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.
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10 Key excerpts on "Denaturation of DNA"
- Thomas M. Devlin(Author)
- 2015(Publication Date)
- Wiley-Liss(Publisher)
Figure 2.16 Migration of bubbles through double-helical DNA. DNA contains short open-stranded sections of DNA that can “move” along the helix. Figure 2.17 Denaturation of DNA. At high temperatures the double-stranded structure of DNA is completely disrupted, with eventual separation of strands and formation of single-stranded open coils. Denaturation also occurs at extreme pH ranges or at extreme ionic strengths. Renaturation (fast) Denaturation Renaturation (slow) Denaturation Random association CHAPTER 2 DNA AND RNA: COMPOSITION AND STRUCTURE • 37 DNA becomes denatured at pH 11.3 as the N–H groups on the bases become depro- tonated, preventing them from participating in hydrogen bonding. Alkaline denaturation is often used to prevent damage to the DNA that can occur at a high temperature or low pH. Denaturation can also be induced at low ionic strengths, because of enhanced inter- strand repulsion between negatively charged phosphates and by various denaturing agents (compounds that can effectively hydrogen bond to the bases while disrupting hydrophobic stacking interactions). A complete denaturation curve similar to that shown in Figure 2.18 is observed at a relatively low constant temperature by variation of the concentration of an added denaturant such as urea. Complementary DNA strands, separated by denaturation, can reform a double helix if appropriately treated. This is called renaturation or annealing. If denaturation is not complete and a few nucleobases remain hydrogen bonded between the two strands, the helix-to-coil transition is rapidly reversible. Annealing is possible even after complementary strands have been completely separated. Under these conditions the renaturation process depends on the DNA strands meeting in a manner that can lead to reformation of the origi- nal structure. Not surprisingly, this is a slow, concentration-dependent process.
eBook - ePub- John A. M. Gall, Jason Payne-James, John A. M. Gall, Jason Payne-James(Authors)
- 2016(Publication Date)
- Wiley-Blackwell(Publisher)
DNA can be damaged in two main ways: through mechanisms that break the hydrogen bonding between the complementary strands, destroying the double helix (DNA denaturation), and by mechanisms that break the sugar phosphate backbone (DNA damage) producing single‐ and double‐stranded breaks, or through other interactions with the molecule, which distort the molecule or remove the bases. It is these latter damaging mechanisms that are more important when wanting to destroy (decontaminate) DNA, shortening or damaging the template molecule so that it cannot be amplified to produce a profile. Separation of the molecule into two strands through denaturation can also increase the vulnerability of the molecule to backbone damage.DNA denaturationHydrogen bonds are relatively weak, allowing the two strands to be pulled apart. This is a necessary process in DNA replication, but the strands can also be separated by mechanical means and by heat. The temperature at which this latter takes place is known as the melting temperature. Three main factors affect this temperature: the GC content, because it is harder to break three hydrogen bonds than two; the salt content, because Na+ ions interact with the negatively charged phosphate ions in the DNA backbone, adding stability by shielding the charge repulsion between the backbone molecules; and the length of the molecule, because the more hydrogen bonds to be broken, the more difficult it is to separate the two strands easily.Chemical denaturants can also break this hydrogen bonding; there are three main mechanisms. Chemicals that raise the pH (e.g., sodium hydroxide) will pull the hydrogen ions away from the nitrogenous base, thus removing the ability for the two strands to bond. Competitive denaturation can occur with some chemicals (e.g., urea and formaldehyde) as these compete with hydrogen ions to bond with the electronegative nitrogenous bases, particularly at high chemical concentrations. Some aldehydes (e.g., formaldehyde) can also form covalent bonds with the electronegative nitrogenous bases and block the formation of hydrogen bonds between complementary strands.DNA damageDNA, even if protected within a cell, can still be damaged by many different physical or chemical agents. While some are endogenous, formed inside the cell through normal metabolic pathways, others are exogenous, coming from the external environment. This damage may be introduced unintentionally or intentionally. It is for the latter purpose (decontamination) that particular agents are considered next.
