Title: Maintenance and expression of genetic information
1 Maintenance and expression of genetic information
Central Dogma DNA RNA
Protein
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3 GAATTGCGCCTTTTG
45-GAATTGCGCCTTTTG-3 3-CTTAACGCGGAAAAC-5
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8Minor Groove
Major Groove
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11DNA can be supercoiled
12Semi-conservative Replication of DNA The
Watson-Crick Model
13Proof of the Watson-Crick Model The
Meselson-Stahl Experiment
14 generations
The Meselson-Stahl Experiment
0
0.3
0.7
1
1.1
1.5
1.9
2.3
3
4.1
0 and 1.9 mixed
0 and 4.1 mixed
15The Meselson-Stahl Experiment
Starting DNA Heavy/Heavy
1st generation All Heavy/Light
2nd generation Two Heavy/Light
Two Light/Light
3rd generation Two Heavy/Light
Six Light/Light
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17DNA Polymerase
18A 3 hydroxyl group is necessary for addition of
nucleotides
19DNA chain growth is driven by PPi
release/hydrolysis
5
5
4
4
3
3
1
2
2
5
5
4
4
3
3
1
1
2
2
20Accuracy of DNA polymerases is essential. --Error
rate is less than 1 in 108 --Due in part to
reading of complementary bases --also contains
its own proofreading activity
21DNA Polymerase contains a Proofreading subunit
22Proofreading by DNA polymerase
23Proofreading by DNA polymerase
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25Both Template strands are copied at a Replication
Fork
26The polarity of DNA synthesis creates an
asymmetry between the leading strand and the
lagging strand at the replication fork
27Protein complexes of the replication fork
28Protein complexes of the replication fork DNA
polymerase DNA primase DNA Helicase ssDNA binding
protein Sliding Clamp Clamp Loader DNA Ligase DNA
Topoisomerase
29DNA primase synthesizes an RNA primer to initiate
DNA synthesis on the lagging strand
30Replication of the Lagging Strand
31DNA ligase seals nicks left by lagging strand
replication
32DNA helicase unwinds the DNA duplex ahead of DNA
polymerase creating single stranded DNA that can
be used as a template
33DNA helicase moves along one strand of the DNA
34ssDNA binding proteins are required to iron out
the unwound DNA
35ssDNA binding proteins bind to the sugar
phosphate backbone leaving the bases exposed for
DNA polymerase
36DNA polymerase is not very processive (ie it
falls off the DNA easily). A sliding clamp is
required to keep DNA polymerase on and allow
duplication of long stretches of DNA
37A clamp loader complex is required to get
the clamp onto the DNA
38Lagging strand synthesis
39Topoisomerase
PCNA
MCM proteins
RPC
40Ahead of the replication fork the DNA
becomes supercoiled
41The supercoiling ahead of the fork needs to be
relieved or tension would build up (like coiling
as spring) and block fork progression.
42Supercoiling is relieved by the action of
Topoisomerases.
Type I topoisomerases Make nicks in one DNA
strands Can relieve supercoiling Type II
topoisomersases Make nicks in both DNA strands
(double strand break) Can relieve supercoiling
and untangle linked DNA helices Both types of
enzyme form covalent intermediates with the DNA
43Topoisomerase I Action
44Topoisomerase I Action
45Topoisomerase II Action
46Topoisomerase II Action
47Topoisomerases as drug targets
Because dividing cells require greater
topoisomerase activity due to increased DNA
synthesis, topoisomerase inhibitors are used as
chemotherapeutic agents. E.g. Camptothecin --
Topo I inhibitor Doxorubicin -- Topo II
inhibitor These drugs act by stablilzing the
DNA-Topoisomerase complex. Also, some
antibiotics are inhibitors of the
bacterial-specific toposisomerase DNA gyrase
e.g. ciprofloxacin
48DNA is replicated during S phase of the Cell Cycle
49In S phase, DNA replication begins at origins of
replication that are spread out across the
chromosome
50Each origin of replicaton Initiates the
formation of bidirectional replication forks
51Origins or replication are strictly controlled so
that they fire only once per cell cycle
Errors lead to overreplication of specific
chromosomal regions. ( gene amplification) This
seen commonly in cancer cells and can be an
important prognostic indicator. It can also
contribute to acquired drug resistance. E.g.
Methotrexate induces amplification of
the Dihydrofolate Reductase locus.
52Errors of DNA Replication and Disease
The rate of misincorporation of bases by DNA
polymerase is extremely low, however repeated
sequences can cause problems.
In particular, trinucleotide repeats cause
difficulties which can lead to expansion of
these sequences. Depending where the repeat is
located expansion of the sequence can have severe
effects on the expression of a gene or
the function of a protein.
53Several mechanisms for the expansion of
trinucleotide repeats have been proposed, but the
precise mechanism is unknown.
From Stryer Looping out of repeats before
replication.
54Several inherited diseases are associated with
expansion of trinucleotide repeat sequences.
Very different disorders, but they share the
characteristic of becoming more severe in
succeeding generations due to progressive expansio
n of the repeats