Maintenance and expression of genetic information
Central Dogma:
DNA
RNA
Protein
GAATTGCGCCTTTTG
5’-GAATTGCGCCTTTTG-3
3’-CTTAACGCGGAAAAC-5’
Minor Groove
Major Groove
DNA can be supercoiled
Semi-conservative
Replication of DNA
The Watson-Crick Model
Proof of the Watson-Crick Model:
The Meselson-Stahl Experiment
The Meselson-Stahl Experiment
# generations
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
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
DNA Polymerase
A 3’ hydroxyl
group is necessary
for addition of
nucleotides
DNA chain growth is driven by PPi release/hydrolysis
5’
4’
3’
5’
4’
1’
2’
3’
2’
5’
5’
4’ 3’
4’ 3’
1’
2’
1’
2’
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
DNA Polymerase contains a Proofreading subunit
Proofreading
by DNA
polymerase
Proofreading
by DNA
polymerase
Both Template strands are copied at a Replication Fork
The polarity of DNA synthesis creates an asymmetry
between the leading strand and the lagging strand
at the replication fork
Protein complexes of the replication fork
Topoisomerase
Protein complexes of the replication fork:
DNA polymerase
DNA primase
DNA Helicase
ssDNA binding protein
Sliding Clamp
Clamp Loader
DNA Ligase
DNA Topoisomerase
DNA primase
synthesizes an
RNA primer
to initiate DNA
synthesis on the
lagging strand
Replication of
the Lagging Strand
DNA ligase seals nicks left by lagging strand replication
DNA helicase unwinds
the DNA duplex
ahead of DNA polymerase
creating single stranded
DNA that can be used
as a template
DNA helicase moves along one strand of the DNA
ssDNA binding proteins are required to “iron out” the unwound DNA
ssDNA binding proteins bind to the sugar phosphate backbone
leaving the bases exposed for DNA polymerase
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
A “clamp loader:”
complex is required
to get the clamp onto
the DNA
Lagging strand synthesis
Topoisomerase
PCNA
MCM
proteins
RPC
Ahead of the replication
fork the DNA becomes
supercoiled
The supercoiling ahead
of the fork needs to be
relieved or tension would
build up (like coiling as
spring) and block fork
progression.
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
Topoisomerase I
Action
Topoisomerase I
Action
Topoisomerase II
Action
Topoisomerase II
Action
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
DNA is replicated during S phase of the Cell Cycle
In S phase, DNA replication begins at origins of replication
that are spread out across the chromosome
Each origin of replicaton
Initiates the formation
of bidirectional
replication forks
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.
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.
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.
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
expansion of the repeats