section 23.3
Dénaturation of DNA
527
FIGURE 23-7
Different states of a covalent DNA circle, (a) A nonsupercoiled covalent
DNA having 36 turns of the helix, (b) An underwound covalent circle
having only 32 turns of the helix, (c) The molecule in (b), but with four
superhelical turns to eliminate the underwinding. In solution, (b) and
(c) would be in equilibrium; the equilibrium would shift toward (b) with
increasing temperature.
superhelices. In bacteria, the underwinding of superhe-
lical DNA is not a result of unwinding before end joining
but is introduced into preexisting circles by an enzyme
called
DNA gyrase,
which is one of a class of enzymes
called
topoisomerases
(Chapter 24). In eukaryotes, the
underwinding is a result of the structure of chromatin,
a DNA-protein complex of which chromosomes are com-
posed. In chromatin, DNA is wound about specific protein
molecules, in a direction that introduces underwinding.
23.3 Dénaturation of DNA
The three-dimensional structures of DNA, RNA, and pro-
teins are determined by weak noncovalent interactions,
principally hydrogen bonds and hydrophobic interactions.
The free energies of these interactions are not much greater
than the energy of thermal motion at room temperature,
so that at elevated temperatures the structures of these
molecules are disrupted. A macromolecule in a
disrupted
state
is said to be denatured; the
ordered state,
which
is presumably that originally present in nature, is called
the native state. A transition from the native to the dena-
tured state is called
dénaturation.
When double-stranded
(native) DNA is heated, the bonding forces between the
strands are disrupted and the two DNA strands separate;
thus, completely denatured DNA is single stranded.
Much information about the structure and stabilizing
interactions has been obtained by studying dénaturation.
Some property of DNA that changes as dénaturation
FIGURE 23-8
Melting curve of DNA showing the melting temperature (Tm) and possible
molecular conformations for various degrees of melting.
proceeds is measured, e.g., the absorption of ultraviolet
light. A change in the ultraviolet absorbance (or some other
property) as a function of temperature is called a
melting
curve
(Figure 23-8). Many reagents either break hydro-
gen bonds or weaken hydrophobic interactions, and are
powerful dénaturants. Thus, dénaturation is also studied
by varying the concentration of a dénaturant at a con-
stant temperature. For DNA, the simplest way to de-
tect dénaturation is to monitor the ability of DNA in a
solution to absorb ultraviolet light at a wavelength of
260 nm. The absorbance of DNA at 260 nm, A260, is
not only proportional to its concentration (as is the case
for most light-absorbing molecules) but also depends
on the structure of the molecule; the more ordered the
structure, the less light is absorbed. Therefore, free nu-
cleotides absorb more light than a single-stranded polymer
of DNA (or RNA), which in turn absorbs more light than
a double-stranded DNA molecule. For example, solutions
of double-stranded DNA, single-stranded DNA, or free
bases, each at 50 /xg/mL, have the following
A2
6
o values:
Double-stranded DNA:
A
2
6o=1.00
Single-stranded DNA:
A
26
o
= 1-37
Free bases:
A
2 6 0
=
1.60
If a DNA solution is heated slowly and the A260 is mea-
sured at various temperatures, a melting curve such as that
shown in Figure 23-8 is obtained. The following features
of this curve should be noted:
1. The A
2 6
o remains constant up to temperatures well
above those encountered by living cells in nature.
