MBB 407/511

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Nov. 29, 2005
MBB 407/511 Lecture 21 Eukaryotic DNA
Replication
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Outline
V. Telomerase
VI. Packaging of Eukaryotic DNA
VII. DNA Topology
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Circular dsDNAs can be replicated completely E.
Coli Eukaryotes Plasmids mitochrondrial
DNA chromosome E. coli Chromosome circular
dsDNA viruses (e.g., SV40) Replication of linear
dsDNAs (e.g., eukaryotic chromosomes) poses an
end replication problem.
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The End Replication Problem
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Adding Telomeres to the Leading Strand Allows
for Addition of a New Okazaki Fragment to the
Lagging Strand
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Packaging of Eukaryotic DNA
Chromatin is 50 DNA/50 protein by weight
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Chromatin Proteins
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DNA Wraps Around Histone Octomers to Form
Nucleosomes
2 H2A 2 H2B 2 H3 2 H4
Beads on a String
Core DNA is protected from DNases
Nucleosomes Two each of H2A, H2B, H3, H4
histone octomer (core) 146 bp core DNA wraps
around a histone core Between each nucleosome
is 20-60 bp linker DNA bound by a molecule of
H1 histone Spaced 200 bp apart (146 bp core
20-60 linker DNA)
Regular spacing between nucleosomes
Why are histones positively charged?
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Histone H1 Induces Tighter DNA Wrapping Around
the Nucleosome
Histone H1 binds two DNA helices
H1 is the Linker Histone Binds the Linker DNA
30-nm Fiber
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A Model for Chromosome Structure
DNA exists in chromatin form during interphase
Solenoid cylindrical coil
DNA is most compact in chromosome form during
metaphase of mitosis
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What is Supercoiling?
10.5 bp
Supercoiling occurs in nearly all chromosomes
(circular or linear)
Relaxed vs Supercoiled DNA
Relaxed DNA has no supercoils
Negatively supercoiled DNA is underwound (favors
unwinding of the helix) (circular DNA isolated
from cells is always negatively supercoiled)
Positively supercoiled DNA is overwound
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L T W
Linking Number (L or Lk) number of times the
two strands are intertwined
Twists (T or Tw) number of helical turns
For a 2,000 bp DNA duplex, T 200 (2,000 bp ?
1 turn/10 bp 200 turns)
Writhes (W or Wr) number of times the duplex
crosses itself (only topologically constrained
DNA molecules can have writhe)
A relaxed DNA molecule has zero writhes. (? For a
relaxed DNA molecule, L T)
Writhes Supercoils
Over- or underwinding results in writhes instead
of twists
Why? Because the strain of writhes is less than
the strain of twists
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Additional Terms Used To Describe Topology
The Linking Number Difference (DL) is the
difference between the linking number of a
DNA molecule (L) and the linking number of its
relaxed form (L0). The equation is DL L Lo.
DL is a measure of the number of writhes
For a relaxed molecule DL 0
The Superhelical Density (s) is a measure of
supercoiling that is independent of length. The
equation is s DL / Lo.
s is a measure of the ratio of writhes to twists
For a relaxed molecule s 0
DNA in cells has a s of 0.06 (for circular
molecules purified from bacteria and eukaryotes)
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Sample Linking Number Questions
1)
A. L T W for relaxed molecule W 0 ? L0
T 5500 bp X 10 bp/turn 550 turns
B. L T W 550 ( 50) 500
C. DL L L0 500 550 50
D. s DL / L0 50 / 550 0.09
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Instead of treating the relaxed 5,500 bp plasmid
DNA molecule above with DNA gyrase, you transfer
it from aqueous solution to 50 ethanol. Under
these conditions, the structure changes from
B-DNA to A-DNA due to the relatively lower water
concentration. (A-DNA has 11 bp/turn).
2)
A. What is the linking number after transfer to
50 ethanol? B. How many helical turns will there
be after transfer to 50 ethanol? C. How many
writhes will there be after transfer to 50
ethanol?
A. L 550 (linking stays the same because
no bonds are broken)
B. 5500 X 11 bp/turn 500 helical turns
C. L T W 550 500 W W 50
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Type I and II Topoisomerases (usually relax
supercoiled DNA)
Rule 1 They change the linking number by
changing the of writhes.
Rule 2 The change the linking number by
breaking one or both strands of the DNA molecule,
winding them tighter or looser, then rejoining
the ends.
Rule 3 They work only on topologically
constrained DNA molecules because only
topologically constrained DNA molecules can have
writhe.
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Type I Topoisomerases
Topo I of E. coli 1) acts to relax only negative
supercoils 2) increases linking number by 1
increments
Topo I of eukaryotes 1) acts to relax positive
or negative supercoils 2) changes linking number
by 1 or 1 increments
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Relaxation of SV40 DNA by Eukaryotic Topo I
Maximum supercoiled
3 min. Topo I
25 min. Topo I
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Type II Topoisomerases
They relax or underwind DNA by cutting both
strands then sealing them. They change the
linking
number by increments of 2 or 2.
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E. Coli vs. Eukaryotic Type II Topoisomerases
The strain of underwinding DNA is relieved by
Negative supercoils or Local
disruption of base pairs
DNA Gyrase
DNA Helicase
Euk. Topo II
Topo II of Eukaryotes 1) Relaxes only negatively
supercoiled DNA 2) Increases the linking number
by increments of 2 3) Requires ATP
Topo II of E. coli (DNA Gyrase) 1) Acts on both
neg. and pos. supercoiled DNA 2) Increases the
of neg. supercoils by increments of 2 3)
Requires ATP
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All Topoisomerases Cleave DNA Using a Covalent
Tyrosine-DNA Intermediate
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The Role of Topoisomerases in DNA Replication
1) Topoisomerases remove positive supercoils that
normally form ahead of the growing replication
fork
DNA gyrase
E. Coli ? DNA gyrase (adds neg. supercoils)
Eukaryotes ? Topo I (relaxes pos. supercoils)
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2) Replicated circular DNA molecules are
separated by type II topoisomerases
Catenated (linked)
Type II topoisomerases decatenate and catenate
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A Review of the Different Topoisomerases
Topo Type E. coli Eukaryotic
I Topo I Topo
I

Cleaves Relaxes only supercoils
Relaxes and supercoils
1 strand
(nicks) Changes linking by 1
Changes linking by 1 or 1
reseals
Requires no cofactors Requires
no cofactors
II Topo II (DNA Gyrase) Topo
II
Cleaves Acts on and supercoils
Relaxes only supercoils
2 strands
(ds cut) Changes linking by
Changes linking by
reseals increments of -2
increments of -2
Catenates and decatenates DNA
Catenates and decatenates DNA
Requires ATP
Requires ATP
Eukaryotic topoisomerases cannot introduce net
supercoils, Therefore, how can eukaryotic DNA
become negatively supercoiled?
Introduces net neg. supercoils

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How Does Eukaryotic DNA Become Negatively
Supecoiled?
Plectonemic supercoils
Solenoidal (Toroidal) supercoils
Q What will happen if you remove the histone
core?
? DNA wrapping around histone cores leads to net
negative supercoils!
A The solenoidal supercoil will adopt a
plectonemic conformation