Download Types of DNA Mutations

DNA Synthesis
5'
3'
Helicase
Gyrase
Primase
SSB
Leading Strand
Lagging Strand
3'
5'
DNA
Pol
III
DNA
Pol
I
3'
5'
DNA Ligase
RNA primers
New DNA
DNA template
Telomere
3'
5'
E. coli DNA Polymerase III
Processive DNA Synthesis
The bulk of DNA synthesis in E. coli is carried out by the
DNA polymerase III holoenzyme.
• Extremely high processivity: once it combines with the
DNA and starts polymerization, it does not come off until
finished.
• Tremendous catalytic potential: up to 2000
nucleotides/sec.
• Low error rate (high fidelity) 1 error per 10,000,000
nucleotides
• Complex composition (10 types of subunits) and large
size (900 kd)
E. coli Pol III: an asymmetrical dimer
Sliding clamp
clamp loader
Polymerase
3'-5' exonuclease
Polymerase
Stryer Fig. 27.30
2 sliding clamp is important for processivity of Pol III
DNA Synthesis
5'
3'
Helicase
Gyrase
Primase
SSB
Leading Strand
Lagging Strand
3'
5'
DNA
Pol
III
DNA
Pol
I
3'
5'
DNA Ligase
RNA primers
New DNA
DNA template
Telomere
3'
5'
Lagging strand loops to enable the simultaneous replication
of both DNA strands by dimeric DNA Pol III
Stryer Fig. 27.33
DNA Ligase seals the nicks
AMP + PPi
O
O
OH
-O
P
O-
O
O
DNA Ligase +
(ATP or NAD+)
P
O
O-
• Forms phosphodiester bonds between 3’ OH and 5’ phosphate
• Requires double-stranded DNA
• Activates 5’phosphate to nucleophilic attack by trans-esterification
with activated AMP
DNA Ligase -mechanism
ENZYME
1.
E + ATP  E-AMP + PPi
(+)H2N
O
P
Ade
O(-)
O
O
2. E-AMP + P-5’-DNA  AMP-O
P
O
O
5'-DNA
OH
OH
OO
3. DNA-3'
OH
+ AMP-O
P
O
O
5'-DNA
DNA-3'
O
O-
+
P
O
OAMP-OH
5'-DNA
DNA Synthesis in bacteria: Take Home Message
1) DNA synthesis is carried out by DNA polymerases
with high fidelity.
2) DNA synthesis is characterized by initiation, priming,
and processive synthesis steps and proceeds in the 5’
3’ direction.
3) Both strands are synthesized simultaneously by the
multisubunit polymerase enzyme (Pol III). One strand is
made continuously (leading strand), while the other one
is made in fragments (lagging strand).
4) Pol I removes the RNA primers and fills the resulting
gaps, and the nicks are sealed by DNA ligase
Eukaryotic vs prokaryotic cells
Prokaryotes:
Eukaryotes:
• no membrane-bound nucleus
• DNA is located in membranebound nucleus
• transcription and translation
are coupled
• Transcription and translation are
separated in space and time
DNA replication in eukaryotes
Similarities with E.coli replication
1. Polynucleotide chains are made in the 5’  3’ direction
2. Require a primer (RNA).
3. Similarities with the E Coli DNA Pol active site and tertiary structure
Differences
1.
2.
3.
4.
Eukaryotic replication is much slower (only 100 nt/sec).
Many replication origins.
DNA is associated with histones.
DNA Polymerases are more specialized, and their interactions
are more complex.
4. Chromosomal DNA is linear -> requires special processing of
the ends.
