DNA Synthesis

1
Chapter 2

• DNA Synthesis

2
Chapter 12
3
Distinguishing between Models of DNA Replication

• Three different models of how DNA might replicate
  were proposed based on DNA structure.
• Semi-conservative replication
• Conservative replication
• Dispersive replication

4
Figure 12.1a-c
Alternative Hypotheses for DNA Synthesis
(a) Hypothesis 1
(b) Hypothesis 2
(c) Hypothesis 3
Semi-conservative replication
Conservative replication
Dispersive replication
Intermediate molecule
5
Distinguishing Between Models of DNA Replication

• The Meselsohn and Stahl experiment determines
  which model is correct.
• 15N was fed to growing E. coli cells to mark DNA,
  then cells were switched to 14N.
• DNA replication is semi-conservative new DNA has
  one 15N strand and one 14N strand.

6
Figure 12.2a,b
7
Figure 12.2a
MESELSON-STAHL EXPERIMENT
Question Is replication conservation or
semi-conservation?
1. Grow E. coli cells in medium with15N as sole
source of nitrogen. Collect sample and purify
DNA.
15N
GENERATION 0 DNA sample
2. Transfer cells to medium containing 14N.
After cells divide once, collect sample and
purify DNA.
3. After cells have divided a second time in 14N
medium, collect sample and purify DNA.
Top of centrifuge tube (lower density)
4. Centrifuge the three samples and compare the
location of the bands. DNA containing 15N is
heavier than DNA containing 14N and forms a
band lower in the tube.
14N
Hybrid
15N
Bottom of centrifuge tube (higher density)
0
1
2
Conclusion?
Generation
8
Figure 12.3a
Structure of dNTPs
P
P
P
CH2
5'
Base
O
3'
OH
9
Figure 12.3b
DNA synthesis reaction
5' end of strand
P
P
Base
Base
CH2
CH2
O
O
P
P
CH2
CH2
Base
Base
O
O
H20

P
3'
P
P
P
OH
P
Synthesis reaction
CH2
Base
P
O
CH2
Base
5'
O
OH
3'
3' end of strand
OH
10
Cell free in vitro DNA synthesis reactions were
used to identify the enzymes involved in DNA
replication.

• Proteins were extracted from E. coli and tested
  for activity in the cell free system.
• Kornberg et al. determine that
• DNA replication requires an enzyme the first
  discovered was DNA polymerase I.
• DNA replication requires a DNA template.

11
Figure 12.4
TESTING TEMPLATE-DIRECTED SYNTHESIS
1. Isolate single strand of ?X174 DNA.
?X174 virus
2. Make copies of ?X174 DNA in vitro using DNA
polymerase I.
Normal ?X174 DNA
Synthetic ?X174 DNA
3. Add synthetic ?X174 DNA to E.coli cells
growing in culture.
E. coli
Synthetic DNA
4. Observe result New generation of ?X174
particles appears. Synthetic ?X174 DNA is
infectious.
Conclusion DNA polymerase I catalyzes
template-directed synthesis
12
Figure 12.5
7500
5000
Amount of radioactive thymidine in DNA (counts
per minute)
2500
0
0
30
60
90
120
150
Time (min)
13
Figure 12.6
Radioactively labeled strand
Unlabeled strand (from before addition of label)
Two labeled strands newly synthesized
Direction that DNA polymerase is moving
14
Cell free in vitro DNA synthesis reactions were
used to identify the enzymes involved in DNA
replication.

• Proteins were extracted from E. coli and tested
  for activity in the cell free system.
• Cairns and de Lucia DNA polymerase I is not the
  main replication enzyme.

15
Cell free in vitro DNA synthesis reactions were
used to identify the enzymes involved in DNA
replication.

• Proteins were extracted from E. coli and tested
  for activity in the cell free system.
• Kohiyama and Kornberg discover DNA polymerase
  III, which is the main replication enzyme.

16
Cell free in vitro DNA synthesis reactions were
used to identify the enzymes involved in DNA
replication.

• Characteristics of replication in vitro in E.
  coli
• At the replication fork, DNA polymerase III
  builds the new strands in the 5-3 direction.
• New nucleotides are only added to 3 hydroxyl
  groups of other nucleotides.
• The new strands are initiated by adding
  nucleotides to a short RNA primer. (Fig. 12.7)

