MOLECULAR GENETICS

1
MOLECULAR GENETICS

• DNA SYNTHESIS

2
OBJECTIVES

• Understand the process of DNA replication
• Be familiar with the kinds of errors that may
  occur during replication of DNA
• Understand how protein structure and function are
  affected by genetic mistakes

3
FUNCTIONS OF DNA

• Functions
• DNA Synthesis
• DNA makes more DNA
• Protein Synthesis
• Transcription?Translation

4
DNA SYNTHESIS
When In the Cell Cycle Is The Amount of DNA
Doubled?
Sister chromatids
5
Cell
Before S-phase
Nucleus
After s-phase
6
MODELS OF DNA REPLICATION

• Three Models of DNA Replication
• Semi-conservative Replication
• Conservative Replication
• Dispersive Replication

7
MODELS OF DNA REPLICATION
Hypothesis A Hypothesis B Hypothesis
C Semi-conservative Replication
Conservative Replication Dispersive
Replication
8
MODELS OF DNA REPLICATION
Two double helices
Double helix
Protein that copies DNA

If DNA replication is semi conservative, each new
double helix has one original and one new strand
9
MODELS OF DNA REPLICATION

• Meselson Stahl Experiment
• Methods
• 15N was fed to growing E. coli cells to
• mark DNA
• Then cells switched to 14N
• Results
• After 2 generations, ½ samples had low density
  other ½ had intermediate density
• Conclusions
• New DNA composed of
• Entirely 14N
• One 15N strand and one 14N strand
• DNA replication is semi conservative

10
DNA SYNTHESIS IS SEMI-CONSERVATIVE

• DNA replication is semi-conservative in that each
  new DNA molecule incorporates an old strand that
  serves as a template

11
MECHANICS OF DNA SYNTHESIS

• Replication Fork
• Place where double stranded DNA opens to form a
  bubble
• Origins of Replication
• Regions on the DNA where synthesis begins

12
MECHANICS OF DNA SYNTHESIS

• DNA Synthesis Requires An Enzyme (Kornberg,
  1950s)

13
MECHANICS OF DNA SYNTHESIS

• DNA Polymerase
• Forms new strand of DNA
• Complementary to template

14
MECHANICS OF DNA SYNTHESIS

• But first, DNA must be primed

15
MECHANICS OF DNA SYNTHESIS

• Primase
• Enzyme that adds initial nucleotides (RNA primer)
  to template strand
• RNA primer later replaced with DNA

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MECHANICS OF DNA SYNTHESIS

• DNA Synthesis
• Initiated when DNA Polymerase (Pol III) binds to
  RNA primer
• Pol III adds nucleosides to growing new strand
• Nucleoside triphosphate (dNTPs)
• Nucleotide with 3 phosphates
• Four kinds (depends on base)
• dATP
• dTTP
• dGTP
• dCTP
• Numbering Carbons

P
P
P
base
5
sugar
1
4
2
3
17
MECHANICS OF DNA SYNTHESIS

• DNA Polymerase (Pol III)
• Catalyzes formation of phosphodiester bonds
  between nucleoside nucleotide
• Breaking bonds b/w phosphates provides energy for
  snythesis rxn

P
P
P
CH2
Base
5'
O
3'
OH
Structure of nucleoside (dNTPs)
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MECHANICS OF DNA SYNTHESIS

• DNA Polymerase adds nucleosides to 3 end of
  growing DNA strand
• Free OH
• New DNA grows from 5?3

5
3 end
Next nucleoside added to this end!!
OH
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MECHANICS OF DNA SYNTHESIS
Growing Strand Of DNA
5
5
base
5

sugar
3
Next nucleoside added to this end!!
P P
H2O
3
OH
3
OH
20
MECHANICS OF DNA SYNTHESIS
NEW

• DNA Strands
• Are Antiparallel!