Eukaryotic DNA has many replication origins
Cell Cycle
Eukaryotic DNA polymerases
Size, kd
Pol 
250
Pol 
39
Pol 
170
Pol 
Pol 
3’- exo
no
Function
Notes
chromosomal DNA
replication
Inhibited by
arabinosyl NTPs
DNA repair
Inhibited by dideoxy
NTPs
yes
chromosomal DNA
replication
Inhibited by
arabinosyl NTPs
200
yes
DNA replication
in mitochindria
Inhibited by dideoxy
NTPs
260
yes
DNA repair
Inhibited by
aphidocolin
Pol 
lesion bypass
Pol 
lesion bypass
Analogy between bacterial and eukaryotic proteins
involved in DNA replication
Bacteria
Eukaryotes
SSB
Pol I polymerase
Pol III polymerase
2 subunit of Pol III
3’ exonuclease of Pol I
 subunit of Pol III
RPA
Pol 
Pol 
PCNA
RnaseH + FEN1
RCF
RPA = Replication protein A
PCNA = proliferating cell nuclear antigen
FEN1 = flap endonuclease
Lagging strand synthesis in eukaryotes
RNA primer
5’
(a)
RPA=Replication protein A
RPA
Pol/primase
10-30 nt
5’
(b)
PCNA
RCF
(c)
RCF = clamp loader
PCNA = sliding clamp
5’
Pol
(d)
5’
Rnase H/FEN1
RnaseH = 5’-nuclease
FEN1 = flap endonuclease
(e)
ligase
(f)
Telomerase preserves chromosomal ends
• The ends of the linear DNA strand cannot be replicated due to the
lack of a primer
• This would lead to shortening of DNA strands after replication
5‘…
3‘…
3'
5'
RNA primer
• Solution: the chromosomal ends are extended by DNA telomerase
This enzyme adds hundreds of tandem repeats of a hexanucleotide
(AGGGTT in humans) to the parental strand:
5‘…
3‘…
3'
5'
AGGGTTAGGGTTAGGGTT…
5‘…
3‘…
3'
5'
AGGGTTAGGGTTAGGGTT…
TCCCAATCCCAATCCCAA…
telomere
Circular DNA does not have ends:
Upstream Okazaki
fragment
RNA primer
Linear DNA:
5‘…
3‘…
3'
5'
RNA primer
Telomerase is a reverse transcriptase that uses it own
RNA as a template for elongation of the 3’ end of DNA
Telomerase mechanism
Telomerase mechanism - continued
Telomeres form G-tetraplex structures
N
N
H2N
N
N
N
H2N
HN
N
H
O
N
O
O
N
NH
N
H
N
O
N
NH2
N
NH2
N
N
G
G
G
G
Telomerase inhibitors
1. Telomerase RNA as a target for antisense drugs
Modified oligonucleotides that hybridize with telomerase RNA, preventing it from being
used as a template for telomere synthesis.
2. G-tetraplexes at chromosomal ends as a drug target.
Porphyrins, anthraquinones: stabilize G-tetraplex structure, inhibit telomerase activity.
Termination of Polymerization:
The Key to Nucleoside Drugs
NH2
O
NH
N
HN
N
O
HO
O
N
HO
N
NH
N
N
O
NH2
N
HO
N
NH
N
NH2
O
HO
O OH
O
N3
OH
AZT
Ziagen
Antiviral
Acyclovir
AraC
Antitumor
Principle of action: 1) cellular uptake
2) activation to 5’-triphosphate
3) incorporation in DNA resulting in chain
termination
Nucleoside inhibitors of reverse transcriptase
replication
Typical flow of genetic information:
DNA
RNA
Proteins
Cellular Action
transcription translation
DNA
Notable exception: retroviruses
RNA
DNA
RNA
Proteins
Reverse
transcription translation
transcription
RNA
Cellular
Action
Reverse transcriptases (RT) are RNA-directed DNA Polymerases
Used by RNA viruses (HIV-I , human immunoblastosis virus,
Rous sarcoma virus)
1. Make RNA-DNA hybrid (use its own RNA as a primer)
2. Make ss DNA by exoribonuclease (RNase H) activity
3. Make ds DNA  incorporate in the host genome
RT
RT
RT
RNAse H
RNA
RNA:DNA hybrid