eBook - PDF- Ilya Prigogine, Stuart A. Rice(Authors)
- 2009(Publication Date)
- Wiley-Interscience(Publisher)
Numerous reviews have sum- marized experimental work in the field.2-5 Theoretical investigations of DNA denaturation have attempted to explain the helix-coil transition through molecular models so that both a qualitative and quantitative understanding of the process can be derived. Current studies have been concerned with the unwinding mechanism of DNA,6*7.13 the therniodynamic energies involved in stabilizing the helix,8ss and base distribution information of DNA.l0-I4 In recent years several review articles on the theory of denaturation have been and a book16 has now appeared. The internal stability of the DNA double helix appears to be due partly to hydrogen bonding between members of the base pairs in the two strands. Each base pair bond, or bonding complex, includes solvent effects, the hydrogen bonds between the complimentary bases (adenine- thymine and guanine-cytosine) and the stabilizing forces resulting from the interaction of adjacent base pairs. The latter “stacking forces” not DNA DENATURATION 131 T, O C Fig. 1. Helix coil transition of M. lysodeikticus DNA in 0.1 SSC (0.015 NaCl + 0.0015M sodium citratcpH = 7.0). only confer stability to the base pair bonds but also correlate adjacent base pairs. This correlation, along with other long-range forces, account for the cooperative nature of the helix-to-coil transition. Experimentally, the DNA denaturation usually is observed by measur- ing the ultr~violet absorbance of a dilute solution of DNA as a function of temperature. A curve of the fraction of broken bonds versus temperature is commonly obtained by monitoring optical absorbance at 260 milli- microns. An increase in the absorbance of light reflects a change in the electron corifiguration of the bases due to the breaking of base pair bonds. Assuming the change in absorbance to be proportional to the fraction of broken bonds, we can calculate a melting curve.
eBook - PDF- Hsueh Jei Li(Author)
- 2012(Publication Date)
- Academic Press(Publisher)
Chapter 2 HISTONE-DNA INTERACTIONS: THERMAL DENATURATION STUDIES HSUEH JEI LI Division of Cell and Molecular Biology State University of New York at Buffalo Buffalo, New York 14214 I. Introduction Both histones and DNA are macromolecules. Their inter-actions are complex and should be examined extensively from all possible angles, using various techniques. DNA is the central molecule in chromatin and all protein*DNA complexes. Any physical chemical method which can reveal information about DNA will have its potential application in the studies of protein-DNA interactions, such as those of histone'DNA in chromatin or of other histone-DNA complexes. Physical methods used in studies of nucleic acids include thermal de-naturation, circular dichroism (CD), nuclear magnetic reson-ance (NMR), x-ray diffraction, viscosity, sedimentation, electron microscopy and neutron diffraction, all of which have been applied to chromatin and histone-DNA complexes. In this chapter, the use of thermal denaturation in the inves-tigation of histone-DNA interactions will be discussed. II. Thermal Denaturation in DNA Denaturation of a double-helical DNA is accompanied by an increase in absorbance. This phenomenon is called hyper-chromism and is attributed to the destruction of stacking interaction among nucleotides in DNA (1-4). Denaturation of DNA can occur by adding acid or base to the solution, or by raising its temperature. In the latter case, it is called thermal denaturation. It was observed that Denaturation of DNA occurs in a small temperature range (5). The tempera-ture at which 50% of native DNA is denatured is called the melting temperature (T m ) of the DNA. The T m is a linear function of the G + C (guanine + cytosine) content of the 37 HSUEH JEI LI DNA (5) and is greatly increased in a solution of higher ionic strength (5-11). Statistical thermodynamics has been used successfully in treating helix-coil transition in DNA (12-16).