OLD
21
MECHANICS OF DNA SYNTHESIS

• New DNA is synthesized in the 5 ? 3 direction
• Nucleosides added to 3 end of growing strand

22
MECHANICS OF DNA SYNTHESIS

• Leading Strand
• New growing strand (continuous) that follows
  replication fork
• Lagging Strand
• Strand that grows (discontinuous) in direction
  away from fork

23
FORMATION OF THE LEADING STRAND
3'
DNA polymerase III
5'
5'
Newly synthesized leading strand
3'
5'
Replication fork
DNA IS UNWINDING OPENING IN THIS DIRECTION
24
MECHANICS OF DNA SYNTHESIS

• Lagging Strand
• Synthesized in short pieces called Okazaki
  fragments, each with their own primer
• Fragments later joined together by DNA ligase

25
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'
26
ROLE OF PROTEINS IN DNA SYNTHESIS

• Proteins In DNA Synthesis
• DNA Polymerase
• Many types
• Primase
• Synthesizes RNA primer
• Helicase
• Unwinds strands of DNA
• Single-Strand Binding Proteins
• Keep original complimentary strands separated
• Ligase
• Links O. Fragments into continuous strand

27
DNA REPLICATION
3
Pol III synthesizes leading strand
1
Helicase opens helix
Primase synthesizes RNA primer
2
4
Pol I excises RNA primer fills gap
5
6
Pol III elongates primer produces Okazaki
fragment
DNA ligase links Okazaki fragments to form
continuous strand
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MUTATION

• Mutation
• Any change in an organisms genome
• Often occurs during DNA replication

A
G
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AN UNCORRECTED GENETIC ERROR
Mutations can occur during DNA synthesis
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
30
MUTATION

• Mutations Can Also Result From
• Mistakes in mRNA synthesis (transcription)
• Exposure to chemicals or radiation
• Errors in meiosis
• Nondisjunction
• Breaks in chromosome

UV Light
31
MUTATION

• Types of Mutations
• Base-pair Substitutions
• Replacement of one or more nucleotides with
  another
• Silent
• Missense
• Nonsense
• Base-pair Insertion or Deletion
• Change in number of nucleotide pairs
• Can result in frame shifting
• Silent
• Missense
• Nonsense

32
MUTATION

• Mutations can be Deleterious, Beneficial, or
  Silent
• In individuals are usually deleterious
• Change in DNA sequence often produces protein(s)
  of abnormal form function
• Cause disease and death
• In populations, they are a source of genetic
  diversity
• Allow evolution to occur

33
MUTATIONS
Not all mutations are deleterious.
EX Silent mutation
34
MUTATION

• Not all mutations are heritable!
• Depends on whether mutation is present in gametes
  or in somatic cells only

35
MOST MUTATIONS ARE DELETERIOUS, SOME ARE
HERITABLE SICKLE CELL ANEMIA
Phenotypes
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
Some DNA point mutations lead to a different
amino acid sequence.
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GENETIC REPAIR MECHANISMS

• Mismatch Repair
• Mechanism by which mismatched nucleotides are
  repaired by DNA Polymerase during DNA synthesis

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MISMATCH REPAIR
3'
5'
Mismatched bases.
A
T
G
T
C
C
T
C
G
C
A
C
A
G
G
DNA polymerase III proofreads and corrects
point mutations during DNA replication.
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
38
GENETIC REPAIR MECHANISMS

• Excision Repair Systems
• Consists of coordinated
• groups of molecules
• Excise stretch of single-stranded DNA, around
  damaged site
• Resynthesize new strand based on intact,
  complementary strand

39
MISMATCH REPAIR IN PROKARYOTES
Methyl group on template DNA strand
Mismatch
40
METHYLATION-DIRECTED MISMATCHED BASE REPAIR
1. Where a mismatch occurs, 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
41
DEFECTS IN GENETIC REPAIR MECHANISMS

• Defects in genes responsible for excision repair
  are frequently associated with cancer
• Xeroderma pigmentosum (XP)
• Individuals are 1000 2000 x as likely to get
  skin cancer

Vulnerability of Cells To UV Light
Normal cells
Cell Survival
Cells from XP Patients
Level UV Light
42
DEFECTS IN GENETIC REPAIR MECHANISMS
XP CELLS HAVE LITTLE OR NOABILITY TO REPAIR
DAMAGE
60
50
Damaged DNA is repaired in normal individuals
40
Amount of radioactive thymidine incorporated
(counts per minute)
30
20
Repair is defective in XP patients
10
0
Dose of UV light
43
UV-induced thymine dimers caused DNA to kink
H
P
O
P
H
O
CH2
CH2
DNA strand with adjacent thymine bases
Thymine
O
O
O
O
UV light
H
CH3
Thymine Dimer
H
CH3
P
H
P
O
O
H
Kink
O
CH2
CH2
O
O
O
Thymine
H
CH3
H
CH3
P
P