DNA

1
DNA replication models
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pH 7 pH 12
Meselson-Stahl experiment1958
15N
DNA single stranded
Double helix intact
Molecules that differ in density can be separated
by centrifugation through density gradient (will
migrate to point in solution where the density
equals their own)
14N
15N, then 14N
Hybrid molecules
3
Strand separation
4
DNA Replication
- propagation of life as we know it requires the
faithful replication of genetic material
(DNA) DNA replication duplication of
chromosomes for the purpose of cell division -
replication requires a template and consists of 3
phases initiation chain elongation termination
Replication is semi-conservative- each daughter
DNA molecule consists of one parental and one
newly synthesized
5
Replication

• Enzymatic synthesis of DNA ALWAYS occurs in the
  5 ? 3 direction.
• Raw Materials for synthesis are

1. A DNA Template 2. deoxynucleotide
tri-phosphates (dNTPs) 3. a protein complex
involving DNA polymerase 4. a DNA or RNA
primer 5. Mg2 ions (cofactor)
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Replication fork- simple view
DNA polymerase
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DNA Synthesis

• - nucleotide gets positioned through H- bonding
  with template
• - 3-OH nucleophilic attack on alpha phosphate of
  incoming dNTP.
• reaction is driven by the removal and splitting
  of the pyrophosphate
• because of requirement for 3-OH and 5 dNTP
  substrate, DNA polymerase can only catalyze
  reaction in the 5 ?3 direction (direction of
  new strand!)

2 phosphates
Figure 24.2
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DNA polymerases require a primer tosynthesize
DNA from a template
An interesting property of ALL DNA polymerases is
that they can only extend a pre-existing DNA (or
RNA) chain by using a primer - for RNA
polymerases to transcribe DNA, they can start
from scratch they do not need a primer
DNA
5
- DNA replication needs a primer extension is in
the 5 to 3 direction - transcription does not
need a primer extension is also in the 5 to 3
direction
5-
3
3-
-5
RNA
-3
5-
-5
3-
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DNA Replication
- in all cells (eukaryotic, prokaryotic) and with
circular or linear DNA molecules, DNA replication
begins at defined sequences termed ORIGINS of
REPLICATION
Origin of Replication
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There often are multiple origins of replication
in a genome Ori C in E. coli 9replication is
bidirectional) - 245 base pairs (bp) long -
contains four DnaA binding sites - DnaA is the
replication initiator protein that controls the
rate limiting step in replication
If you just look at this part it looks like the
replication fork in the text
leading strand
5
3
5
3
3
5
3
5
lagging strand
RNA Primers
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Okazaki model of discontinuouschain growth in
DNA replication
helicase
DNA pol I
primase
DNA ligase
DNA pol III
Figure 24-4
cartoon animations http//www.uark.edu/campus-res
ources/mivey/m4233/DNAstruc.htm
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Replication fork- complex view
Single-strand DNA binding protein
Sliding clamp
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On diagrams the Origin of Replication is
identified as ( O ) - replication typically
starts in both directions - there are some cases
where synthesis is in one direction only or where
one direction has a significantly later start
time -marker frequency gradients count number
of copies of marker gene tells you uni- vs.
bi-directional
Figure 24-9
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Bacteriophage lambda DNA
- linear DNA must circularize by base pairing of
sticky ends, undergo ligation, and then
replication can begin - partial replication
yields a theta structure - electron micrographs
of replicating lambda DNA established that
replication was bi-directional from a fixed
origin - Most prokaryotic chromosomes are
circular DNA and replicate bidirectionally from
one origin
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Bacterial replicationa new round is initiated
before the first round is complete
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Multiple origins
Initiation of replication is controlled by DNA
sequence (origin) proteins that bind to the
origin Replicon -all of the DNA replicated from
one origin controlled by the proteins that act at
the origin A single chromosome is not always a
single replicon in bacteria it is in humans
several dozen/ chromosome
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If humans did not have multiple origins of
replication, then replication of the genome from
a single origin with two forks would take several
weeks
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DNA Polymerases in bacteria

• enzymes catalyzing polynucleotide chain
  elongation
• 1950 Arthur Kornberg discovered DNA pol I
• In fact, there are 5 polymerases discovered to
  date, although we will focus only on 3 enzymes
• Turns out they have distinct roles in the cell,
  only one (pol III) is the major polymerase
  involved in chain elongation during replication

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Types of E. coli DNA Polymerases
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DNA Polymerase I
This is the best understood of the DNA polymerases
proteolytic cleavage yields the 67 kDa Klenow
Fragment
-C
N-
Normally DNA polI contains all 3 domains
36 kD
67 kD
Klenow Fragment of DNA Pol I (Used widely in labs
since it avoids DNA degradation mediated by 5
exo)
- 3 exonuclease degrades single-stranded DNA
from 3 end - 5 exonuclease degrades base paired
DNA from the 5 terminus -polymerase adds
nucleotides
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Specific functions of Klenow fragment
-can be used to fill in DNA ends after cleavage
by a restriction enzyme
5
3
5
3
3
5
3
5
restriction enzyme cleavage
Klenow dNTPs
restriction enzyme cleavage site
5 overhang
filled-in DNA
EcoRI enzyme
5
3
5
3
NNNGAATTCNNN NNNCTTAAGNNN
NNNG NNNCTTAA
AATTCNNN GNNN
e.g., EcoRI site
3
5
3
5
- Klenow fragment can also repair the ends of
DNA which are not flush, or blunt, through its 3
exo activity
5
3
5
3
3
5
3
5
blunt DNA
(3 overhang)
Klenow (no dNTPs)
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5 exonuclease activity

• 2 important roles
• Excision of RNA primers on lagging strandand
  replacement with DNA i.e. by nick translation
• 5 exo activity removes ribonucleotides, just as
  polymerase function replaces them with
  deoxyribonucleotides
• DNA repair
• Can cleave both DNA and RNA at single
  nucleotides- can remove and replace damaged
  nucleotides
• More detail later!

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DNA Polymerase I Carries Out Nick Translation in
E. coli
T
A