution). It is not
always easy to
distinguish between
deleterious and
beneficial
10
Somatic cells can
accumulate mutations over
a lifespan that may impact
health
DNA repair mechanisms
safeguard DNA sequences
from random mutation
DNA mismatch repair
06_21_Errors [Link]
Replication error rate (DNA polymerase) is 1 in 107 basepairs
DNA mismatch repair corrects 99% of these for an overall rate of 1 in 109
11
DNA mismatch repair
proteins recognize the
newly synthesized strand
and selectively excise and
resynthesize this strand
Recognition probably
involves nicks in the newly
synthesized strand
Common chemical reactions known
to cause DNA damage
12
DNA damage can lead to mutation
substitution deletion
General mechanism
of DNA repair
1. Recognition and excision
2. Replacement
3. Joining
13
Recombination
Rearrangement of DNA sequences can provide variation
Like mutation, recombination can be either:
• Neutral
• Beneficial
• Deleterious
Types of recombination:
• Homologous
• Non-homologous
Homologous recombination
Requires alignment of homologous sequences
Used for DNA repair, shuffling genes during meiosis (so
from each parent, on each chromosome, you get some
genes from grandfather and some from grandmother)
14
Homologous recombination
06_29_Homol.[Link]
No alteration of sequence
at the site of exchange
Nonhomologous recombination
(called site-specific recombination in the text)
DNA exchange that is independent of sequence
No homology is required
Usually involves insertion or deletion of DNA
15
Transposons are common mobile genetic elements
Transposase is a recombination protein necessary for
transposon mobility. Recognizes transposon ends (red)
Transposon movement mechanisms
Amplification of transposon
16
Retrotransposons move via an RNA intermediate
L1 element (15% human genome)
Alu element (11% human genome)
Many transposons present in the human
genome are not capable of further movement
Nonhomologous
recombination is a
key step in the
lysogenic phase of
viral reproduction
Lysogenic phase
Lytic phase
17
Retroviruses found
only in eukaryotic cells
06_39_retrovirus.jpg
Acyclovir -
Frontline therapeutic for
Herpesvirus - looks like
a G base and the top of
the ribose sugar
No 3 OH, so
next nucleotide can t
be added to the chain -
Stops herpesvirus DNA
replication
18
Azidothymidine -
Frontline therapeutic for
HIV - looks like a T
base and a ribose with
a modified 3 OH
No 3 OH, so
next nucleotide can t
be added to the chain -
Stops HIV DNA
synthesis
Useful text questions
6-2
6-3
6-4
6-5
6-6
6-7
6-8
6-10
6-12, 13, 14, 15, 16, 18
19
Be able to:
• Explain the first experiment that showed that DNA replication is semi-
conservative
• Draw a piece of DNA, labeling the 5 and 3 ends of each strand
• Draw the DNA opened up for replication, with the RNA primers
attached, and label the 5 and 3 ends of the RNA primers
• Draw the new leading strand
• Draw the next RNA primers, labeling the 5 and 3 ends
• Draw the DNA being made on the lagging strand
• Describe how the RNA primer is removed, and explain how ligase seals
the gap
• Name, and describe the function of, the key enzymes in replication
• Describe what telomerase does
• Sketch two mechanisms of DNA repair
• Outline what recombination does
• Describe what a transposon is, and how it can copy itself
• Sketch the molecular mechanism used by a retrovirus
• Describe how acyclovir and AZT work
20