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FCH 532 Lecture 11
Chapter 29: Nucleic Acid Structures
Linking number

The linking number L of DNA, a topological property, determines the degree of
supercoiling;

The linking number defines the number of times a strand of DNA winds in the
right-handed direction around the helix axis when the axis is constrained to lie
in a plane;

If both strands are covalently intact, the linking number cannot change;

For instance, in a circular DNA of 5400 basepairs, the linking number is
5400/10=540, where 10 is the base-pair per turn for type B DNA.
The twist and writhe
1.
Twist T is a measure of the helical winding of the DNA strands around
each other. Given that DNA prefers to form B-type helix, the preferred
twist = number of basepair/10;
2.
Writhe W is a measure of the coiling of the axis of the double helix. A
right-handed coil is assigned a negative number (negative
supercoiling) and a left-handed coil is assigned a positive number
(positive supercoiling).
3.
Topology theory tells us that the sum of T and W equals to linking
number: L=T+W
4.
For example, in the circular DNA of 5400 basepairs, the linking number
is 5400/10=540
1.
If no supercoiling, then W=0, T=L=540;
2.
If positive supercoiling, W=+20, T=L-W=520;
The relation between L, T and W
Positive supercoiling
The relation between L, T and W
Negative supercoiling
L, T and W calculation
1.
A relaxed circular, double stranded DNA (1600
bps) is in a solution where conditions
favor 10 bps per turn. What are the L, T,
and W?
2.
During
replication,
part
of
this
DNA
unwinds (200 bps) while the rest of the DNA
still favor 10 bps per turn. What are the
new L, T, and W?
1600 bps
L=1600/10=160
W=0 (relaxed)
T=L-W =160
1400 bps
200 bps
L=160
T=(1600-200)/10=140
W=L-T=+20
L, T, and W characterize
superhelical DNA
•L= linking number = number of
times one strand wraps around the
other. It is an integer for a closed
circular DNA.
•T = twists/turns in the DNA ( No.
bp/10.4; positive for right-handed
DNA
•W = writhes =number of turns of
the helix around the superhelical
axis
T = 26
W=0
L=T+W
What kind of number is L??
Figure 29-20 Two ways of introducing
one supercoil into a DNA with 10
duplex turns.
Topoisomerases change the
linking number of superhelical
DNA
Type I topos change L in units of one by breaking a single strand
of DNA and allowing the duplex to unwind.
Type II topos change L in units of two by breaking both strands
and allowing a pass-through of both strands of the double helix.
Type I topoisomerases (nicking-closing
enzymes)
DNA (n turns) + topoisomerase
>covalent DNA-enzyme intermediate>
dsDNA (n-1 turns) + topoisomerase
The formation of a covalent DNAenzyme complex preserves the free
energy of the phosphodiester bond
in DNA as a phosphodiester
bond between DNA and Tyrosine.
Model of DNA topo I (Ec N-terminus)
Note relative size of the enzyme
compared to the cross section of
the DNA helix, and how the
enzyme encircles and holds the
DNA.
Tyrosine (Y)
Position of active site tyr.
How does it contact DNA?
How do we know it is this
amino acid?
Page 1127
Figure 29-26 X-Ray structure of the Y328F mutant of E.
coli topoisomerase III, a type IA topoisomerase, in
complex with the single-stranded octanucleotide
d(CGCAACTT).
Type IA Topoisomerase
1.
2.
3.
4.
5.
6.
7.
Recognize ss region of DNA and gap opening between domains
I and II.
DNA cleaved with newly formed 5’end covalently linked to Tyr
and the segment with the newly formed 3’ end is noncovalently
bound to the protein.
Unbroken strand passed through the opening formed by the
cleaved strand to enter protein’s central hole.
Unbroken strand is trapped by the partial closing of the gap
2 cleaved ends of the cut strand are rejoined in reversal of
cleavage reaction
Gap between domains I and III reopens to allow the the rejoined
strand to leave the enzyme-the unbroken strand has been
passed through the break.
Enzyme returns to original state-If strands formed a negatively
supercoiled duplex DNA, L is increased by 1.
Figure 29-27 Proposed mechanism for the strand
passage reaction catalyzed by type IA topoisomerases.
Page 1128
For (-) supercoiled DNA-L is increased by 1
For circles, they have been catenated or decatenated.
Type IB Topoisomerase
•
•
•
•
Controlled rotation mechanism
Human topo I is a Type IB enzyme.
Uses Tyr 723 to form phophTyr bond.
Y723F is catalytically inactive mutant, although Y723 would be
positioned to nucleophilically attack the P atom of the P-O5’ bond.
• Sequence independent binding.
• Rotation occurs about sugar-phosphate bonds in uncleaved strand
(opposite the cleavage site).
• Positive charged amino acids hold the DNA strand in place.
Figure 29-28 X-Ray structure of the N-terminally
truncated, Y723F mutant of human topoisomerase I in
complex with a 22-bp duplex DNA.
Page 1129
Loosely held
strand, free to
rotate
Tightly held
scissile strand
Page 1129
Figure 29-29
Controlled
rotation mechanism for
type IB topoisomerses.
a)
Binding of supercoiled
DNA
b)
Formation of
noncovalent complex
c)
Upstream, cleaved
product attached to the
enzyme
d)
Downstream portion
rotates DNA, small
rocking motions of
enzyme.
e)
Covalent intermediate
with decreased linking
number.
f)
Ligation of cleaved
strand to intact strand
g)
Release of DNA