eBook - ePub- Michael J Waring(Author)
- 2018(Publication Date)
- Royal Society of Chemistry(Publisher)
Chapter 4Thermal Denaturation of Drug–DNA Complexes†Jonathan B. Chaires James Graham Brown Cancer Center, University of Louisville, 505 S. Hancock St., Louisville, KY 40202, USAEmail: [email protected]4.1 Introduction
Only a few months after Watson and Crick presented their model for the DNA double helix,1 the alkaline and thermal Denaturation of DNA, as monitored by changes in UV absorbance, was reported.2 This seminal work explicitly recognized that the observed hyperchromism resulted from “…the destruction of a secondary molecular structure constituted by labile bonds involving the puric and pyrimidic rings”, and specifically referred to the Watson–Crick structure. Soon after, the Doty laboratory published a remarkable and prescient series of studies on the acid, alkaline and thermal Denaturation of DNA, employing a wide array of biophysical tools to show that the duplex strands separated upon denaturation.3 –10That line of research lead directly to the well-known paper by Marmur and Doty11 that related the DNA melting temperature (T m ) to the GC content of the DNA. Concurrently, statistical mechanical theories for the effect of preferential ligand binding on the helix-to-coil transition were developed,12 ,13albeit with explicit reference to protein transitions rather than to DNA melting. These theories were general, however, and were equally applicable to DNA transitions. Only five years after the appearance of the Watson–Crick model, calorimetry was used to study the enthalpy of the acid Denaturation of DNA.14Against this background, thermal denaturation became a commonly used tool for the study of drug–DNA complexes from the early 1960s onward. The attraction of melting studies lies in their simplicity and readily available, inexpensive, instrumentation. Melting provides a simple and unambiguous demonstration of drug binding to DNA. Small molecules that bind preferentially to the DNA duplex stabilize the structure and elevate its T m . Intercalators and groove-binders both recognize particular features of duplex DNA, and consequently raise its Tm . Apart from a simple qualitative demonstration of binding to DNA, melting studies can be analyzed to obtain quantitative information about the binding interaction. A variety of approaches for such quantitative analysis have appeared over the years,15 –19and new approaches continue to be developed.20
eBook - PDF- Luis Vazquez, A R Bishop, S Jimenez(Authors)
- 1995(Publication Date)
- World Scientific(Publisher)
First sudies of energy transfer in an inhomogeneous DNA molecule have been presented by Techera et al. 5 and Muto 6 , while first attempts to consider the lo-cal denaturation of an inhomogeneous ring-shaped DNA molecule were presented by Muto 7 9 . The molecule of DNA is a double helix built from two antiparallel linear poly-mers, built from four different monomers (nucleotides) and the base in one side is complementary to the base in the other side. Each monomer is built from a constant part (phospate and desoxiribose groups) and a variable part, being this last one of the four bases: adenine, guanine, thymine and cytosine. Adenine and guanine are purines and are larger than the two pyrimidic bases, thymine and cytosine. Guanine (G) is associated to cytosine (C), being linked by three hydrogen bonds. Adenine (A) is associated to thymine (T), being linked by two hydrogen bonds. We will consider C and G as strong bases (5), and A and T as the weak ones (w). From a biochemical point of view, the denaturation occurs when the two strands of the DNA helix readily come apart, because the hydrogen bonds between its paired 132 bases are disrupted. This can be accomplished by heating a solution of DNA or by adding acid or alkali to ionize its bases. The denaturation temperature depends markedly on its base composition. Here, we are mainly interested in the dynamics of inhomogeneous circular strands of DNA, with particular attention to the open state distribution in the different regions of the DNA strand. DNA sequences of the following type are considered XXXX . .. XXYYY ... YYYXX ... XXXX , where X stands for W, S or M type (Af corresponds to the averaged value of a homogeneous chain, and when it refers to the full homogeneous chain the symbol H is used), and YYY...YYY represents altemances of the following types: SWW, SSWW and randoms.
eBook - PDF- Alexander Vologodskii(Author)
- 2015(Publication Date)
- Cambridge University Press(Publisher)
An interesting version of DNA melting is observed after large temperature jumps, beyond the melting interval (Spatz & Crothers 1969). During the first stage a certain number of melted regions form very quickly, without unwinding of the denatured regions. This stage resembles the melting of circular DNA molecules, where unwinding of complementary strands is impossible owing to topological restrictions (see Section 6.5.3.3). Such melting without strand unwinding decreases the entropy of the denatured state. Therefore, it becomes possible only at temperatures that are much higher than the equilibrium melting temperatures. During the second, much slower stage of the process, shrinking of remaining helical regions follows unwinding of the melted regions. Renaturation of long melted DNA molecules has even more obstacles. First, the concentration of the complementary strands is low and it slows down the helix nucleation. Second, when the temperature is decreased, a significant number of intramolecular hairpins form quickly. Although the free energy of conformations with these hairpins is much higher than the free energy of the perfect duplexes, they represent a kinetic obstacle for the duplex formation, especially at lower temperatures. Third, zipping of the complementary strands requires their rewinding, which is a slow process as well owing to the friction. Since formation of intramolecular hairpins precedes the zippering, they have to be denatured during the zippering stage of the process. Clearly, the hairpin denaturation is slower at lower temperature, and it slows down the entire process. On the other hand, a certain stability of regular double-stranded segments is needed for efficient zippering. As a result, the renaturation rate reaches its maximum at temperatures that are below T m by 20–30 °C (Fig. 4.8) (Marmur & Doty 1961, Wetmur & Davidson 1968).
