Download Components of RNA and DNA RNA Is More Labile Than DNA

RNA and DNA Consist of Polymerized Nucleotides
5’-end
O
O
O
O
5
O
4
3
O
O
O
P O 5’–PO
4
O
P O
O
O
O
Base
Base
O
O
O
Base
O
O
O
O
O
P O
Base
1
2
P O
O
P O
O
O
O
O
3’–OH
O
P O
O
Base
2’–OH
O
Base
O
3’-end
Ribonucleic acid (RNA)
Deoxyribonucleic acid (DNA)
G. Magnusson 2003
Components of RNA and DNA
O
O
O
O
P O
N
O
O
O
N
O
O
N
P O
N
O
O
O P O
O
O P O
O
O P O
O
N
O
N
N
O
O
N
N
N
N
N
dCMP
O
O
O P O
O
N
O
O
N
O
O
N
GMP
N
O
O
N
N
O
O
N
O
O
CMP
O
O
P O
O
dAMP
O
O
O
N
O
O
AMP
N
N
N
O
O
N
O
N
N
O
O
O P O
O
N
N
dGMP
O
UMP
N
N
O
O
dTMP
G. Magnusson 2003
RNA Is More Labile Than DNA
RNA is labile in a water solution because the phosphate groups
can form phosphodiester bonds with both the 2’ and 3’ –OH
groups of ribose.
O
O
O
O
O
P O
O
O
P O
O
Base
Base
O
O
P O
O
O
O
P
O
O
O
O
Base
+
O
O
O
O
P
O
O
O
O
Base
O
Base
O
O
P O
O
O
O
Base
O
O
O
G. Magnusson 2003
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The Strands of DNA are Anti-Parallel
5’-end
3’-end
O
O
O
O
P O
O
A
T
O
O
O
O P
P O
O
O
G
C
O
O
O
O
O
O
O
O
O P
P O
O
O
O
O
3’-end
A
T
O
O P
O
O
O
5’-end
G. Magnusson 2003
Structure of Double-Stranded DNA
B-form of DNA
Base-pairing of B DNA
10 base pairs
3.4 nm
G. Magnusson 2003
The Size of Cellular DNA
Escherichia coli (a bacterium) has one DNA molecule
(chromosome) with a size of ca. 106 nucleotides
Homo sapiens cells have 46 chromosomes, each consisting
of one DNA molecule, with a total size of ca. 109 nucleotides
G. Magnusson 2003
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Nuclear DNA Forms Chromatin
Histones are among the most conserved proteins in eukaryotes
H2B
H4
H4
H3
H3
H2A
The nucleosome core is
composed of an octamer
of histone molecules. Two
each of the histones H2A,
H2B, H3 and H4. H1 is a
linker histone.
G. Magnusson 2003
Synthesis of DNA
Enzymes catalysing the synthesis of DNA: DNA polymerases.
DNA polymerases have some general properties
q DNA molecules are extended at the 3’-end. DNA is
synthesised in the 5’ to 3’ direction
q Deoxynucleoside triphosphates (dATP, dCTP, dGTP, dTTP)
are used as building blocks
q The polymerisation reaction is directed by A–T, G–C
base pairing to a template strand
Primer
5’
3’
Template strand
G. Magnusson 2003
Polymerisation of deoxynucleotides
O
O P O
O
Growing strand
’Primer’
N
O
N
O
N
N
O
O
P O
O
P
O
O
Mg2+
P
G N
N C
N
N
O
N
O
O
O
O
Template strand
T
O
N
O
O
O
N
N
N
Active site of
DNA polymerase
O
O
N
O P O
O
O
N
A N
O
N T N
O
N
N
A
N
N
G. Magnusson 2003
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Fidelity of DNA synthesis
In mammals, the error frequency of DNA synthesis
must be <10-9
The base pairing mechanism leads to one error in
~104 polymerised nucleotides
G. Magnusson 2003
Reasons for Incorrect Base-pairing
H
H
H
N
N
Correct base-pairing
N
N
N
N
N
N
H
N
N
Tautomer of A can base pair with C instead of T
G. Magnusson 2003
Correction of Replication Errors
Proofreading activity of DNA polymerases
5’
Correct synthesis
3’
Template strand
5’
Misincorporated nucleotide
3’
Template strand
3’ to 5’ exonuclease activity of DNA polymerase catalyses
the removal by hydrolysis of 3’-terminal nucleotides that are
not base-paired to the template strand.
5’
3’
Template strand
With this mechanism the error frequency of DNA synthesis is
decreased to ~10-6.
G. Magnusson 2003
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Correction of Replication Errors
DNA polymerase I from E. coli has two catalytic activities,
although the enzyme consists of a single polypeptide chain.
Pol
Pol
Exo
Exo
View of a DNA polymerase bound to a primer-template junction in its synthesis (left) and proofreading (right) modes
Pol denotes the polymerase active site; Exo, the 3’ to 5’exonuclease active site.
From Baker & Bell (1998) Cell, 92:295
G. Magnusson 2003
Correction of Replication Errors
Postreplication mismatch repair
5’
3’
Template strand
Methylated base
In DNA, some of the C (and A) bases are methylated. Methylation
is a slow process. Newly replicated DNA is undermethylated.
Mismatch repair enzymes recognise mismatched nucleotides and
remove the nucleotide in the undermethylated strand.
5’
3’
Template strand
The mismatch repair system increases the fidelity of DNA
replication to ~10-9.
G. Magnusson 2003
Replication of Chromosomal DNA
Semiconservative DNA replication
+
A half-replicated DNA molecule
What happens at the
replication fork?
G. Magnusson 2003
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The Replication Fork, a Simplified View
5’
3’
Template strand
Newly synthesised DNA
Lagging strand
Discontinuous DNA synthesis
DNA polymeras a/Primase
DNA chain ~100 nucleotides
RNA primers ~10 nucleotides
DNA helicase
Topoisomerase I
RF-A, single-strand DNA
binding protein
Leading strand
Continuous DNA synthesis
DNA polymerase d with
sliding clamp (PCNA)
5’
3’
G. Magnusson 2003
Reaction Catalysed by DNA Topoisomerase I
Tyr
Topoisomerase I
DNA
OH
Tyr
H2N
O
O
Rotation around
intact DNA strand
5’
O
P
O
O
Tyrosine
Tyr
HO
3’
G. Magnusson 2003
The Replication Fork, a Simplified View
5’
3’
Lagging strand
Discontinuous DNA synthesis
DNA polymeras a/Primase
RNA primers ~10 nucleotides
Template strand
Newly synthesised DNA
DNA chain ~100 nucleotides
DNA helicase
Topoisomerase I
Leading strand
Continuous DNA synthesis
DNA polymerase d with
sliding clamp (PCNA)
RF-A, single-strand DNA
binding protein
5’
3’
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Lagging Strand Synthesis
5’
3’
5’
3’
5’
3’
4, 5
3
1, 2
DNA polymerase
RNAase H
DNA ligase
1.
2.
3.
4.
5.
RNA primer synthesis: DNA pol a/Primase
DNA chain synthesis: DNA pol a/Primase
Removal or RNA primer: RNAase H
Extension of DNA chain to close the gap: DNA polymerase
Joining of DNA chains: DNA ligase
5’
3’
G. Magnusson 2003
Reactions Catalysed by DNA Ligase
O
1
E–lys–N+– P
E–lys–NH2 + ATP
N
O
O
O
O
N
O
+ O P
O P
O
N
O
O
N
N
O
OH
2
O
O
P
O
O
O
O
E–lys–N+– P
N
O
O
O
O
N
N
O
O
OH P
O O
P
O O
N
N
N
N
N
O
N
O
N
O
O
O
G. Magnusson 2003
Reactions Catalysed by DNA Ligase
O
3
E–lys–NH3+
O
O
OH P
O O
O
P
O O
O
N
N
O P O
O
N
N
+
O
N
O
O
O
P
O
N
O
O
N
N
N
N
O
O
G. Magnusson 2003
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Replication of Chromosomal Ends (Telomers)
RNA primer
New DNA
5’
3’
5’
3’
Template strand
Degradation
of RNA primer
Loss of genetic information at chromosomal ends is prevented
by the addition of nontemplated nucleotides by telomerase
New RNA primer
5’
3’
5’
3’
Telomerase product
G. Magnusson 2003
Mechanism of Telomerase Activity
Telomerase is a DNA polymerase that uses an RNA template.
This template is a subunit of the enzyme and consists of an RNA
molecule with a AAUCCC sequence repeat.
Telomerase product
3’ GGGTTAGGGTTAGGGTTA
AAUCCCAAUCCCAAU
5’
Telomerase RNA
5’
3’
In the new-born, the telomeres have a length ~10~4 nucleotides
with ca. 102 unpaired nucleotides at the 3’-end.
G. Magnusson 2003
Initiation of DNA-Synthesis at an Origin
From Bogan et al. (2000) J. Cell. Physiol. Vol. 184, 139
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Frequent Spontaneous DNA Damage
O
P
O O
O
Frequency of individual lesions of
nucleotides is suggested by the
size of the arrows.
N
O
NH2
N
A
N
N
O
O
P
O O
Depurination is the most frequent
lesion ~104/day/cell.
NH2
O
Deamination of C to form U occurs
~103 times per day in a cell.
N
C
N
O
O
O
P
O O
N
O
O
N
G
N
N
O
N
O
P
O O
O
O
N
T
N
O
O
G. Magnusson 2003
UV-Induced Damage to DNA
Absorption of UV-radiation (200–320 nm) by DNA induces two major
types of potentially mutagenic lesions. Skin cells not protected by
pigmentation accumulate ~104 lesions per hour when exposed to
strong sun-light.
O
O
N
N
N
O
PO4
N
O
OH
N
N
N
O
PO4
O
DNA backbone
PO4
PO4
Cyclobutane thymine dimer
kinks DNA 7°–9°
N
O
PO4
PO4
Thymine (6–4) pyrimidone
photoproduct kinks DNA 40°
G. Magnusson 2003
Mechanisms of DNA Repair
In the cell, DNA damage
is recognised by specific
proteins either as damage
to individual bases or as
distortion of the double
helix.
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Base Excision Repair of DNA
DNA-glycosylase
5’
3’
Damaged base e.g. uracil
resulting from deamination
of a C
e.g. uracil-DNAglycosylase
Apurinic/apyrimidinic
endonuclease
Excision
endonuclease
DNA polymerase b
DNA ligase
G. Magnusson 2003
Repair of Lesions Distorting the DNA Helix
Global
genome
repair
5’
3’
The XP factors were identified as
a result of defective function in
cells of individuals with the
inheritable disease Xeroderma
Pigmentosum
DDB1 XPE
XPC
hHR23B
Unwinding
Transcription
coupled
repair
TFIIH
RNA
pol
XPF TFIIH
XPA
XPA
XPG
ERCC1
II
RPA
RPA
Stabilised high
specificity complex
Incision 3’ and 5’
to lesion
Excision of
27–29 nucl.
Repair
of patch
DNA pol
DNA ligase
G. Magnusson 2003
Ames’ Mutagen Test
• Salmonella bacteria requiring histidine
for growth
• Use strains with several different types of
mutations in the his genes
• Treat bacteria with test chemical, directly
or after metabolic activation in liver cell
extract
• Plate bacteria on agar in histidine
deficient medium
• Count colonies and calculate mutation
frequency
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Mouse Carcinogen Test
• Treat animals with test substance. For substances
with low potency, use high doses or many animals
• TD50 (mg/kg bodyweight/day during lifetime)
calculated as the dose of chemical that would
decrease the number of tumour-free animals by 50%
• Tumours have to be analysed by a pathologist
• TD50 for various substances has a range of >107
G. Magnusson 2003
Risk Assessment
• All environmental risks cannot be avoided
• There is no inherent difference in the toxicity
of natural and synthetic substances
• Human exposure has to be considered in the
assessment of risk
• HERP (human exposure rodent potency).
Percentage of the rodent potency (TD50)
received by a human during lifetime exposure
G. Magnusson 2003
HERP
HERP (%)a
• 3.6 Beer, 500 ml (etyl alcohol, 23 ml/day)
• 0.4 Typical indoor air (formaldehyde, 600 µg/day)
• 1 x 1 0- 6 Lindaneb (32 ng/day)
aA
HERP value of 100 means that the exposure is identical
to the TD50 in rodent tests.
bLindane is a mutagenic insecticide that is banned from use in
Swedish agriculture.
G. Magnusson 2003
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