17
Figure 12.7a
Formation of the leading strand
3'
DNA polymerase III
5'
5'
Newly synthesized leading strand
3'
5'
Replication fork
18
Figure 12.7b
Formation of lagging strand
3'
5'
Lagging strands
5'
3'
3'
DNA polymerase III
5'
3'
5'
Okazaki fragments
5'
3'
DNA polymerase III beginning synthesis of new
fragment
3'
Gap
5'
19
Figure 12.8
Completely single stranded
3'
5'
Polymerase inactive
G
A
A
T
C
T
G
C
Completely double stranded
3'
5'
G
A
A
T
C
T
G
C
Polymerase inactive
C
T
T
A
G
A
C
G
5'
3'
Single strand as template plus 3' end to start
synthesis
5'
3'
G
A
A
T
C
T
G
C
Polymerase active
C
T
T
OH 3'
5'
20
Figure 12.8
Completely single stranded
3'
5'
Polymerase inactive
G
A
A
T
C
T
G
C
Completely double stranded
3'
5'
G
A
A
T
C
T
G
C
Polymerase inactive
C
T
T
A
G
A
C
G
5'
3'
Single strand as template plus 3' end to start
synthesis
3'
5'
G
A
A
T
C
T
G
C
Polymerase active
C
T
T
5'
OH 3'
21
Cell free in vitro DNA synthesis reactions were
used to identify the enzymes involved in DNA
replication.

• Characteristics of replication in vitro in E.
  coli
• The leading strand is the new growing strand that
  follows the replication fork.
• The lagging strand grows in the direction away
  from the replication fork and is synthesized in
  short pieces called Okazaki fragments, each with
  their own primer.
• Different enzymes catalyze each step of the
  process. (Fig. 12.9)

22
Figure 12.9
3
Pol III synthesizes leading strand
2
1
Helicase opens helix
Topoisomerase nicks DNA to relieve tension from
unwinding
Primase synthesizes RNA primer
4
5
Pol I excises RNA primer fills gap
6
7
Pol III elongates primer produces Okazaki
fragment
DNA ligase links Okazaki fragments to form
continuous strand
23
Figure 12.10
24
Laboratory Analysis of DNA Sequences

• DNA sequence analysis is used to compare genes
  within and between species to determine function
  andevolutionary relatedness.

25
Laboratory Analysis of DNA Sequences

• Two basic methods used in sequence analysis of
  DNA
• Polymerase Chain Reaction amplifies DNA from
  primers, produces numerous copies. (Fig. 12.11a,b)

26
Figure 12.11a
Primers are required to run PCR
CCCCATGCTTACAAGCAAGT
Primer
3'
5'
3'
5'
Primer
Region of DNA to be amplified by PCR
ATCCTATGGTTGTTTGGATGGGTG
27
Figure 12.11 steps 1-3
POLYMERASE CHAIN REACTION
3'5'
1. Start with a solution containing template DNA,
synthesized primers, and an abundant supply of
the four dNTPs.
5'3'
3'
5'
2. Denaturation Heating leads to denaturation of
the double-stranded DNA.
3'
5'
3. Primer binding At cooler
temperatures,the primers anneal to the template
DNA by complementary base pairing.
5'
3'5'
5'3'
5'
28
Figure 12.11 steps 4-6
5'
5'3'
3'5'
3'5'
4. Extension During incubation, DNA polymerase
synthesizes complementary DNA strand starting at
the primer.
5'3'
5'3'
5. Repeat cycle of three steps (2-4) again,
doubling the copies of DNA.
6. Repeat cycle again, up to 20-30 times, to
produce millions of copies of template DNA.
29
Figure 12.11 steps 1-3
1. Start with a solution containing template DNA,
synthesized primers, and an abundant supply of
the four dNTPs.
2. Denaturation Heating leads to denaturation of
the double-stranded DNA.
3. Primer binding At cooler temperatures, the
primers anneal to the template DNA by
complementary base pairing.
30
Figure 12.11 steps 4-6
4. Extension During incubation, DNA polymerase
synthesizes complementary DNA strand starting at
the primer.
5. Repeat cycle of three steps (2-4) again,
doubling the copies of DNA.
6. Repeat cycle again, up to 20-30 times, to
produce millions of copies of template DNA.
31
Laboratory Analysis of DNA Sequences

• Two basic methods used in sequence analysis of
  DNA
• Dideoxysequencing a method for determining the
  exactnucleotide sequence of any DNA. (Fig.
  12.12)

32
Figure 12.12a,b
33
Figure 12.12c
Different-length strands can be lined up by size
to determine DNA sequence.
34
Mutation and DNA Repair Mechanisms

• Mutations are created by chemicals, radiation,
  errors in meiosis and mistakes in DNA
  replication.
• Mutations can be deleterious, beneficial, or
  silent. (Fig. 12.15, 12.17)
• Mutations in an individual are usually
  deleterious, may cause disease and death. (Fig.
  12.16a,b)
• Mutations in a population are a source of genetic
  diversity that allows evolution to occur.