eBook - PDF- J.S. Willis(Author)
- 1997(Publication Date)
- Elsevier Science(Publisher)
The lamellar-to-hexagonal transition often occurs at high temperatures, which makes it attractive as a possible rate limiting transition, but there is no evidence that it is important in hyperthermic killing, although it has not been investigated in detail. Order-disorder transitions also occur in DNA and RNA. The main transitions in DNA are unlikely to play a role in killing since they are normally reversible and occur at high temperatures (in the range of 85-90 C). However, localized melting of destabilized regions could be im- portant. Structured RNA, such as double stranded regions, undergoes a melting transition similar to DNA. Likely RNA targets are tRNA and other forms of compact RNA such as rRNA and the small, nuclear ribonucleoprotein (snRNP) complexes. Very little is known about the stability of RNA, but isolated yeast tRNAphe melts through four transi- tions over the temperature range of 30-90 C (Biltonen and Freire, 1978). With the recent discoveries of the large number of enzymatic functions performed by RNA and its likely structural role in many protein-RNA complexes, it must be considered as a potential target much like protein. The most likely target for heat shock is protein. The denaturation or partial unfolding of proteins during heat exposure, resulting in damage due to the inactivationof crucialcellularfunctionsor the disruptionof cellularstructures, is most consistent with the large body of experimental observations. The process of protein denaturation and its detection in cells is discussed below. IV. PROTEIN DENATURATION A. Molecular Characteristics of Protein Denaturation Proteins undergo an order-disorder transition from the native to the unfolded or denatured state referred to as protein denaturation. This
- J. A. V. Butler, J. T. Randall(Authors)
- 2016(Publication Date)
- Pergamon(Publisher)
It has often been considered that denaturation is analogous to the transformation from the crystalline to the amorphous state, although the process of denaturation appears rather to be the modification of a highly specific structure to give rise to a more random arrangement. (19) ' (40) Such a change in the molecular configuration allows, for instance, the exposure of SH groups, which may be unavailable due to steric hindrance in the native protein. (81) As we have already seen, changes in particle size, caused by dissociation or aggregation, are the most direct manifestation of a structural transformation. Dissociation is quite a frequent phenomenon. For instance, at room temperatures and outside thepH range 4-9, the serum albumin molecule splits into smaller particles, the first step (into half molecules) being reversible, while on heating amandin, part of the molecule dissociates into very small particles. (24) Nevertheless, practically nothing is known about the mechanism of such behaviour. As explained above, the mechanism of aggregation has been more fully elucidated, particularly in the case of insulin. (106) ' (126) > (151) » (218) Here each molecule undergoes only small structural changes during fibril formation, the globular units being reversibly linked endwise; the heat of dissociation falls with increase of charge. The variation of 209 REVERSIBLE DENATURATION OF PROTEINS viscosity of serum albumin with ρΉ. in acid media has been inter-preted^ 2 ^ in terms of reversible associations: A -f H + ^ ± A H + and nXK+ ^± (AK + )n. More generally, it has been shown that, if denatura-tion is accompanied by aggregation, the apparent (directly measured) free energy of activation includes a term corresponding to the proba-bility of the particles colliding and aggregating, so that the free energy of activation of the true denaturation may be 3 or 4 times smaller.
eBook - PDF- M Volkenstein(Author)
- 2012(Publication Date)
- Academic Press(Publisher)
Thus, careful investigation of the melting curves of DNA yields valuable information about its structure. This method may also be useful in RNA studies. Lifshitz studied theoretically the dependence of the form of the DNA melting curve on the sequence of the pairs A-T and G-C [108]. He showed that in an ideal case the investigation of this curve can yield valuable information about the DNA FIG. 8.20 Dependence of the relative changes in [x] (closed circles) and in the optical density (open circles) on Τ at 0.075M. (1) Herring sperm DNA; (2) T2 phage DNA; (3) calf thymus DNA; (4) Ε. coli DNA. 8.4 The Thermodynamics of Helix-Coil Transitions 517 FIG. 8.21 Dependence of the relative changes in g on the fraction of melted base pairs. (1) Herring sperm DNA; (2) calf thymus DNA; (3) Ε. coli DNA; (4) T2 phage DNA. primary structure. However, the precision of the current ex-periments is not sufficient for such investigation. The thermodynamic parameters of the Denaturation of DNA were determined microcalorimetrically by Privalov [94,109,110]. Heat of denaturation depends strongly on the pH. If the pH in-creases from 7.0 to 9.7, T m decreases from 84.8°C to 66.3°C, ΔΗ from 9650 to 7140 cal mole* 1 , and AS from 27 to 21 eu. If the pH decreases from 5.4 to 3.2, T m decreases from 84°C to 55°C f ΔΗ from 9400 to 4000 cal mole 1 , and AS from 25.6 to 12.4 eu. T m changes considerably with ionic strength, whereas ΔