truktra a Topolgia DNA

1
truktúra a Topológia DNA

• Doublehelix - 51 rokov
• Formy DNA,
• topoizomerázy,
• interakcia DNA-proteín,
• izolácia nukleových kyselín

The modern era of molecular biology began in 1953
when James D. Watson and Francis H. C. Crick
proposed that DNA has a double-helical structure.
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Three notions converged in the construction of
the double helix model for DNA by Watson and
Crick in 1953

• X-ray diffraction data showed that DNA has the
  form of a regular helix, making a complete turn
  every 34 Å (3.4 nm), with a diameter of 20 Å (2
  nm). Since the distance between adjacent
  nucleotides is 3.4 Å, there must be 10
  nucleotides per turn.
• The density of DNA suggests that the helix must
  contain two polynucleotide chains. The constant
  diameter of the helix can be explained if the
  bases in each chain face inward and are
  restricted so that a purine is always opposite a
  pyrimidine, avoiding partnerships of
  purine-purine (too thick) or pyrimidine-pyrimidine
  (too thin).
• Irrespective of the actual amounts of each base,
  the proportion of G is always the same as the
  proportion of C in DNA, and the proportion of A
  is always the same as that of T. So the
  composition of any DNA can be described by the
  proportion of its bases that is G C. This
  ranges from 26 to 74 for different species.

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Watson-Crick base pairing, complementary base
pairs, vodíkové mostíky - slabé interakcie
The double helix maintains a constant width
because purines always face pyrimidines in the
complementary A-T and G-C base pairs. The
sequence in the figure is T-A, C-G, A-T, G-C
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Double helix - dvojzávitnica
The two strands of DNA form a double helix.
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• Watson and Crick proposed that the two
  polynucleotide chains in the double helix
  associate by hydrogen bonding between the
  nitrogenous bases. G can hydrogen bond
  specifically only with C, while A can bond
  specifically only with T. These reactions are
  described as base pairing, and the paired bases
  (G with C, or A with T) are said to be
  complementary.
• Tm - Melting temperature of DNA dependent on
• GC content and ionic concentration
• Tm 69,3 0,41 (GC)
• (at 0,165M NaCl and pH 7,0)
• Denaturation and renaturation of DNA are the
  basis of nucleic acid hybridization

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Each base pair is rotated 36 around the axis of
the helix relative to the next base pair. So 10
base pairs make a complete turn of 360. The
twisting of the two strands around one another
forms a double helix with a narrow groove (12 Å
across) and a wide groove (22 Å across) The
double helix is right-handed the turns run
clockwise looking along the helical axis. These
features represent the accepted model for what is
known as the B-form of DNA. It is important to
realize that the B-form represents an average,
not a precisely specified structure. DNA
structure can change locally. If it has more base
pairs per turn it is said to be overwound if it
has fewer base pairs per turn it is underwound.
Local winding can be affected by the overall
conformation of the DNA double helix in space or
by the binding of proteins to specific sites.
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Rôzne formy DNA
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Formy DNA
V rekombinantých dejoch, interakciách DNA-DNA,
DNA-RNA, DNA-proteín
V spórách bacilov
Vo väcine prípadov
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Genomika - túdium genómov, projekty sekvenovania
genómov velkého mnostva organizmov, vrátane
ludského
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Topologické manipulácie s DNA
Supercoiling describes the coiling of a closed
duplex DNA in space so that it crosses over its
own axis.Topological isomers are molecules of
DNA that are identical except for a difference in
linking number.Twisting number of a DNA is the
number of base pairs divided by the number of
base pairs per turn of the double helix.Writhing
number is the number of times a duplex axis
crosses over itself in space. Topological
manipulation of DNA is a central aspect of all
its functional activities recombination,
replication, and (perhaps) transcription as
well as of the organization of higher-order
structure. All synthetic activities involving
double-stranded DNA require the strands to
separate. However, the strands do not simply lie
side by side they are intertwined. Their
separation therefore requires the strands to
rotate about each other in space.
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Some possibilities for the unwinding reaction
Separation of the strands of a DNA double helix
could be achieved by several means.
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Superpiralizácia

