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Biochemistry for Pharmacy Students
NUCLEIC ACID STRUCTURE &
FUNCTION
Pál Bauer
Dept. of Medical Biochemistry
Rm. 4515, EOK
Email: [email protected]
OBJECTIVES
• To learn the structures and properties of
nucleotides, the building blocks of nucleic
acids
• To describe how the structure of DNA
relates to functions of the genetic
machinery.
• To explain how DNA is synthesized.
• To describe how mutations in DNA can lead
to genetic diseases.
• To explain how recombinant DNA technology
can be applied to the diagnosis and therapy
of human genetic diseases (discussion
sessions).
HISTORICAL PERSPECTIVE
The beginnings of nucleic acid biochemistry & genetics
occurred in the 1860s:
- Frederic Miescher – Swiss biochemist who
discovered nucleic acids.
- Gregor Mendel – Austrian monk who founded the
science of genetics.
Nucleic acid structure & metabolism is directly relevant to
cancer, gout, many genetically inherited diseases, AIDS
and other infectious diseases.
Frederick Meischer
studied salmon
Gregor
Mendel
studied
garden
peas
• Sugars and phosphates
– Ribose in RNA
– Deoxyribose in DNA
• Partial hydrolysis products
– Nucleotides (contains phosphates)
– Nucleosides (no phosphates)
Names of nucleosides
Base
Ribonucleoside
Deoxyribonucleoside
Adenine
Adenosine
Deoxyadenosine
Guanine
Guanosine
Deoxyguanosine
Cytosine
Cytidine
Deoxycytidine
Uracil
Uridine
Deoxyuridine
Thymine
Thymine riboside
Thymidine
Nucleosides: numbering system
• The intra-nucleotide linkage
Base composition of double stranded DNA
•
•
•
[pyrimidines] = [purines]
Content: A = T, G = C
DNA base compositions are the same for
different tissues of the same organism.
Watson-Crick base pairing
Crick
Watson
A Reminder of the Differences between
Eukaryotes and Prokaryotes
Animal Cell
(eukaryote)
E. coli
Bacterial Cell
(prokaryote)
Plant Cell
Fig. 2-7
(eukaryote)
Model of the Nuclear Envelope
Artwork by Don Guzy
5’
• The Watson-Crick
Structure
3’
•
•
•
•
Right-handed helices
Anti-parallel
Base-pairing
Open structure
accessible to water
• Stacking forces
between planar
paired bases give a
rigid structure
5’
3’
Denaturation of DNA
By pH, heat, solvents, urea, amides
• Helix-coil transition
• Hyperchromic effect
• Melting temperature
Denaturation of
double-stranded
DNA
DNA IS THE GENETIC MATERIAL IN CELLS
Indirect evidence
a. High DNA content of chromosomes
b. 260 nm is a very mutagenic wavelength; bases
maximally absorb light energy at 260 nm
c. Constancy of [DNA] / cell (germ cells)
and 2x [DNA] / cell (somatic cells)
Direct Evidence
a. Transformation of bacteria with DNA
Requires both DNA functions:
replication and expression
b. Transfection with viral nucleic acids
c. In vitro expression of DNA
(from bacteriophage T4)
d. Synthesis of active DNA in vitro
Pneumococci
DNA CONTENT
OF SOME CELLS AND VIRUSES
Source of DNA
Viruses
SV40
Papilloma (wart)
Adenoviruses
Herpesviruses
Poxviruses
Haploid size of genome,
base pairs____
5 x 103 (5 kb)
8 x 103
2.1 x 104
1.56 x 105
2.4 x 105
Cells
Escherichia coli
Yeast
Drosophila
Human
Frogs
Onion Plant
Fern Plant
Animal mitochondria
Plant chloroplast
4.5 x 106
1.3 x 107
1.6 x 108
3.2 x 109
6 x 109
18 x 109
160 x 109
(4,500 kb)
(3.2x106 kb)
(6 x 106 kb)
1.5 x 104 (15 kb)
1 x 105
The total length of the circular E. coli chromosome is 1.7 mm,
whereas the length of an E. coli cell is 2 μm.
partially lyzed
E. coli cell
Human haploid genome
22 autosomal chromosomes (chromosomes 1-22)
plus X and Y chromosomes
3.2 x 109 total bp
• Histones and chromosome structure
a. Nucleosomes
The doublestranded DNA
is wrapped
around the
outside of each
nucleosome twice.
