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Review of
directionality in
DNA
Now, for DNA replication.
Meselson and Stahl:
DNA replication is semiconservative
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• Theoretically, 3 ways a DNA molecule could give rise to 2
new DNA molecules:
• Semi-conservative means that each time DNA is replicated,
the new double stranded molecules consist of one old strand
and one new strand.
• Conservative would result in a molecule with 2 old strands
and one with 2 new ones.
• Dispersed, each new DNA molecule would be a
combination of old and new pieces.
Illustration of 3 models
http://en.wikipedia.org/wiki/Image:DNAreplicationModes.png
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Meselson and Stahl Experiment
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http://users.rcn.com/jkimball.ma.
ultranet/BiologyPages/M/Mesels
on_Stahl.html
•DNA was produced in cells grown with N-15, a
“heavy” isotope of nitrogen.
•When DNA was placed into an ultracentrifuge, it
migrated closer to the bottom because of its greater
density.
•What happened when N-15 labeled cells were
allowed to keep growing in the presence of N-14?
Conclusion of experiment
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Meselson_Stahl.html
After 1 generation, all the DNA molecules of intermediate
density.
After 2 generations, half of them intermediate, the other
half light.
These results consistent with semi-conservative replication.
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“It has not escaped our notice that
the specific pairing we have
postulated immediately suggests
a possible copying mechanism
for the genetic material."
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-Watson and Crick
http://www.sciencetechnologyaction.com/lessons2.php?s
tudyid=6&edition=1
DNA replication
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• Origin of DNA replication: particular site on DNA where
copying of the DNA always starts.
– Replication is bidirectional
– In each direction, there is a replication fork.
– Most bacterial DNA is circular, so there is one Origin and
one terminus
• Replicon: a length of DNA molecule replicated after
initiation from one origin. Examples:
– Bacterial DNA, plasmids, segments of eukaryotic
chromosomes.
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DNA replication
terminology
Speed of replication:
Bacteria: 1500 bp per second
Eukaryotes: 10-100 bp per second
YET
in fruit flies, only 15-30 minutes to
replicate all the DNA, similar to
E. coli. How? Multiple origins.
E. coli, a typical impatient bacterium
• E. coli takes 30 minutes to replicate all its DNA, yet it can
double every 20 minutes. How does it do this?
• Starts a round of DNA replication before finishing the
previous round.
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DNA polymerases (bacteria):
Enzymes that synthesize DNA
• Kornberg discovers DNA pol I (1956)
– Demonstrates enzyme faithfully copies DNA (1960)
• DNA pol II and III discovered
– Pol I: cleaves out Okazaki fragments (see below)
• Most abundant of the 3.
– Pol II: repairs DNA damage
– Pol III: main DNA replicating enzyme
• Pol III is a complex, multi-component enzyme
complex (has a quaternary structure)
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A couple of words on terminology
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• A chemical reaction in which molecules are
combined to make a products is a synthesis reaction.
• DNA is synthesized in cells, but we can direct DNA
synthesis in a test tube also. PCR, sequencing both
involve DNA synthesis.
• DNA replication is a natural biological process in
which a DNA molecule is copied in a cell.
– Replication is a specific act of synthesis.
What every DNA polymerase needs
• A template of DNA
– Enzymes copy a single strand of DNA
– Can’t work without something to copy from
• A primer
– A primer is a polynucleotide with a “free 3´OH end”
– In normal DNA replication, this is RNA
• A substrate
– To make DNA, a polymer, monomers are needed
– Nucleotide triphosphates (NTPs) are the monomers
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Adding and removing bases:
Directionality
• DNA synthesis is ALWAYS in a 5´ to 3´ direction
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– See next slide.
• All 3 DNA pols have a 3´ to 5´ exonuclease activity
– Nuclease: enzyme activity that cuts nucleic acids
– Exo- means cuts from an end
– 3´ to 5´ means the opposite direction from synthesis
• “proofreading” ability; polymerase can “backspace” to
remove a base put it by mistake.
• DNA pol I has a 5´ to 3´ exonuclease activity
– Cuts off DNA bases in same direction as synthesis
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**
*
*
Initiation of DNA replication
*
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* Helicases
unwind the
DNA
•DNA pol requires a primer to add to: Primase makes an RNA
•Synthesis is 5´ to 3´, and antiparallel.
•Leading strand; synthesis follows replication fork.
Problems due to antiparallel nature of DNA
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In this picture, replication of the
lower strand of DNA can
proceed as the “replication
fork” moves from right to left
because the direction of
synthesis of new DNA
is 5’ to 3’.
What about the other strand?
The one made without a hitch is
called the “leading strand”, the
other is the “lagging strand”.
Okazaki fragments
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Because of requirement for 5’to 3’ synthesis, lagging strand
must repeatedly top and start; needs an RNA primer each time.
Cleaning up Okazaki’s
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Ligase needed
DNA Pol I cuts out RNA primers, replaces them with DNA.
Uses both the 5´ to 3´ exonuclease and polymerase activities.
Facts about eukaryotic DNA synthesis
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• Multiple replicons
– Makes up for slower synthesis & larger amounts of DNA
• 6 polymerases
– Greek letters instead of Roman numerals: Pol α – ε
– Pol α creates RNA primer, adds some DNA, falls off
• “low processivity”
– Pol δ takes over
• Faster, and has proofreading ability
• Pol ε does same thing under different conditions
Intro to Chromosome structure
Arm
http://www.med.uiuc.edu/m1/genetics
/images/webun1/Chromosome.gif
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There’s some odd
DNA synthesis
that happens at the
telomeres because
the DNA is linear
in eukaryotes.
The Telomere problem
With each round of replication, DNA would get shorter.
users.rcn.com/.../BiologyPages/ T/Telomeres.html
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Solution to the telomere problem
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• An enzyme, telomerase, adds multiple copies of a
short sequence to the end of the telomere. It can then
be shortened without losing any actual chromosomal
DNA, and new copies can be added anytime.
• But how can new DNA be added
to “blunt-ended” DNA without a
template?
Telomerase contains a Guide RNA.
First, telomerase adds in 5’ to 3’ direction using an
RNA molecule in the enzyme as the template.
Leap-frogging: 3 bases, then 6.
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Telomerase- more
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• Remember the original problem: shortening of a
copy of the chromosome because of lagging strand:
Addition of bases by telomerase
lengthens the 3’ end; what about
the problem 5’ end?
More synthesis with a hairpin turn causes the end of
the “blue” strand (above) to basepair with itselfSort of….
Telomere DNA synthesis completed
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Added sequence
wraps around,
makes odd Gquartet base
pairing with
itself; then
synthesis can
occur in 5’ to 3’
direction to fill
the gap.
http://www.ndsu.nodak.edu/instruct/mcclean/plsc431/eukarychrom/telomere.gif
G-quartet base-pairing
www.biochemsoctrans.org/. ../bst0290692a01.gif
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