• Supercoiling of the DNA double helix results when
  it is coiled about itself in space (like twisting
  a rubber band). The supercoiling creates a
  tension in the double helix that changes its
  structure.
• Positive supercoiling, when the DNA is twisted in
  space in the same sense as the strands are wound
  around one another, causes the double helix to be
  more tightly wound.
• Negative supercoiling, when the DNA is twisted in
  space in the opposite sense from the internal
  winding of the strands, causes the double helix
  to be less tightly wound. Negative supercoiling
  can be thought of as creating tension in the DNA
  that is relieved by unwinding the double helix.

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Topologické izoméry

• A closed molecule of DNA can be characterized by
  its linking number, the number of times one
  strand crosses over the other in space. Closed
  DNA molecules of identical sequence may have
  different linking numbers, reflecting different
  degrees of supercoiling. Molecules of DNA that
  are the same except for their linking numbers are
  called topological isomers.
• The linking number is made up of two components
  the writhing number (W) and the twisting number
  (T).

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twisting number, writhing number

• The twisting number, T, is a property of the
  double helical structure itself, representing the
  rotation of one strand about the other. It
  represents the total number of turns of the
  duplex. It is determined by the number of base
  pairs per turn. For a relaxed closed circular DNA
  lying flat in a plane, the twist is the total
  number of base pairs divided by the number of
  base pairs per turn.
• The writhing number, W, represents the turning of
  the axis of the duplex in space. It corresponds
  to the intuitive concept of supercoiling, but
  does not have exactly the same quantitative
  definition or measurement. For a relaxed
  molecule, W 0, and the linking number equals
  the twist.

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• We are often concerned with the change in linking
  number, L, given by the equation
• DL DW DT
• The equation states that any change in the total
  number of revolutions of one DNA strand about the
  other can be expressed as the sum of the changes
  of the coiling of the duplex axis in space (DW)
  and changes in the screwing of the double helix
  itself (DT). In a free DNA molecule, W and T are
  freely adjustable, and any DL (change in linking
  number) is likely to be expressed by a change in
  W, that is, by a change in supercoiling.
• A decrease in linking number, that is, a change
  of DL, corresponds to the introduction of some
  combination of negative supercoiling and/or
  underwinding. An increase in linking number,
  measured as a change of DL, corresponds to a
  decrease in negative supercoiling/underwinding.
• We can describe the change in state of any DNA by
  the specific linking difference, DL/L0, where
  L0 is the linking number when the DNA is relaxed.
  If all of the change in linking number is due to
  change in W (that is, DT 0), the specific
  linking difference equals the supercoiling
  density. In effect, as defined in terms of DL/L0
  can be assumed to correspond to superhelix
  density so long as the structure of the double
  helix itself remains constant.

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• Ak DNA molekula v B konformácii obsahuje 200
  bázových párov, T20 a W -2
• twisting number T - dvojzávitnicové císlo
  writhing number W -císlo superpiralizácie
• Potom linking number L - celkové císlo vinutia
  je
• L T W 20 (-2) 18

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DNA topoizomerázy

• The linking number is related to the actual
  enzymatic events by which changes are made in the
  topology of DNA. The linking number of a
  particular closed molecule can be changed only by
  breaking a strand or strands, using the free end
  to rotate one strand about the other, and
  rejoining the broken ends. When an enzyme
  performs such an action, it must change the
  linking number by an integer this value can be
  determined as a characteristic of the reaction.
  Then we can consider the effects of this change
  in terms of W and T.
• DNA topoisomerases catalyze conversions of this
  type. Some topoisomerases can relax (remove) only
  negative supercoils from DNA others can relax
  both negative and positive supercoils. Some can
  introduce negative supercoils.

