Download Properties of Agarose

DNA structure:
Tertiary Structure: topology of plasmids
Properties of Agarose
Lecture 10: 1
10/18/2006
Methylation of agarose
The agarose polysaccharide also contains uncharged methyl groups.
The extent of natural methylation is directly proportional to the gelling temperature.
Unexpectedly, synthetically methylated agaroses have lower, rather than higher,
gelling temperatures, and the degree of synthetic methylation is inversely
proportional to the gelling temperature.
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 2
10/18/2006
Properties of Agarose
Melting and gelling temperature
The energy needed to melt an agarose gel increases as the gel concentration increases.
The gelling temperature of an agarose gel is also influenced by the gel concentration.
For this reason, gelling or remelting temperatures are expressed at a given agarose
concentration. This property is of practical value since it is possible to vary gelling
and melting parameters by using lower or higher concentrations of agarose. The
dependence of gelling and melting temperatures on concentration is most
pronounced at concentrations less than 1%.
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 2
10/18/2006
Properties of Agarose
Advantages of agarose
• Agarose forms a macroporous matrix which allows rapid diffusion of high
molecular weight (106 dalton range) macromolecules without significant
restriction by the gel.
• Agarose gels have a high gel strength, allowing the use of concentrations
of 1% or less, while retaining sieving and anticonvective properties.
• Agarose is nontoxic and, unlike polyacrylamide, contains no potentially
damaging polymerization by-products. There is no free radical
polymerization involved in agarose gelation.
• Rapid staining and destaining can be performed with minimal
background.
• Agarose gels are thermoreversible. Low gelling and melting temperature
agaroses permit easy recovery of samples, including sensitive heat-labile
materials.
• Agarose gels may be air dried.
DNA structure:
Tertiary Structure: topology of plasmids
current
Constant current
Lecture 10: 4
10/18/2006
Watts
When the current is held constant, the samples
will migrate at a constant rate.
voltage and wattage will increase as the
resistance increases, resulting in an
increase in heat generation during the run.
If a break occurs in the system such as a
damaged lead or electrode or a buffer
leak, the resistance of the gel will be greatly
increased. This will cause a large
increase in wattage and voltage resulting in the
generation of excessive heat. It is
even possible for the system to get hot enough
to boil, or start the apparatus to scorch or
burn.
voltage
Constant voltage
Constant voltage
When voltage is set constant, current and
wattage will decrease as the resistance
increases, resulting in a decrease of heat and
DNA migration. Since the heat generated will
decrease, the margin of safety will increase over
the length of the run. If a problem develops and
the resistance increases dramatically, the current
and wattage will fall since the voltage cannot
increase. Even if the apparatus fails, the worst
that is likely to happen is that the resistance will
increase so much that the power supply will not
be able to compensate, and it will shut off.
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 5
10/18/2006
Agarase
β-Agarase I digests agarose, releasing trapped DNA and producing
carbohydrate molecules which can no longer gel. β-Agarase I can be
used to purify both large (> 50 kb) and small (< 50 kb) fragments of
DNA from gels. The remaining carbohydrate molecules and βAgarase I will not, in general, interfere with subsequent DNA
manipulations such as restriction endonuclease digestion, ligation,
and transformation.
Q2: What is the molecular weight of β-Agarase I?
A2: 30,000 Daltons
Q3: What type of agarose will β-Agarase I digest?
A3: Only low melting point agarose is suitable for β-Agarase I
digestion as the solution must be liquid at the incubation
temperature of 42°C. If the temperature falls below 42°C during the
reaction time, even low melting point agarose will begin to congeal
and be undigestable.
DNA structure:
Tertiary Structure: topology of plasmids
Five Possible Conformations are listed below in order of
electrophoretic mobility (speed for a given applied voltage)
from slowest to fastest:
1. "Nicked Open-Circular" DNA has one strand cut.
2. "Linear" DNA has free ends, either because both strands
have been cut, or because the DNA was linear in vivo.
3. "Relaxed Circular" DNA is fully intact with both strands
uncut, but has been enzymatically "relaxed" (supercoils
removed).
4. "Supercoiled Denatured" DNA is like supercoiled DNA
(see below), but has unpaired regions that make it slightly
less compact; this can result from excessive alkalinity
during plasmid preparation.
