Chemistry
Denaturation of DNA
Denaturation of DNA refers to the process in which the double-stranded DNA molecule unwinds and separates into two single strands, usually due to the disruption of hydrogen bonds between the base pairs. This can occur through factors such as high temperature, extreme pH, or exposure to certain chemicals. Denaturation can result in the loss of the DNA's biological function.
Written by Perlego with AI-assistance
6 Key excerpts on "Denaturation of DNA"
Learn about this page
Index pages curate the most relevant extracts from our library of academic textbooks. They’ve been created using an in-house natural language model (NLM), each adding context and meaning to key research topics.
eBook - ePub- Alexander McLennan, Andy Bates, Phil Turner, Michael White(Authors)
- 2012(Publication Date)
- Taylor & Francis(Publisher)
The denaturation of the ends of the molecule, and of more mobile AT-rich internal regions, will destabilize adjacent regions of helix, leading to a progressive and concerted melting of the whole structure at a well-defined temperature corresponding to the mid-point of the smooth transition, and known as the melting temperature (T m) (Figure7. 2 a). The melting is accompanied by a 40% increase in absorbance. T m is a function of the G + C content of the DNA sample, and ranges from 80°C to 100°C for long DNA molecules. Figure 2.(a) Thermal denaturation of double-stranded DNA (cooperative) and RNA; (b) renaturation of DNA by fast and slow cooling. Hybridization The thermal Denaturation of DNA may be reversed by cooling the solution. In this case, the rate of cooling has an influence on the outcome. Rapid cooling allows only the formation of local regions of dsDNA, formed by the base pairing or annealing of short regions of complementarity within or between DNA strands; hence the decrease in A 260 is rather small (Figure 2b). On the other hand, slow cooling allows time for the wholly complementary DNA strands to find each other, and the sample can become fully double-stranded, with the same absorbance as the original native sample. This annealing or renaturation of regions of complementarity between different nucleic acid strands is known as hybridization. Hybridization is crucial to the analysis of specific sequences of DNA, since just about the only way to detect a specific DNA sequence is by using the corresponding complementary sequence as a ‘probe’ (Sections Q3 and S2). B3 DNA supercoiling Key Notes Closed–circular DNA DNA frequently occurs in nature as closed-circular molecules, where the two single strands are each circular and linked together. The number of links is known as the linking number (Lk). Supercoiling Supercoiling is the coiling of the DNA axis upon itself, caused by a change in the linking number from Lk°, the value for a relaxed closed circle
eBook - ePub- Chang-Hui Shen(Author)
- 2019(Publication Date)
- Academic Press(Publisher)
m , and then they rapidly let go. The midpoint of the curve indicates the point at which half of the DNA population is denatured and the other half is still in double helix form. Denaturation and renaturation can occur with the combinations of DNA-DNA, DNA-RNA, and RNA-RNA as the intermolecular or intramolecular interaction.Fig. 1.15 The melting temperature of E. coli DNA. The temperature at the midpoint of the curve is approximately 87°C.Secondary Structure of DNA
Through advanced techniques, it has been shown that DNA molecules can have dynamic changes in secondary and tertiary structures, so that they can regulate expression of their linear sequence information. Unusual secondary structures, such as slipped structures, cruciform, and triple-helix DNA, are generally sequence-specific. Slipped structures usually occur at tandem repeats and are usually found upstream of regulatory sequences in vitro. Cruciform structures are paired stem-loop formations. They can be found in vitro for many inverted repeats in plasmids and bacteriophages. In a triplex helix, a third strand of DNA joins the first two to form triplex DNA. Triplex helix DNA occurs at purine-pyrimidine stretches in DNA and is favored by sequences containing a mirror repeat symmetry.Tertiary Structure of DNA
Many naturally occurring DNA molecules are circular, with no free 5′ or 3′ end. For example, prokaryotic DNA is circular, and this DNA forms a supercoil. To understand how supercoiled and relaxed circular DNA differ, you can take a covalently closed, circular DNA molecule, cleave both strands in one place, and then rotate the DNA end around several times before covalently closing the circle again. You can imagine this by taking a rubber band with two hands and twisting it together. The resulting DNA circle is supercoiled and will wind around itself. This is because rotating the DNA end changes the helical twist of the DNA from its preferred state containing about 10.5 base pairs per turn to one with a fewer number of base pairs per turn. When the DNA is ligated (meaning joined together), the untwisted portion of DNA will tend to spring back to adopt its favored state, with 10.5 base pairs per turn. This, however, causes the plasmid to wrap around itself, and the closed DNA circle is now supercoiled. An initial unwinding of the DNA (which is the clockwise direction and is equivalent to separating the DNA strands) leads to accumulation of negative supercoils, whereas an initial twisting of the DNA in the counterclockwise direction leads to formation of positive supercoils. Negative and positive supercoils simply differ in direction of rotation.