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Druhy DNA topoizomeráz

• Topoisomerases are divided into two classes,
  according to the nature of the mechanisms they
  employ.
• Type I enzymes act by making a transient break in
  one strand of DNA.
• Type II enzymes act by introducing a transient
  double-strand break.
• Vysoká súvislost s onkologickými chorobami !!! -
  - -
• Ale tie aj pri oprave pokodenej DNA !

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The best characterized type I topoisomerase is
the product of the topA gene of E. coli, which
relaxes highly negatively supercoiled DNA. The
enzyme does not act on positively supercoiled
DNA. Mutations in it cause an increase in the
level of supercoiling in the nucleoid (and may
affect transcription). In addition to the
relaxation of negative supercoils in duplex DNA,
the enzyme interacts with single-stranded DNA. It
may like negative supercoils because they tend to
stabilize single-stranded regions, which could
provide the substrate bound by the enzyme. When
E. coli topoisomerase I binds to DNA, it forms a
stable complex in which one strand of the DNA has
been nicked and its 5phosphate end is covalently
linked to a tyrosine residue in the enzyme. This
suggests a mechanism for the action of the
enzyme it transfers a phosphodiester bond in DNA
to the protein, manipulates the structure of the
two DNA strands, and then rejoins the bond in the
original strand.
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The reaction changes the linking number in steps
of 1. Each time one strand is passed through the
break in the other, there is a DL of 1. In a
free supercoiled molecule, the
interchangeability of W and T should let the
change in linking number be taken up by a change
of DW 1, that is, by one less turn of negative
supercoiling
The type I topoisomerase also can pass one
segment of a single-stranded DNA through another.
This single-strand passage reaction can introduce
knots in DNA and can catenate two circular
molecules so that they are connected like links
on a chain. We do not understand the uses (if
any) to which these reactions are put in vivo.
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• Type II topoisomerases generally relax both
  negative and positive supercoils. The reaction
  requires ATP probably one ATP is hydrolyzed for
  each catalytic event.
• The reaction is mediated by making a
  double-stranded break in one DNA duplex, and
  passing another duplex region through it.
• A formal consequence of two-strand transfer is
  that the linking number is always changed in
  multiples of two. The topoisomerase II activity
  can be used also to introduce or resolve
  catenated duplex circles and knotted molecules.

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Bacterial DNA gyrase is a topoisomerase of type
II that is able to introduce negative supercoils
into a relaxed closed circular molecule. The
supercoiled form of DNA has a higher free energy
than the relaxed form, and the energy needed to
accomplish the conversion is supplied by the
hydrolysis of ATP. In the absence of ATP, the
gyrase can relax negative but not positive
supercoils, although the rate is more than 10
slower than the rate of introducing
supercoils. V termofilných baktériách
existuje tie reverzná DNA gyráza - spôsobuje
pozitívnu superpiralizáciu
DNA gyrase may introduce negative supercoils in
duplex DNA by inverting a positive supercoil.
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Elektrónová mikroskopia príklad interakcie
proteín-DNA
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Atomic Force Microscopy
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Izolácia DNA
Izolácia a purifikácia vektorovej DNA -
najcastejie tzv. plazmidovej DNA, ktorá má v
priemere od 2 - 100 kb 1. Izolácia
nukleoproteínu 2. Deproteinizácia 3. Purifikácia
- Helinského metóda ultracentrifugácie v
gradiente CsCl - nárocná metóda z financného aj
casového dôvodu V súcasnosti KITY súpravy na
izoláciu akejkolvej NK - nie sú práve
najlacnejie, ale sú najrýchlejie
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Birnboim a Doly (1979) izolácia plazmidov
alkalickou lýzou(denaturácia a renaturácia
DNA)Izolácia a purifikácia donorovej DNA - z
akéhokolvek zdroja (vírusy, baktérie, rastliny,
ivocíchy)Problémy - napr. vysoký obsah
polysacharidov (krobu) u rastlinných
zdrojovZdroje nukleových kyselín - brzlíková
DNA, DNA zo spermií lososa