5. * "Supercoiled" (or "Covalently Closed-Circular") DNA is
fully intact with both strands uncut, and with a twist built
in, resulting in a compact form.
Lecture 10: 6
10/18/2006
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 7
10/18/2006
Supercoils: a duplex is twisted in space around its own axis.
Negative supercoils: the DNA is twisted about its axis in the direction
opposite to the intrinsic winding of the helix, thus releasing the torsional
pressure or unwinding the helix.
Positive supercoils: the DNA is supercoiled in the same direction in
The same direction as the intrinsic winding of the double helix.
The linking number (L): the number of times that the two strands of the
double helix of a closed molecule cross each other in total.
L= W + T
T: the twisting number, the total number of turns of the duplex
W: the writhing number, the turning of the axis of the duplex in space.
For relaxed, W = 0.
∆L = ∆W + ∆T, ∆L>0: positive supercoiling; ∆L<0: negative supercoiling
Some 10-50 kcal/mol are needed to separate 10 bp.
Lecture 10: 8
10/18/2006
DNA structure:
Tertiary Structure: topology of plasmids
Relationship of writhing number and twist numbers
L= W + T
DNA structure:
Tertiary Structure: topology of plasmids
Introducing one supercoil into a DNA with 10 duplex turns
Lecture 10: 9
10/18/2006
DNA structure:
Tertiary Structure: topology of plasmids
Two forms of supercoils: a toroidal helix
Lecture 10: 10
10/18/2006
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 11
10/18/2006
DNA supercoility is afftected by intercalating agents such as ethidium bromide
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 12
10/18/2006
A number of natural and essential cellular processes can
have adverse effects on DNA superstructure.
1) the double-helical structure of DNA contorts and supercoils
when unwound by polymerases or helicases.
2) Replication of the two complementary
DNA strands, or recombination between DNA duplexes, can
generate chromosomal knots and catenanes.
Failure to resolve these problems can promote misregulation
of gene expression and chromosomal breakage, and can have
severe consequences for cell viability.
JAMES WANG
Mallinckrodt Professor of Biochemistry and Molecular Biology
Harvard University
Interaction between DNA and an Escherichia coli protein omega.
J Mol Biol. 1971 Feb 14;55(3):523-33
Discovering Topoisomerase
The late 1960s were an interesting time to work on campus,
according to Wang, a professor of chemistry at Berkeley from 19661977, and currently a professor of molecular and cell biology at
Harvard. "There were frequent demonstrations on campus, and once
the campus was tear-gassed by a helicopter. For weeks a 'stink
bomb' left a repugnant smell in our elevator, and one day a band of
demonstrators snaked through the chemistry buildings to look for
'military lasers' that never existed," he recalled in Reflections on an
accidental discovery.
Wang discovered topoisomerase while at Berkeley in 1971. His
finding attracted a lot of attention because scientists now had a way
to separate isomers of DNA that differed only topologically.
"Because of my physicochemical background, all enzymes seemed
rather mysterious and would better be left to others with the right
aptitude for messier systems. Therefore, shortly after my arrival in
Berkeley, I decided to try a chemical approach of using a reagent
carbodiimide for the desired water splitting. I failed miserably in that
expedition.
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 14
10/18/2006
"The finding of topoisomerase was completely accidental. I was studying negative
supercoiling of DNA and had one single cell preparation that was not in agreement with the
others. In experimental sciences, a common dictum is 'repeat the experiment if the result
makes no sense.' Is a strange observation reproducible to allow rigorous scrutiny by the
methods of science?
"When I went back to my notebook to see what was different about this one sample, I
noticed that I had spun the cell lysate in the centrifuge for much longer than usual and at a
higher temperature. The longer time was due to the fact that I suddenly had to take my
daughter to the hospital and had set the machine on 'hold'. The increased temperature was
most likely due to the fact that I didn't set the temperature correctly-centrifuges were very
bulky and demanding back then-as I rushed off to the hospital."
This single sample lacked negative supercoiling, and it was reasonable for Wang to assume
that an activity in the cell, at this incubation, was capable of removing the supercoiling.
"After months with the chromatography columns in the cold room, I isolated the active
fraction and found that it could indeed relax DNA.