Type II Topoisomerase
•
•
•
•
Strand passage mechanism
Also known as DNA gyrase
Heterotetramer with A2B2 subunits. A=GyrA, B=GyrB
Prokaryotic Topo II catalyzes the stepwise negative supercoiling of
DNA with the concomitant hydrolysis of an ATP to ADP + Pi.
• DNA gyrases are inhibited by antibiotics (novobiocin, ciprofloxacin
[Cipro]).
• Change linking number by 2.
• Cuts both strands of duplex, passes the duplex through the break
and reseals.
Page 1131
Figure 29-31a
Structures of topoisomerase
II. (a) X-Ray structure of the 92-kD segment of the yeast
topoisomerase II (residues 410–1202) dimer.
Proposed
mechanism of
topo II
Arg residues
Type II Topoisomerase
1.
2.
3.
4.
5.
G-segment (Gate segment) binds to enzyme inducing
conformational change
ATP binds
T-segment (transported segment) binds causing conformational
change that cleaves the G’segment with the A’ subunit. The Tsegment is transported through the break in the central hole.
The G-segments are resealed and T-segment is released.
Interface reforms with ATP hydrolysis.
Page 1131
Figure 29-32 Model for the enzymatic mechanism of
type II topoisomerases.
Prokaryotic Topoisomerase II is a
DNA gyrase
• In the presence of ATP
DNA gyrase can
create supercoils; it
can relax supercoils in
the absence of ATP
Relaxed circle
+AMP,
PPi
ATP
Supercoiled
circle
Page 1130
Figure 29-30 A demonstration that DNA gyrase acts by
cutting both strands of a duplex, passing the duplex
through the break, and resealing it.
Inhibitors of DNA gyrase inhibit
DNA replication
• Two antibiotics,
oxolinic acid and
novobiocin inhibit
replication.
E. coli- NovS
topoII
NovS
E. coli- NovS
topoII
NovR
Mutants resistant to novobiocin
have a novobiocin-resistant topo
II activity in vitro, thus proving
that the lethal activity of the drug is its inhiition of DNA topo II
activity in vivo. This demonstrates that topo II is an (essential/nonessential) enyme for cell viability.
Summary
• DNA exists in different topological forms in
vivo and in vitro
• DNA topoisomerases catalyze the
interconversion of DNA forms
• Negative superhelicity (underwinding) helps
proteins bind DNA by favoring unwinding of
the helix.
DNA synthesis
Page 1137
Figure 30-1 Action of DNA polymerase. DNA
polymerases assemble incoming deoxynucleoside
triphosphates on single-stranded DNA templates such
that the growing strand is elongated in its 5¢ ® 3¢
direction.
Page 1137
Page 1137
Figure 30-2 Autoradiogram and its interpretive drawing
of a replicating E. coli chromosome.
Figure 11.4 The theta model for replication of a circular DNA
model.
Note that the DNA
structures resemble
the Greek letter
theta (q).
The two replicating
forks advance in
opposite directions.
Page 1138
Figure 11.3 A photographic image (obtained by
autoradiography) of replicating E. coli chromosome.
DNA contained 3H-labeled thymidine (b-emitter). Note
two replication forks (arrows).
DNA Replication in Bacteria vs. Eukaryotes (mammals)
•
DNA replication in bacteria involves a single origin of
replication site.
•
•
50,000 base pairs/minute DNA synthesis rate in bacteria.
DNA replication in eukaryotes involves multiple ARS
elements (autonomously replicating sequence)
replication sites.
•
Only 2,000 base pairs/minute DNA synthesis rate in
eukaryotes.
•
108 base pairs per chromosome (23 chromosomes) =>
1 month to duplicate if only one replication start point.
Figure 11.5. Proposed
pathway for replication of
eukaryotic DNA.
There are several origins of
replication (a)
A pair of replication forks
begins at each origin (b)
As the forks advance in
opposite directions, the
bubbles coalesce to form
two double-stranded DNA
molecules (c, d, e).
The action of DNA polymerase I
Discovered in 1957 by Arthur Kornberg, et al.
General reaction catalyzed by DNA polymerase I:
dNTP + (dNMP)n  (dNMP)n+1 + PPi
dNTP = deoxyribonucleoside triphosphates, dATP, dGTP,
dCTP, dTTP
(dNMP) = preformed DNA with n or n+1 mononucleotides
PPi = pyrophosphate
Figure 11.6 The action of DNA polymerase I
Preformed DNA performs two roles:
One as template (red), which carries the message to be copied.
One as a primer (purple) for attachment of added nucleotides.
Figure 10.7 Phosphodiester bonds linking mononucleotides into nucleic acids.
• The phosphodiester
bonds are between
the 3’ carbon and the
5’ carbon of the
second nucleotide.
• This gives direction to
the nucleic acids!!!
• One end has a free 5’
OH
• The other end has a
free 3’ OH
• The 3’,5’ phosphodiester
bonds are highlighted
with green
Figure 11.6 The action of DNA polymerase I
The incoming deoxythymidine triphosphate (dTTP, blue) is held in
position by complementary hydrogen bonds to adenine in the
template strand.
The new phosphoester bond is formed adding a base at the 3’ end of
the growing strand.
Extra energy is provided by hydrolysis of pyrophosphate.
Table 11.1 Comparison of E. coli DNA polymerases
•
Primary replicating enzyme in E. coli cells is thought
to be DNA pol III: faster, more complex structure.
•
DNA pol I and II probably serve in editing and repair of
DNA.
For the test
1.
2.
3.
4.
5.
6.
7.
Homework problems.
Quiz problems.
You should know key experiments: Griffith, Hershey-Chase,
Messelson-Stahl, etc.
Do not need to know codons (table will be provided).
Nucleic acid structures
Supercoiling
Differences in topoisomerases