eBook - ePubBiophysics for Beginners
A Journey through the Cell Nucleus
- Helmut Schiessel(Author)
- 2021(Publication Date)
- Jenny Stanford Publishing(Publisher)
Eq. 4.39 with α = 0 and f = 0.88 pN, cause a length loss of 18 nm.4.4 DNA MeltingWhen a gene is transcribed by RNA polymerase, Fig. 1.3, or when the whole genome is duplicated by DNA polymerases, Fig. 1.2, the two strands of the DNA double helix need to be separated. Experimentally, it is very easy to induce the separation of the two strands by heating a solution containing double-stranded DNA chains. One can measure the fraction θb , of paired bases through the characteristic light adsorption of double-stranded DNA at 260 nm. At low temperatures all the bases are paired, θb , = 1, whereas at high temperatures all the bases are unbound, θb = 0. At intermediate temperatures, typically around 70 to 90° C the thermal denaturation or melting of DNA occurs. In general, the actual melting curve θb = θb (T) of long DNA chains looks complicated, exhibiting a multi-step behavior where sharp jumps are separated by plateaus of various lengths. This reflects the heterogeneity of the by sequence. Remember that AT pairs are bound via two hydrogen bonds and are thus weaker than GC pairs with their three hydrogen bonds, see Fig. 4.4. As a result, stretches of the DNA double helix that have a high AT content open first and form what are known as denaturation loops or bubbles. The melting curve thus contains information about the sequence of the molecule under study. It is not possible to avoid these sequence effects: If one uses DNA with a homogeneous sequence instead, e.g., only A’s on one strand and T’s on the other, one has the problem that a given base of the A-strand can be paired with any base of the T-strand.In the following we will not go into the sequence dependence any further; see Ref. (Blossey, 2006 ) for an insightful discussion on that subject. Instead, we discuss here an idealized problem where all the base pairs have the same binding energy and each monomer can be bound to one specific matching base on the other strand. We ask ourselves how such an idealized DNA molecule would melt, especially in the limit of an infinitely long chain. In principle we can think of three possibilities: (a) There is no phase transition and θb , goes smoothly from one to zero. (b) The curve θb = θb (T) is continuous but goes at some finite temperature TM to zero and stays zero for T ≥ TM . Some higher-order derivative of θb has a jump or a singularity at T = TM . DNA melting would then correspond to a continuous phase transition. (c) The θb
eBook - ePub- Raymond S. Ochs(Author)
- 2021(Publication Date)
- CRC Press(Publisher)
melting temperature) is much lower for AT-rich strands than for GC-rich strands.FIGURE 16.8 Melting curves for DNA. Naturally occurring DNA has a midpoint in the melting curve (Tm) between poly-AT (low Tm) and poly-GC (high Tm). The results show experimentally that GC pairs are more stable than AT pairs.AT-rich regions exist at DNA replication origins, suggesting that the hydrogen-bonded bases in these segments are intrinsically easier to break. There are three hydrogen bonds in the GC pair and just two in the AT pair, but hydrogen bond energy differences are not the entire reason for the greater strength of the GC pairs. GC pairs also have greater stacking interaction energy. As a result of the increased hydrogen bonding, the GC pair is more planar, increasing the aromaticity of the dual ring system, accounting for the increase in the stacking interaction.16.3 Supercoiling
Like twisting a coiled phone cord, the DNA helix can exist in supercoils. When the number of strand crossovers increases, the result is called a positive supercoil. Helices that have a decreased number of crossovers have a negative supercoil. Enzymes that catalyze changes in DNA supercoiling are called topoisomerases.Bacteria contain two similar enzymes: topoisomerase I and topoisomerase II. The first catalyzes the hydrolysis of one of the phosphodiester bonds in one chain, moves a DNA strand through the opening, and then rejoins the chain. Hydrolysis of a phosphodiester bond joining the backbone sugars of nucleic acids causes a breakage, commonly called cutting (sometimes whimsically illustrated with miniature scissors). The mechanism of the reaction involves two sequential nucleophilic substitutions, shown in Figure 16.9 . A tyrosine hydroxyl group of the enzyme displaces a portion of the strand, forming an enzyme-bound intermediate. Subsequently, the 5'-OH group of ribose attacks the same phosphate group to rejoin the chain. Except for the change in topology (Figure 16.10