ss DNA
ds DNA
HIV Life Cycle
1 = Entry in CD4+ lymphocytes
2 = Reverse transcription
3 = Integration
4 = Transcription
5 = Translation
6 = Viral Assembly
Termination of Polymerization:
Nucleoside Drugs
NH2
O
NH
N
HN
N
O
HO
N
O
HO
N
N
N
N
O
NH2
N
HO
N
NH
N
NH2
O
HO
O OH
O
N3
OH
AZT
Ziagen
(zidovudine)
(abacavir)
Acyclovir
Antiviral
AraC
Antitumor
Other examples: dideoxycytidine, dideoxyinosine
Principle of action:
1) cellular uptake
2) activation to 5’-triphosphate
3) competition with normal substrate and
incorporation in DNA resulting in chain
termination
Anti-HIV drug Ziagen was discovered at the U of M
College of Pharmacy
HN
N
N
HO
N
N
NH2
Ziagen (abacavir)
1998
Robert Vince, Professor
Department of Medicinal Chemistry
Nucleoside Drugs Must Be Converted to
Triphosphates to be Part of DNA and RNA
O
P O
HO
HO
HO
O
Base
Ki nase
ATP
O
OH
OH
Monophosphate
ATP
O
O
O
HO P O P O P O
HO
OH OH
O
OH
Base
Base
Ki nase
O
O
HO P O P O
HO
OH
ATP
Triphosphate
• Compete with normal substrate for RT binding
• Cause chain termination
Ki nase
O
OH
Diphosphate
Base
DNA Chain termination by Nucleoside Analogs
Primer
Strand
O
O
P
Base
O
O-
O
Template
Strand
3'
OH
O
-O
P
O-
O
O
P
O-
O
O
P
O
Base
5'
O-
Mg 2+
Ziagen
No 3’OH!
Mechanisms of selectivity
1.
Activated drug is recognized and incorporated in
DNA only by reverse transcriptase, not by cellular
DNA polymerases (RNA viruses).
•
•
viral polymerases usually have lower fidelity
(no proofreading)
Mammalian DNA polymerases are more accurate
2. The drug is phosphorylated by viral kinase, not
by cellular kinases (e.g. AZT).
Mechanisms of resistance and possible solutions:
1.
2.
The drug cannot enter cells or is pumped out rapidly.
The drug is rapidly deaminated to inactive form or normal substrate is
overproduced.
3. The drug is no longer recognized by kinases and is not
activated to triphosphate form.
Possible solution:
Use activated phosphate form of nucleosides (Viread)
4. Activated drug is not incorporated in DNA by mutant reverse
transcriptase (usually HIV RT mutations at codons 184,65,69, 74, and 115).
Possible solution:
Use a mixture of several RT inhibitors (e.g. zidovudine (AZT) +
lamivudine (3TC) = Combivir®) or a mixture of different mechanisms
of action (e.g. non-nucleoside RT inhibitors, protease inhibitors).
Nucleoside inhibitors of DNA polymerase as
anticancer drugs
NH2
N
N
O
HO
O OH
OH
AraC (1--D-arabinofuranosylcytosine)
• used for treating acute myelocytic leukemia
• activated to triphosphate form by cellular kinases
• causes inhibition of DNA synthesis, repair, and DNA
fragmentation
• very toxic
DNA Damage, Mutations, and
Repair
See Stryer p. 768-773
DNA Mutations
1.
Substitution mutations: one base pair for another,
e.g. T for G
• the most common form of mutation
• transitions; purine to purine and pyrimidine to
pyrimidine
• transversions; purine to pyrimidine or pyrimidine to
purine
2. Frameshift mutations
•
Deletion of one or more base pairs
•
Insertion of one or more base pairs
Spontaneous mutations due to DNA
polymerase errors
•
•
Very low rate of misincorporation (1 per 108 - 1 per 1010)
Errors can occur due to the presence of minor tautomers
of nucleobases.
H3C
O
H2N
N
NH
N
N
N
N
O
T
amino
A
Rare imino tautomer of A
10-4
Normal base pairing
Mispairing
Consider misincorporation due to a rare tautomer of A
2nd
replication
1st
replication
5’ A
3’ T
A
T
A(imino)
C
A(imino)
T
G
C
A
T
Normal replication
Final result: A  G transition (same as T  C in the other strand)
Induced mutations result from DNA damage
Sources of DNA damage: endogenous
1. Deamination
2. Depurination: 2,000 - 10,000 lesions/cell/day
3. Oxidative stress: 10,000 lesions/cell/day
Sources of DNA damage: environmental
1. Alkylating agents
2. X-ray
3. Dietary carcinogens
4. UV light
5. Smoking
Normal base pairing in DNA and an example of
mispairing via chemically modified nucleobase
O
N
N
o
h
N
NH
h
N
NH2 O
G
OR
n
N
N
NH2
N
N
N
O
NH
HN
h
NH2
O
O6-AlkG
C
T
G A
G
C
G
T
A
T
DNA oxidation
Reactive oxygen species: HO•, H2O2, 1O2, LOO•
O
O
H3C
H3C
HO
NH
N
O
N
NH
N
N
O
thymine glycol
O
H
O
N
HO
NH
NH2
N
NH
O
N
N
NH2
8-oxo-G
•10,000 oxidative lesions/cell/day in humans
Deamination
NH2
N
O
N
N
N
N
N
A
O
N
N
G
N
NH2
O
N
N
Mechanism:
Hypoxanthine
N
HO
O
N
N
NH
N
NH2
NH
NH2
NH
N
H
Xanthine
N
O
N
N
H2O
N
- NH3
N
O
HO
NH2
N
NH
NH
O
N
C
O
N
N
N
Uracil
H NH
A
NH N
N
O
O
G
N
C
Rates increased by the presence of NO (nitric oxide)
N
NH
N
Depurination to abasic sites
O
N
O
O
O
N
O
NH
N
H2O
NH2
O
O
OH
O
Abasic site (AP
site)
2,000 – 10,000/cell/day
N
N
H
NH
N
NH2
UV light-induced DNA Damage
O
H3C
O
NH
NH
N
O
H3C
N
O
O
CH3
O
NH
O
O P OO
N
O
…CC…
O
O
O
CH3
NH
O
O P OO
N
O
O
Pyrimidine dimer
Easily bypassed by Pol  (eta) in an error-free manner
Deletions and insertions can be caused by intercalating agents
Stryer Fig. 27.44
Metabolic activation of carcinogens
N7-guanine adducts
G  T transversions
Stryer Fig. 27.45
Chemical modifications of DNA in mutagenesis and anticancer therapy
carcinogen or drug (X)
detoxification
metabolic activation
excretion
reactive metabolite (X-)
DNA
DNA adducts
X
X
repair
replication
*
intact DNA
cell death
Anticancer
*
mutations
Cancer
Importance of DNA Repair
• DNA is the only biological macromolecule
that is repaired. All others are replaced.
• More than 100 genes are required for DNA repair,
even in organisms with very small genomes.
• Cancer is a consequence of inadequate DNA repair.
DNA Repair Types
• Direct repair
– Alkylguanine transferase
– Photolyase
• Excision repair
– Base excision repair
– Nucleotide excision repair
– Mismatch repair
• Recombination repair
Direct repair
• DNA photolyase (E. Coli)
O
O
H3C
H3C
NH
NH
N
5'
N
O
5'
O
CH3
O
N
O
CH3
O
NH
O
O P OO
O
NH
O
O P OO
N
O
O
3'
O
3'
O
O6-alkylguanine DNA alkyltransferase (AGT)

Directly repaires O6-alkylguanines (e.g. O6-Me-dG, O6-Bz-dG)
In a stoichiometric reaction, the O6 alkyl group is transferred to a
Cys residue in the active site. The protein is inactivated and
degraded.

O
N
N
CH3
O
N
N
N
NH
AGT-CH2-SH
NH2
O6-methylguanine
N
N
AGT-CH2-S
NH2
CH3
AGT protein is highly conserved
hydrophobic side-chains
form alkyl-binding pocket
helix-turn-helix
motif
Excision Repair
Takes advantage of the double-stranded (double information)
nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
Antiparallel DNA Strands contain the same
genetic information
5'
3'
5'
3'
5'
3'
3'
A :: T
A :: T
A :: T
G ::: C
G
G ::: C
T :: A
T :: A
T :: A
5'
Original DNA duplex
3'
5'
DNA duplex with
one of the nucleotides
removed
3'
5'
Repaired DNA duplex
Excision Repair
Takes advantage of the double-stranded (double information)
nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
Base excision repair (BER)
• Used for repair of small damaged bases in DNA (AP
sites, methylated bases, oxidized bases…)
H
N
O
N
N
O
O
O
OH
N
NH
NH
O
8-oxo-G
O
NH2
Abasic site (AP
site)
N
NH2
N
H
O
N
Xanthine
N
N
N
Me
N3-Me-Ade
• Human BER gene hogg1 is frequently deleted in lung
cancer
Base Excision Repair
Base2-ppp
O
O
O
Base1
O
O P OO
O
Base2
O
O P OO
O
O
O P OO-
O
O
O P OO
O
R
O
Base1
Base1
O
OH
O
O P OO
O
(b)
Base3
O
O P OO-
O
Base1
O
O P OO
O
OH
(a)
Base3
O
Base2
(c), (d)
OH
O P OO
O
Base3
O
O P OO-
O
O P OO
O
O
O P OO-
AP site
a) modified base is excised by N-glycosylase
b) the abasic site is cleaved by AP endonuclease/lyase
c) the resulting gap is filled by Polymerase b
d) DNA Ligase seals the nick
Base3
BER enzyme AlkA complex with DNA
Stryer Fig. 27.48
Uracil DNA glycosylase removes deaminated C
No Me group
NH2
O
N
N
BER
C
NH
O
N
Cytosine
O
Not normally present in DNA
Uracil
However, deamination of 5-Me-C produces thymine:
O
NH2
H3C
H3C
N
N
O
Cytosine (C)
NH
N
BER
O
Thymine (T)
Net result: G:T base pair
Normal DNA base
Nucleotide Excision Repair
•
Corrects any damage that both distorts the DNA molecule and
alters the chemistry of the DNA molecule (pyrimidine dimers,
benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links).
O
H3C
O
NH
N
5'
O
O
O P OO
N
O
HO32N
CH
Pt
NH
H2N
N
Cl
Cl
O
O
3'
•
HO
O
NH
H2N N OHNH
O N
2
HOPt
OH H2N OH2
HO
OH
H
N
NH2
N
-GGH2N
H2N
N
Pt
N
O
N
N
NH
N
NH2
Xeroderma pigmentosum is a genetic disorder resulting
in defective NER
Nucleotide excision repair (NER)
Mammalian
Enzyme
exinuclease
Pol /
DNA ligase
Mismatch Repair Enzymes
Nucleotide mismatches can be corrected after DNA synthesis!
Repair of nucleotide mismatches:
1. Recognize parental DNA strand (correct base) and daughter
strand (incorrect base)
Parental strand is methylated:
H3C
NH2
HN
N
N
N
O
N
Me
N
N
2. Replace a portion of the strand containing erroneous nucleotide
(between the mismatch and a nearby methylated site –up to 1000 nt)
Mismatch Repair in E. coli
Stryer Fig. 27.51
Recombination repair
DNA Synthesis in bacteria: Take Home Message
1) DNA synthesis is carried out by DNA polymerases
with high fidelity.
2) DNA synthesis is characterized by initiation, priming,
and processive synthesis steps and proceeds in the 5’
3’ direction.
3) Both strands are synthesized simultaneously by the
multisubunit polymerase enzyme (Pol III). One strand is
made continuously (leading strand), while the other one
is made in fragments (lagging strand).
4) Pol I removes the RNA primers and fills the resulting
gaps, and the nicks are sealed by DNA ligase
Genetic diseases associated with defective DNA repair
Xeroderma Pigmentosum
NER
Hereditary nonpolyposis
colorectal cancer
MMR
Cockrayne’s syndrome
NER
Falconi’s anemia
DNA ligase
Bloom’s syndrome
BER, ligase
Lung cancer (?)
BER