The nucleosomes
are regularly
spaced along the
DNA.
electron micrograph of chromatin
electron micrograph of
supercoiled nucleosomes
in chromatin.
A typical phone cord is coiled
like a DNA helix, and the
coiled cord can itself coil in
in a supercoil. If twisted
tight enough, the supercoils
will themselves form an even
higher order of supercoiling.
Double-stranded DNA helices
also form supercoils of
supercoils.
Two drawings depicting the different
levels of DNA supercoiling that provide
DNA compaction in a eukaryotic
chromosome. The levels take the form
of coils upon coils.
nucleosomes
histones
DNA replication
3’
5’
5’
5’
3’
5’
3’
3’
5’
3’
5’
3’
5’
3’
5’
All DNA polymerases catalyze elongation of
the primer strand in a 5’ to 3’ direction,
copying the template strand in a 3’ to 5’
direction. This means that the “leading
strand” can be synthesized continuously,
but the “lagging strand” must be
synthesized discontinuously.
A problem is that DNA
polymerases must have a primer
strand with a 3’ OH from which
to begin DNA synthesis. So, where
do these primers come from when
a DNA polymerase synthesizes new
Okazaki fragments in the lagging
strand?
Replication does not usually begin at the end of a DNA molecule; it
begins in the middle of the DNA.
5’
3’
3’
5’
5’
3’
3’
5’
(DNA pol I, II and III)
DNA
12.4 Schematic model of the
proofreading function of DNA
polymerase
Figure 12-21
An example of error
correction by the 3’ to 5’
exonuclease (proofreading)
activity of DNA polymerase I.
A mismatched base pair (a
C-A mismatch) impedes the
movement of DNA polymerase I
to the next site. Sliding backward, the enzyme removes its
mistake with the 3’ to 5’ exonuclease activity & then
resumes its 5’ to 3’
polymerase activity.
c. DNA Polymerase II
• Mutants in the E. coli gene for this DNA
polymerase are not lethal. Also is a repair
enzyme.
• Requires duplex DNA template and primer.
• Utilizes a template strand and elongates a primer
strand, similar to DNA polymerase I.
d. DNA Polymerase III (pol C gene)
Mutants are ts (temperature sensitive),
i.e., conditionally lethal.
The 3-D structure of E. coli DNA polymerase I.
The active site for the polymerase activity and the
3’ to 5’ exonucelase activity is deep in the crevice
at the far end of the bound DNA. The template strand
is dark blue.
a. Illustration of the 5’ to 3’
exonuclease activity of E. coli
DNA polymerase I (sometimes
called a “nick translation”
activity).
An RNA or DNA strand
paired with a DNA template
strand is simultaneously
degraded by the 5’ to 3’
exonuclease activity & is
replaced by the polymerase
activity of the same enzyme.
b. The origin of replication – the ori C locus
Accessory proteins
dna B gene product
Probably this protein is membrane-associated
and recognizes the initiation sequence on DNA.
c. RNA primers
DNA polymerases require a preformed primer;
RNA polymerases do not.
d. Details of the events:
1. Accessory protein binds (dna B protein)
2. "Primase," an RNA polymerase (dna G)
It does not require a primer; forms a short
piece of RNA complementary to the DNA
template strand.
3. DNA binding proteins
cell
membrane
cell
membrane
5’
5’
3’
5’
5’
3’
5’
3’
Additional information about the overall
process of DNA replication
The 5’ end of RNA primer is usually 5’ pppA... or 5’ pppG….
DNA polymerase III dissociates the primase that synthesizes
the RNA primer.
The 5' → 3' nuclease of DNA polymerase l, or another enzyme
called RNase H, removes the RNA primer. RNase H
hydrolyzes RNA in DNA/RNA base-paired regions.
DNA polymerase I (and maybe DNA pol II?) fill the gaps left
by the removal of the RNA primers.
The complete but "nicked" (broken) daughter strands that result
after the gaps are filled in are then joined by DNA
ligase.
• DNA ligase (animal enzyme)
• The unwinding problem: 5000 turns/min in E. coli
(helicases and topoisomerases)
Helicases: unwind the two strands of DNA & cause
supercoiling ahead of the fork.
Topoisomerase I: relaxes supercoils
a. Enzyme recognizes right-handed superhelices
introduced during DNA replication.
b. Small DNA segment unwinds, as strand
breaks, the superhelix relaxes.
c. Enzyme, still attached to broken strand,
reseals the break.
d. The topoisomerase is both a nuclease and a
ligase. (The nucleolytic activity can be viewed as the
reverse of ligase action.)
SUMMARY OF DNA REPLICATION IN BACTERIA
Map of the E. coli genome showing some of the genes that encode
proteins involved in DNA replication, repair and recombination.
DNA REPLICATION in EUKARYOTIC CELLS is SOMEWHAT
MORE COMPLICATED and INVOLVES ADDITIONAL DNA
POLYMERASES & MULTIPLE REPLICATION ORIGINS
Greek
Name
Gene
Name
Proposed Main Function
alpha
POLA
DNA Replication
beta
POLB
Base Excision Repair
gamma
POLG
Mitochondrial Replication
delta
POLD1
DNA Replication
epsilon
POLE
DNA Replication
zeta
POLZ
Bypass Synthesis
eta
POLH
Bypass Synthesis
theta
POLQ
DNA Repair
iota
POLI
Bypass Synthesis
kappa
POLK
Bypass Synthesis
lambda
POLL
Base Excision Repair
 mu
POLM
Non-Homologous End Joining
sigma
POLS
Sister Chromatid Cohesion
•
The number of known eukaryotic DNA
polymerases has doubled in the last
several years as researchers discover
additional enzymes with DNA
polymerizing activity.
•
Most of the newly discovered DNA
polymerases are involved in repair of
DNA, rather than DNA replication.
•
The DNA polymerases alpha, delta and
epsilon are the most clearly involved in
DNA replication.
•
DNA polymerases delta and epsilon
have proof-reading 3’->5’ exonuclease
activity; the other DNA polymerases do
not.
Multiple replication origins occur in the large eukaryotic chromosomes.
Mutation
Types and rates of mutation
Type
Genome
mutation
Mechanism
chromosome
missegregation
(e.g., aneuploidy)
Frequency________
10-2 per cell division
Chromosome
mutation
chromosome
rearrangement
(e.g., translocation)
6 X 10-4 per cell division
Gene
mutation
base pair mutation
10-10 per base pair per
(e.g., point mutation,
cell division or
or small deletion or
10-5 - 10-6 per locus per
insertion
generation
Mutation rates* of selected genes
Gene
Achondroplasia
Aniridia
Duchenne muscular dystrophy
Hemophilia A
Hemophilia B
Neurofibromatosis -1
Polycystic kidney disease
Retinoblastoma
New mutations per 106 gametes
6
to 40
2.5 to
5
43
to 105
32
to
2
to
44
to 100
60
to 120
5
to 12
57
3
*mutation rates (mutations / locus / generation) can vary
from 10-4 to 10-7 depending on gene size and whether
there are “hot spots” for mutation (the frequency at most
loci is 10-5 to 10-6).
Many polymorphisms exist in the genome
• the number of existing polymorphisms is ~1 per 500 bp
• there are ~5.8 million differences per haploid genome
• polymorphisms were caused by mutations over time
• polymorphisms called single nucleotide polymorphisms
(or SNPs) are being catalogued by the Human
Genome Project as an ongoing project
Types of base pair mutations
normal sequence
CATTCACCTGTACCA
GTAAGTGGACATGGT
transition (T-C to A-G)
CATCCACCTGTACCA
GTAGGTGGACATGGT
transversion (T-A to G-C)
CATGCACCTGTACCA
GTACGTGGACATGGT
base pair substitutions
transition: pyrimidine to pyrimidine
transversion: pyrimidine to purine
deletion
CATCACCTGTACCA
GTAGTGGACATGGT
insertion
CATGTCACCTGTACCA
GTACAGTGGACATGGT
deletions and insertions can involve one
or more base pairs
Spontaneous mutations can be caused by tautomers
Tautomeric forms of the DNA bases
Adenine
Cytosine
AMINO
IMINO
Tautomeric forms of the DNA bases
Guanine
Thymine
KETO
ENOL
Mutation caused by tautomer of cytosine
Cytosine
Normal tautomeric form
Guanine
Cytosine
Rare imino tautomeric form
Adenine
• cytosine mispairs with adenine resulting in a transition mutation
Mutation is perpetuated by replication
C G
C G
• replication of C-G should give daughter strands each with C-G
C G
C A
• tautomer formation C during replication will result in mispairing
and insertion of an improper A in one of the daughter strands
C A
T A
• which could result in a C-G to T-A transition mutation in the next
round of replication, or if improperly repaired
Chemical mutagens
Deamination by nitrous acid
O
Attack by oxygen free radicals
leading to oxidative damage
N
• many different oxidative modifications occur
• by smoking, etc.
• 8-oxyG causes G to T transversions
NH
NH
N
NH2
guanine
O
H
N
NH
O
NH
N
NH2
8-oxyguanine (8-oxyG)
• the MTH1 protein degrades 8-oxy-dGTP preventing misincorporation
• mutation of the MTH1 gene causes increased tumor formation in mice
Ames test for mutagen detection
• named for Bruce Ames
• reversion of histidine mutations by test compounds
• His- Salmonella typhimurium cannot grow without histidine
• if test compound is mutagenic, reversion to His+ may occur
• reversion is correlated with carcinogenicity
Thymine dimer formation by UV light
Summary of DNA lesions
Missing base
Acid and heat depurination (~104 purines
per day per cell in humans)
Altered base
Ionizing radiation; alkylating agents
Incorrect base
Spontaneous deaminations
cytosine to uracil
adenine to hypoxanthine
Deletion-insertion
Intercalating reagents (acridines)
Dimer formation
UV irradiation
Strand breaks
Ionizing radiation; chemicals (bleomycin)
Interstrand cross-links
Psoralen derivatives; mitomycin C
Tautomer formation
Spontaneous and transient
Correlation between DNA repair
activity in fibroblast cells from
various mammalian species and
the life span of the organism
100
human
elephant
Life span
cow
10
hamster
rat
mouse
shrew
1
DNA repair activity
Defects in DNA repair or replication
All are associated with a high frequency of chromosome
and gene (base pair) mutations; most are also associated with a
predisposition to cancer, particularly leukemias
• Xeroderma pigmentosum
• caused by mutations in genes involved in nucleotide excision repair
• associated with a >1000-fold increase of sunlight-induced
skin cancer and with other types of cancer such as melanoma
• Ataxia telangiectasia
• caused by gene that detects DNA damage
• increased risk of X-ray
• associated with increased breast cancer in carriers
• Fanconi anemia
• caused by a gene involved in DNA repair
• increased risk of X-ray and sensitivity to sunlight
• Bloom syndrome
• caused by mutations in a a DNA helicase gene
• increased risk of X-ray
• sensitivity to sunlight
• Cockayne syndrome
• caused by a defect in transcription-linked DNA repair
• sensitivity to sunlight
• Werner’s syndrome
• caused by mutations in a DNA helicase gene
• premature aging
Transition
• Transition
vs. transversion
Exchange of a purine with a purine or pyrimidine with a
pyrimindine base. More common than transversion. Often the
result of tautomeric shifts.
– GC  AT transition
• causal agents: e.g. base analog 5’bromouracil
– AT  GC transition
• causal agents: e.g. base analog 2-aminopurine
• Transversion
Exchange of a purine with a pyrimidine base and vice versa
– GC  TA or GC  CG transversion
– AT  CG or AT  TA transversion
Methyl-directed mismatch
repair
1. Mismatch
within 1 kb of
5’
CH3
CH3
MutS, MutL, ATP
ADP+Pi
5’
CH3
MutL
MutH, ATP
ADP+Pi
CH3
5’
3’
5’
3’
CH3
MutS
2. MutS and MutH bind to
mismatched spots along
the DNA (except C-C)
3. DNA on both sides of the
Mitsmatch runs through
MutS:MutL complex
CH3
MutS
MutH
MutL MutH
CH3
methylated GATC
4. MutH binds to MutL and
to GATC
CH3
5. Endonuclease of MutH
cleaves unmethylated DNA
at hemimethylated GATC
Mismatch repair in E. coli
Scenario 1
Mismatch is at the 5’ end of
cleavage site
1. Unmethylated DNA is
unwound via DNA helicase II
2. The 3’-5’ exonuclease activity
of exonuclease I or exo X
degrades DNA through the
mismatch
5’
3’
ATP
ADP+Pi
5’
3’
3. DNA polymerase III
synthesizes the new DNA
strand
4. DNA ligase closes the
remaining nick.
CH3
5’
3’
CH3
CH3
CH3
MutS-MutL
DNA helicase II
exonuclease I
or
exonuclease X
3’
5’
CH3
3’
5’
DNA polymerase III
SSBs
CH3
3’
5’
Mismatch repair in E. coli
Scenario 2
Mismatch is at the 3’ end of
cleavage site
1. Unmethylated DNA is
unwound via DNA helicase II
2. The 5’-3’ exonuclease activity
of exonuclease VII or RecJ
nuclease degrades DNA
through the mismatch
5’
3’
ATP
ADP+Pi
5’
3’
3. DNA polymerase III
synthesizes the new DNA
strand.
4. DNA ligase closes the
remaining nick.
CH3
5’
3’
CH3
CH3
CH3
MutS-MutL
DNA helicase II
exonuclease VII
or
RecJ nuclease
3’
5’
CH3
3’
5’
DNA polymerase III
SSBs
CH3
3’
5’
•
•
•
•
•
Base excision repair
not restricted to a short time post replication
similar in most organisms (bacteria – mammals)
recognizes abnormal bases in the DNA
usually less expensive than mismatch repair
requires four enzymes
1.
2.
3.
4.
DNA glycosylases
AP-endonucleases
DNA polymerase I
DNA ligase
•
DNA
glycosylases
G P
Relatively small
P
O
enzymes (20 – 30 KDa)
• Recognize abnormal bases
– deaminated bases
– alkylated bases
• Remove base via cleavage at
the glycosidic bond between
the deoxyribose and the base
• Cleavage creates apurinic and
apyrimidinic (AP sites)
U
P
G
O
P
O
O
P
A
O
Before
P
A
O
After
DNA glycosylases
Enzyme
Uracil DNA glycosylase
Hypoxanthin DNA glycosylase
3- methyladenine I DNA glycosylase
3- methyladenine II DNA glycosylase (*)
Formamidopyrimidine DNA glycosylase
Units / mg protein (x 10 - 3)
not adapted
3,800
2.1
3.6
0.22
4.2
adapted
3,500
2
3.6
4.1
3.9
Legend:
Adapted = incubation with 1 g/ml NNG for 1 hour
Not adapted = untreated control
Source: Karran et al. (1983), Nature 296, 770 - 773
AP-endonucleases
5’ P P P P
P P P
• recognize AP-sites
A G G C A G C
• cleave phosphodiester
bonds near the AP site
T C C
T C G
and generate a 5’ –
3’ P P P P P P P
phosphate and 3’AP endonuclease
hydroxyl
• In E. coli this enzyme
also has 3’-5’ exonuclease P P P P P P P
activity
A G G C A G C
• The 3’-OH functions as a
T C
C G
primer
5’
P
P
3’
P
P
Base excision repair (BER)
P
P
P
P
P
P
A
G
G
C Uracil DNA glycosylase
A
T
U
C
G
T
P
P
P
P
P
G
P
P
G
C
C
G
P
P
P
AP endonuclease
P
P
A
P
G
T
G
C
P
P
C
C
P
P
DNA polymerase I
G
P
DNA ligase
P
P
A
P
G
T
P
G
C
G
P
P
•
Defects
in
BER
In humans BER involves:
– DNA polymerase beta
– The Xrcc1 geneproduct
– Ape1 (first step in removing the damaged base)
• BER deficiencies have been implicated with:
– cancer
– neurodegenerative diseases
– aging
•
•
•
•
•
•
Nucleotide
excision
repair
Recognizes large distortions in the DNA structure
Repairs UV-damaged DNA
Multisubunit enzyme
Cleaves two phosphodiester bonds upstream and
downstream of the lesion
Generally generates fragments of 12 to 13 nts
Requires four different enzymes
1.
2.
3.
4.
Exinuclease
DNA helicase
DNA polymerase
DNA ligase
Nucleotide excision repair in E. coli
Enzyme
Protein
Function
UvrA (MW= 104,000)
scans DNA, binds to UvrB
UvrB (MW = 78,000)
scanner; binds DNA cleaves
phosphate bond at 3' end, 5
positions downstream of lesion
UvrC (MW = 68,000)
binds UvrB & DNA cleaves
phosphate bond at 5' end, 8
positions upstream of lesion
Exinuclease
DNA helicase
DNA
polymerase
DNA ligase
UvrD
DNA polymerase I
(= PolA )
Lig
removes DNA fragment
fills emerging gap
seal nick
Nucleotide excision repair in E. coli
Mechanism
• The (UvrA)2:UvrB complex
scans DNA
• UvrA dimer dissociates from
pryimidine dimer. UvrB binds
DNA and cuts at 3’ end.
• UvrC associates with UvrB and
cuts DNA at 5’ end of the
P
pyrimidine dimer
• UvrD DNA helicase removes
the DNA fragment
• DNA polymerase I fills the gap
• DNA ligase seals the remaining
nick.
UvrA
UvrA
UvrB
exinuclease
P
ATP
P
P
OH
DNA pol. I
UvrD DNA
helicase
P
DNA ligase
• Cause:
Xeroderma pigmentosum
– Defect in human nucleotide excision repair (NER)
– 16 polypeptides involved in NER
– NER is the only pathway to remove pyrimidine dimers
in humans
• Symptoms:
– Very light sensitive
– High risk of sun-light induced skin cancer
– Neurological abnormalities (high rate of oxidative
metabolism in neurons)
• Observation:
Direct repair
– UV-damaged bacteria that were subsequently incubated
in daylight recovered better than those kept in the dark
• Photoreactivation
–
–
–
–
requires DNA photolyases
requires visible light at 300 – 500 nm
aka “light repair”
Contrast: dark repair (BER, NER, mismatch repair)
• Structure
–
–
–
–
DNA photolyases
MW = ~ 54,000 (in E. coli)
Generally contain 2 chromophores
Chromophore No. 1: always FADH Chromophore No. 2: folate (in E. coli and yeast)
• N5,N10-methenyltetrahydrofolylpolyglutamate
• Function
– bind to pyrimidine dimers
– resolve pyrimidine dimers into original bases
1.
Mechanism
outline
Absorption of a photon by MTHFpolyGlu
2. Transfer of excitation energy to FADH –
3. Excited FADH – transfers elecctron to the pyrimidine
dimer (unstable dimer radical)
4. Shift of electrons breaks cyclobutane ring
5. Electron is transferred back to the flavin radical to
regenerate FADH –
6
O -methylguanine
• Mutagenic behavior
– pairs with thymine rather than cytosine
• Specific Repair Mechanism
–
–
–
–
–
differs from base excision repair
requires O6-methylguanine methyltransferase
one time reaction (“suicide enzyme”)
expensive repair mechanism
methylated methyltransferase is a transcriptional
activator of its own gene
O6-methylguanine DNA
methyltransferase
active Enz.
active Enz.
Cys-SH
O-CH3
Cys-S-CH3
N
N
O
N
N
H
H
H 2N
N
N
R
O6-methylguanine nucleotide
Source: adapted from Lehninger pg. 957
H 2N
N
N
R
guanine nucleotide
Transition
• Transition
vs. transversion
Exchange of a purine with a purine or pyrimidine with a
pyrimindine base. More common than transversion. Often the
result of tautomeric shifts.
– GC  AT transition
• causal agents: e.g. base analog 5’bromouracil
– AT  GC transition
• causal agents: e.g. base analog 2-aminopurine
• Transversion
Exchange of a purine with a pyrimidine base and vice versa
– GC  TA or GC  CG transversion
– AT  CG or AT  TA transversion
6
O -methylguanine
• Mutagenic behavior
– pairs with thymine rather than cytosine
• Specific Repair Mechanism
–
–
–
–
–
differs from base excision repair
requires O6-methylguanine methyltransferase
one time reaction (“suicide enzyme”)
expensive repair mechanism
methylated methyltransferase is a transcriptional
activator of its own gene
O6-methylguanine DNA
methyltransferase
active Enz.
active Enz.
Cys-SH
O-CH3
Cys-S-CH3
N
N
O
N
N
H
H
H 2N
N
N
R
O6-methylguanine nucleotide
Source: adapted from Lehninger pg. 957
H 2N
N
N
R
guanine nucleotide
Inaccurate DNAE. coli
repair
recA- strain
E. coli recA+ strain
intensive UV exposure
Numerous survivors
High mutation rates
Few survivors
Low mutation rates
Error-prone repair
•
Activated upon:
–
–
•
severe DNA damage
disruption of DNA replication
“SOS-response”
–
–
1.
2.
3.
4.
5.
Inaccurate repair mechanism
Requires at least 14 proteins in E. coli
Din proteins (damage induced)
Rec poteins (recombination)
Umu proteins (UV-mutagenesis)
Uvr proteins (UV-resistance)
Others: SulA, HimA, Ssb, and PolB
Genes involved in the SOS response
Gene
pol B
uvr A & uvr B
umu C & umu D
sul A
sul B
recA
din B
ssb
uvr D
him A
rec N
din D
din F
Protein function encoded:
DNA polymerase II (polymerization subunit)
ABC exinuclease
DNA polymerase V
inhibits cell division via interaction with FtsZ
recA protease, recombination and repair
DNA polymerase IV
single-stranded binding protein
DNA helicase II (DNA -unwinding protein)
subunit of integration host factor
required for recombinational repair
?
?
Damaged DNA during replication
• UV-damaged DNA results in collapse of the replication
fork during DNA replication
• 2 scenarios
– Unrepaired lesions
Unrepaired DNA breaks
12.4 DNA damage and repair and their
role in carcinogenesis
• A DNA sequence can be changed by copying errors
introduced by DNA polymerase during replication and by
environmental agents such as chemical mutagens or
radiation
• If uncorrected, such changes may interfere with the ability of
the cell to function
• DNA damage can be repaired by several mechanisms
• All carcinogens cause changes in the DNA sequence and
thus DNA damage and repair are important aspects in the
development of cancer
• Prokaryotic and eukaryotic DNA-repair systems are
analogous
12.4 General types of DNA damage and
causes
12.4 Proofreading by DNA polymerase
corrects copying errors
Figure 12-20
12.4 Schematic model of the
proofreading function of DNA
polymerase
Figure 12-21
12.4 Chemical carcinogens react with DNA
directly or after activation, and the
carcinogenic effect of a chemical correlates
with its mutagenicity
Figure 12-22
12.4 Base deamination leads to the
formation of a spontaneous point
mutation
Figure 12-23
12.4 Mismatch repair of single-base
mispairs
Figure 12-24
12.4 Chemically modified bases, such as
thymine-thymine dimers, are corrected by
excision repair
Figure 12-25
12.4 Excision repair of DNA by the E.
coli UvrABC mechanism
Figure 12-26
12.4 End-joining repair of nonhomologous
DNA
Figure 12-28
12.5 Recombination between
homologous DNA sites
• Recombination provides a means by which a genome can
change to generate new combinations of genes
• Homologous recombination allows for the exchange of blocks
of genes between homologous chromosomes and thereby is
a mechanism for generating genetic diversity
• Recombination occurs randomly between two homologous
sequences and the frequency of recombination between two
sites is proportional to the distance between the sites
12.5 The cross-strand Holliday structure
is an intermediate in recombination (part
I)
Figure 12-29
12.5 The cross-strand Holliday structure
is an intermediate in recombination (part
II)
Figure 12-29