eBook - ePub- Athel Cornish-Bowden(Author)
- 2013(Publication Date)
- Wiley-Blackwell(Publisher)
Chapter 11 Temperature Effects on Enzyme Activity11.1 Temperature denaturation
In principle, the theoretical treatment discussed in Section 1.8 of the temperature dependence of simple chemical reactions applies equally to enzyme-catalyzed reactions, but in practice there are several complications that must be properly understood if any useful information is to be obtained from temperature-dependence measurements.§ 1.8, pages 15–21First, almost all enzymes become denatured if they are heated much above physiological temperatures, and the conformation of the enzyme is altered, often irreversibly, with loss of catalytic activity. Denaturation is chemically a complicated and only partly understood process, and only a simplified account will be given here. In this section I shall limit it to reversible denaturation, assuming that an equilibrium exists at all times between the active and denatured enzyme and that only a single denatured species needs to be taken into account. However, I emphasize that limiting it to reversible denaturation is for the sake of simplicity, not because irreversible effects are unimportant in practice.Denaturation does not involve rupture of covalent bonds, but only of hydrogen bonds and other weak interactions that are involved in maintaining the active conformation of the enzyme. Although an individual hydrogen bond is far weaker than a covalent bond (about 20 kJ · mol−1 for a hydrogen bond compared with about 400 kJ · mol−1 for a covalent bond), denaturation generally involves the rupture of many of them. More exactly, it involves the replacement of many intramolecular hydrogen bonds with hydrogen bonds between the enzyme molecule and solvent molecules. The standard enthalpy of reaction, ΔH 0 ′, is often very high for denaturation, typically 200–500 kJ · mol−1 , but the rupture of many weak bonds greatly increases the number of conformational states available to an enzyme molecule, and so denaturation is also characterized by a large standard entropy of reaction, ΔS 0
eBook - ePub- Subrata Pal(Author)
- 2019(Publication Date)
- Academic Press(Publisher)
The transition from a state with two separate strands to a double helix leads to a decrease in entropy. Yet, formation of double-stranded DNA is thermodynamically favorable over single-stranded DNA. It may be initially thought that the formation of double helix is primarily driven by hydrogen bonds between the paired bases. Nonetheless, prior to pairing, the edges of the unpaired bases are already involved in hydrogen bonding interactions with surrounding water molecules in the aqueous solution. Although the hydrogen bonds between unpaired bases in single-stranded DNA and water are enthalpically less favorable than those between paired bases in double-stranded DNA, annealing between two strands essentially replaces one set of hydrogen bonds with another; the overall contribution of hydrogen bonding to the stability of the double helix is only modest.The major contribution to the stability of the double helix comes from base stacking. The bases are flat and relatively water-insoluble molecules. In the double-stranded structure, they stack over each other almost perpendicularly to the direction of the helical axis. Polar bonds are located on the edges of the bases, whereas their top and bottom surfaces are relatively nonpolar. Hence, when DNA is single-stranded, water forms ordered structures around the bases. Formation of duplex DNA releases the ordered water molecules, resulting in an increase in entropy. Base stacking also contributes enthalpically to the stability of double-stranded DNA by facilitating van der Waals interactions between the instantaneously formed dipoles on the hydrophobic surface of the bases.Major and minor grooves
Although in the double helical structure of DNA the bases project inward, they are accessible through two grooves of unequal width—the major and the minor. To understand the basis of formation of the grooves, let us look at the geometry of the base pairs (Fig. 5.8 ). In each case, the glycosidic bonds that connect the bases to the sugars make an angle (~ 120° on the narrower side or 240° on the wider side) between themselves. As the base pairs stack on top of each other, being rotated by ~ 36° at each step (Fig. 5.7
