Maintenance and expression of genetic information

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Maintenance and expression of genetic information
Central Dogma DNA RNA
Protein
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GAATTGCGCCTTTTG
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5-GAATTGCGCCTTTTG-3 3-CTTAACGCGGAAAAC-5
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Minor Groove
Major Groove
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DNA can be supercoiled
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Semi-conservative Replication of DNA The
Watson-Crick Model
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Proof of the Watson-Crick Model The
Meselson-Stahl Experiment
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generations
The Meselson-Stahl Experiment
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0.3
0.7
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1.1
1.5
1.9
2.3
3
4.1
0 and 1.9 mixed
0 and 4.1 mixed
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The 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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DNA Polymerase
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A 3 hydroxyl group is necessary for addition of
nucleotides
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DNA chain growth is driven by PPi
release/hydrolysis
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5
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2
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5
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1
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2
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Accuracy 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
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DNA Polymerase contains a Proofreading subunit
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Proofreading by DNA polymerase
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Proofreading by DNA polymerase
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Both Template strands are copied at a Replication
Fork
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The polarity of DNA synthesis creates an
asymmetry between the leading strand and the
lagging strand at the replication fork
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Protein complexes of the replication fork
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Protein complexes of the replication fork DNA
polymerase DNA primase DNA Helicase ssDNA binding
protein Sliding Clamp Clamp Loader DNA Ligase DNA
Topoisomerase
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DNA primase synthesizes an RNA primer to initiate
DNA synthesis on the lagging strand
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Replication of the Lagging Strand
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DNA ligase seals nicks left by lagging strand
replication
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DNA helicase unwinds the DNA duplex ahead of DNA
polymerase creating single stranded DNA that can
be used as a template
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DNA helicase moves along one strand of the DNA
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ssDNA binding proteins are required to iron out
the unwound DNA
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ssDNA binding proteins bind to the sugar
phosphate backbone leaving the bases exposed for
DNA polymerase
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DNA 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
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A clamp loader complex is required to get
the clamp onto the DNA
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Lagging strand synthesis
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Topoisomerase
PCNA
MCM proteins
RPC
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Ahead of the replication fork the DNA
becomes supercoiled
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The supercoiling ahead of the fork needs to be
relieved or tension would build up (like coiling
as spring) and block fork progression.
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Supercoiling 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
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Topoisomerase I Action
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Topoisomerase I Action
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Topoisomerase II Action
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Topoisomerase II Action
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Topoisomerases 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
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DNA is replicated during S phase of the Cell Cycle
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In S phase, DNA replication begins at origins of
replication that are spread out across the
chromosome
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Each origin of replicaton Initiates the
formation of bidirectional replication forks
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Origins 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.
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Errors 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.
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Several mechanisms for the expansion of
trinucleotide repeats have been proposed, but the
precise mechanism is unknown.
From Stryer Looping out of repeats before
replication.
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Several 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