PROPERTIES OF DNA II.

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PROPERTIES OF DNA II.
Supercoiling and Topoisomerases
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The E. coli chromosome 2 puzzles
1. It was circular and twisted
2. Chromatograph showed evenly spaced bands
Relaxed
Highly supercoiled
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Supercoiled DNA
Twist right
Topoisomerase II
?L 0
?L 2
Twist left
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What is Supercoiling?
Circular DNA can exist either as a flat
or twisted molecule. Twisting occurs
when double helical chains cross over
Flat (relaxed)
Supercoiled (two twists)
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Characteristics of Supercoiled DNA
1. Property of closed circular DNA
2. E. coli DNA has one LEFT twist every 200 bases
3. ATP energy is required to form supertwists
4. DNA forms that differ only in number of
twists are called topoisomers
5. Name is derived from topography referring
to structural features
viewed from above.
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Determining Supercoiling in DNA
L T W
L Linking number
T Turns or twists in the DNA
W Writhing number referring to the number
of crossovers
Right twist positive Left twist negative
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Measuring Supercoiling
Example Assume a B-DNA with 105 base pairs
There are 10.5 bp in one turn of B-DNA
(a turn refers to the helical distance of one
strand crossing the axis and returning to
original position)
T 10
If the DNA is relaxed, then
10.5 bp
W 0
L 10
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Rule As long as the DNA stays as an unbroken
duplex DNA circle, L is a constant.
Rule To add or remove twists in circular DNA
requires breaking the strands of the DNA
Rule Because L is a constant in unbroken duplex
DNA, any change in T, i.e., ?T, must be
accompanied by an equal and opposite change in
super coiling.
?T ?W (favors unwinding)
Or
?W ?T
Rule Topoisomers are DNA molecules that
are interconvertible without breaking covalent
bonds.
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Generating Topoisomers
If the circular DNA with 10 turns is broken and
one turn is opened, when the molecule is
resealed, a relaxed molecule will now
have
T 9 W 0 and
L 9
the relaxed (flat) molecule now has
105 bp in only 9 turns
11.67 bp per turn
STRAINED
HIGH ENERGY
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Question How can a circular flat DNA molecule
with 105 bases in 9 turns (L9) be arranged to
have the least strain and lowest energy
Answer Keep T at 10 but add one negative
supercoil
SOLUTION
L 9
L 9
T 9
T 10
LOW ENERGY because of 10 bp/turn and one negative
twist
HIGH ENERGY because of 11 bp/turn
W 0
W - 1
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Topoisomerases
What are topoisomerases?
Ans A family of enzymes that control
supercoiling
Why are they important?
Ans DNA functions in replication and
transcription depend on topological state.
So how do topoisomerases work?
Ans The two DNA strands must separate.
Topoisomerase II promotes () coiling which aids
strand separation.
Why does this happen?
Ans ? W is matched by ? T and DNA unwinds
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Does a ? W favors unwinding of the DNA strands?
Ans Yes
What do topoisomerases do?
Ans Topoisomerase create and remove supercoils
in DNA.
Can they change linking number?
Ans Yes. Type-1 nicks and closes one site at a
time. Each nick removes one negative turn.
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What about Type II?
Type II requires ATP and cuts and reseals both
strands at the same time. Type II loops around
the DNA much like a ring, and catalyzes the
breaks at select locations. It then passes a
segment through the loop and reseals.
Can Type II form negative supercoils?
Yes. Only the bacterial enzyme (known as DNA
gyrase) has the ability to supercoil DNA.
What about Eukaryotes
In eukaryotes negative supercoils arise during
packing the DNA into nucleosomes, not by
topoisomerases
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Could you show me a picture of Type II
topoisomerase?
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The Topoisomerases
Topoisomerase I 1. Single strand nicks 2.
Negative coil to relaxed 3. Active tyrosine
Topoisomerase II 1. Double strand nicks 2.
Creates supercoiling 3. Requires ATP energy9
kcal/mol of supercoils
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Topoisomerase I
1. Relaxes negatively coiled
Tyrosine
3 -OH
HO
97 kD monomer
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Topoisomerase II
1. Relaxes negative and positive supercoils
2. Bacterial enzyme (DNA gyrase)creates negative
supercoils
3. Cuts both strands
4. Requires ATP energy
5. Multimeric enzymes with pairs of tyrosines
6. Create Staggered cuts
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Topoisomerase II
Bind and cut
ATP
Thread second strand through cut
ADP Pi
Reseal First