"The manuscript was held up for quite some time as the journal's reviewers took their time
to 'believe' the results. I can imagine their bewilderment with this strange report of an
unprecedented enzyme by someone with no track record in enzymology. For one whole
year, I would wonder whether one day the whole thing would turn out to be an artifact. After
one year, however, I myself was fully convinced. It probably took many others a few more
years to accept the findings," Wang said.
DNA structure:
Tertiary Structure: topology of plasmids
Detection of topoisomearse by agarose gel electrophoresis
Lecture 10: 15
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DNA structure:
Tertiary Structure: topology of plasmids
What can Topoisomerase I do to the DNA?
Lecture 10: 16
10/18/2006
DNA structure:
Tertiary Structure: topology of plasmids
Topoisomerase II (DNA gyrase)
Lecture 10: 17
10/18/2006
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 18
10/18/2006
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 19
10/18/2006
Topoisomerases: A class of enzymes that alter the topology (supercoiling)
of double-stranded DNA. The topoisomerases act by transiently cutting one
or both strands of the DNA.
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 20
10/18/2006
The intact holoenzyme is a 97K protein with three Zn(II) atoms in tertacysteine
motifs near its carboxy-terminus. Topoisomerase I appears to reverse
supercoiling by transiently breaking a segment of single-stranded DNA, passing
an intact single- or double-stranded strand of DNA through the gate, then
rejoining the broken segment.
This is not an active process in the sense that energy in the form of ATP is not
spent by the topoisomerase during uncoiling of the DNA; rather, the torque
present in the DNA drives the uncoiling. Type I enzymes can be further
subdivided into type IA and type IB, based on their chemistry of action. Type IA
topoisomerases change the linking number of a circular DNA strand by units of
strictly 1, wherease Type IB topoisomerases change the linking number by
multiples of 1.
Type II topoisomerases cut both strands of the DNA helix simultaneously. Once
cut, the ends of the DNA are separated, and a second DNA duplex is passed
through the break. Following passage, the cut DNA is resealed. This reaction
allows type II topoisomerases to increase or decrease the linking number of a
DNA loop by 2 units, and promotes chromosome disentanglement.
Lecture 10: 21
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DNA structure:
Tertiary Structure: topology of plasmids
DNA topoisomerases fall into two categories — type I and type II. For the type
I enzymes, the DNA strands are transiently broken one at a time; for the type II
enzymes, by contrast, a pair of strands in a DNA double helix are transiently
broken in concert by a dimeric enzyme molecule.
TABLE 1 Topoisomerase classifications and characteristics
Catalytic domains
DNA cleavage
ATP hydrolysis
IA
IB
IIA
IIB
toprim/CAP
—
IB/Int
—
toprim/CAP
GHKL
toprim/CAP
GHKL
No
No
Single strand
3’
+/- n
h-topo I, v-topo I
topo V
Yes
Yes
Double strand
5’
+/-2
topo II, topo IV
DNA gyrase
Yes
Yes
Double strand
50’
+/- 2
topo VI
Mechanistic properties
Metal dependent?
Yes
ATP dependent?
No
DNA cleavage
Single strand
Cleavage polarity
5’
∆Lk
+1
Representative
E. coli topo I & III
Enzymes
reverse gyrase
DNA structure:
Tertiary Structure: topology of plasmids
Lecture 10: 22
10/18/2006
(a) 5Y-CAP domain from S. cerevisiae
topo II. Active site tyrosine and arginine
residues are shown inyellow.
(b) 5Y-CAP domain from Methanococcus
jannaschii topo VI-A
(c) Toprim domain from M. jannaschii
topo VI-A (102), showing the acidic cluster
and a bound Mg2+ ion.
(d) Type IB/Int catalytic domain from
vaccinia topo I (v-topo I). The active site
Tyr274 is pointed away from the other
active site residues Arg223 and His265,
while Arg130 and Lys167 are disordered.
(e) GHKL domain from E. coli DNA
gyrase, a type IIA topo (159). Bound
ADPNP is shown in blue and a bound
Mg2+ ion in black.
(f ) Transducer domain from E. coli DNA
gyrase, showing the critical Lys337 residue.
DNA structure:
Tertiary Structure: topology of plasmids
Catalytic mechanisms of topoisomerases
Lecture 10: 23
10/18/2006