35
Figure 12.15
A
A
C
T
G
G
C
Wild type
T
T
G
A
C
C
G
A
A
C
T
G
G
C
A
A
C
T
A
G
C
MUTANT
3'
5'
T
T
G
A
T
C
G
T
T
G
A
T
C
G
A
A
C
T
G
G
C
DNA replication
DNA replication
T
T
G
A
C
C
G
A
A
C
T
G
G
A
A
C
T
G
G
C
C
5'
3'
Wild type
Parental DNA
T
T
G
A
C
C
G
T
T
G
A
C
C
G
First generation progeny
A
A
C
T
G
G
C
Wild type
T
T
G
A
C
C
G
Second generation progeny
36
Figure 12.17 upper
Mutation type
Definition
Example
Consequence
G
Original sequence
ACC
CAT
Insertion
Addition of any number or nucleotides due to an
error in DNA synthesis
Addition of 1 or 2 bases disrupts reading frame.
Usually results in a dysfunctional gene product.
ATA
GAT
GTA
ACC
CGA
Mutant sequence
ATA
GAT
TGT
A
A
Deletion of 1 or 2 bases disrupts reading frame.
Usually results in a dysfunctional gene product.
Original sequence
ACC
CAT
Deletion
Removal of any number of nucleotides due to an
error in DNA synthesis
ATA
GAT
GTA
ACC
ATG
Mutant sequence
ATA
GTC
TA
37
Figure 12.17 lower
Mutation type
Definition
Example
Consequence
Genes
A
A
A
A
A
Produces an extra copy of one or more genes. If
point mutations occur in extra DNA, it can
produce a new product.
Gene duplication
Addition of a small chromosome segment due to an
error during crossing over at meiosis I.
A
B
B
B
B
B
B
C
C
D
C
C
C
D
C
D
D
D
D
Mutant
Chromosome inversion
Change in a chromosome segment when DNA breaks in
two places, flips, and rejoins.
Changes gene order along chromosome. Other types
of chromosome breaks can lead to deletion or
addition of chromosome segments.
A
A
A
B
B
C
B
C
C
D
D
D
38
Figure 12.16
DNA point mutation can lead to a different amino
acid sequence.
Phenotype
Start of coding sequence
CAC
GTG
GAC
TGA
GGA
CTC
CTC
DNA sequence
GTG
CAC
CTG
ACT
CCT
GAG
GAG
Normal
Amino acid sequence
Normal red blood cells
Histidine
Threonine
Glutamic acid
Glutamic acid
Valine
Leucine
Proline
CAC
GTG
GAC
TGA
GGA
CTC
CAC
DNA sequence
GTG
CAC
CTG
ACT
CCT
GAG
GTG
Mutant
Amino acid sequence
Sickled red blood cells
Threonine
Histidine
Glutamic acid
Leucine
Valine
Valine
Proline
39
Mutation and DNA Repair Mechanisms

• DNA Repair Mechanisms
• DNA polymerase I proofreads and corrects point
  mutations during replication.
• Other excision repair systems scan newly formed
  DNA and correct remaining mutations. (Fig.
  12.13a,b)
• Repair enzymes identify the correct template
  strand by its methyl groups. (Fig. 12.14a,b)
• Defects in repair system enzymes are implicated
  in a variety of cancers. (Fig. 12.18a-c)

40
Figure 12.13
3'
5'
Mismatched bases.
A
T
G
T
C
C
T
C
G
C
A
C
A
G
G
G
5'
OH 3'
5'
Polymerase III can repair mismatches.
3'
A
T
G
T
C
C
T
C
G
C
A
C
A
G
G
5'
OH 3'
T
G
OH
41
Figure 12.14a
Methylated DNA loop
Methyl group on template DNA strand
Mismatch
42
Figure 12.14b
METHYLATION-DIRECTED MISMATCHED BASE REPAIR
1. Where a mismatch occurs, the correct base is
located on the methylated strand the incorrect
base occurs on the unmethylated strand.
Mismatch
2. Enzymes detect mismatch and nick unmethylated
strand.
3. DNA polymerase I excises nucleotides on
unmethylated strand.
4. DNA polymerase I fills in gap in 5'    3'
direction.
5. DNA ligase links new and old nucleotides.
Repaired Mismatch
43
Figure 12.18a
UV-induced thymine dimers caused DNA to kink
H
P
O
P
H
N
O
CH2
CH2
O
N
N
O
Thymine
O
N
O
DNA strand with adjacent thymine bases
UV light
H
CH3
Thymine dimer
H
CH3
P
H
P
O
O
H
Kink
N
N
O
CH2
CH2
O
N
Thymine
O
O
N
H
CH3
H
CH3
P
P
44
Figure 12.18b
Vulnerability of cells to UV light damage
100
Percentage of cells surviving
10
1
Dose of UV light
45
Figure 12.18c
Ability of cells to repair damage
60
50
40
Amount of radioactive thymidine incorporated
(counts per minute)
30
20
10
0
Dose of UV light
46
Applying Ideas, Question 2
3. One round of DNA replication in
non-radioactive solution
1. One round of DNA replication in radioactive
solution
2. Mitosis
47
Applying Ideas, Question 4
Wild type
100
10
uvrA
Percentage of cells surviving
1
recA
0.1
uvrA and recA
0.01
Dose of UV light