Download DNA Replication and Repair

Meselson & Stahl
 In 1950, Matthew Meselson and Franklin Stahl
grew E.coli bacteria in a 15N rich environment.
 They then transferred the E.coli into
a 14N rich environment.
 After three generations, Meselson and Stahl
used centrifugation to isolate the DNA of each
generation of E.coli and compare their densities
Meselson & Stahl
Their results:
 The first generation (grown in 15N)
had heavy DNA
 The second generation (15N E.coli
grown in 14N) had intermediate DNA
 The third generation (still in 14N)
had both intermediate and light DNA
Conclusion:
 E.coli separate their DNA using semiconservative
replication!
Semiconservative Replication
 Even though Meselson and Stahl thought it only
applied to bacteria, other researchers found that it
occurs in eukaryotes as well!
But what is Semiconservative Replication?
 Individual strands of DNA stay intact and are used as
templates for replication of new strands of
complimentary DNA
 This means each new strand of DNA is half “old” and
half “new”.
Replication & Repair
Separation
 DNA helicase is an enzyme
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that unwinds the DNA by
breaking the hydrogen bonds
between base pairs
To keep it apart, single-stranded binding proteins
(SSBP) bind to the strands to prevent base pairs from
re-annealing (pairing of complimentary strands of
DNA through hydrogen bonding)
DNA gyrase relieves tension from unwinding
When an enzyme reaches a point of untwisted, singlestranded DNA, we call it a replication fork.
John Cairns found that DNA replicated bidirectionally.
Evidence points to bidirectional replication
Label at both replication forks
Replication & Repair
Building Complimentary Strands
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An RNA primer anneals to the template strand and is
synthesized by primase (enzyme).
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DNA polymerase III binds to the RNA primer and
elongates the strands of DNA
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It synthesizes a new DNA strand in the 5’ to 3’ direction
by adding complimentary nucleotides to the 3’ end of
the growing strand.
Arthur Kornberg found the first DNA polymerase
enzyme and was the first to discover they only added
nucleotides in the 5’ to 3’ direction.
Replication & Repair
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DNA is always synthesized in the 5’ to 3’ direction
Since DNA strands are antiparallel, only one strand
can be synthesized continuously
The leading strand is synthesizes towards the
replication fork, and therefore is continuous
The lagging strand however, is being synthesized
away from the replication fork
 lagging strand
 leading strand
Replication & Repair
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RNA primers are constantly being added to the
lagging strand to allow the forming of pieces of
synthesized DNA called Okazaki Fragments (after
Reija Okazaki who discovered them)
DNA polymerase I or Rnase H removes the RNA
primers (from both the strands) and replaces them
with the appropriate nucleotides.
DNA ligase joins the Okazaki fragments together into
one strand by creating a phosphodiester bond.
As both strands are synthesized, they automatically
twist back into a double helix.
Replication & Repair
Quality Control: Repair!
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If mistakes in replication are found, DNA polymerase
I and III can act as an exonuclease
They will excise an incorrectly paired nucleotide
Mistakes must be made immediately in order to
prevent them from being copied in future replications
Replication and Repair Video!
http://www.youtube.com/watch?v=teV62zrm2P0
DNA Replication Tutorial
 http://www.wiley.com/college/pratt/0471393878/stude
nt/animations/dna_replication/index.html
 This is awesome!!! Do this!!!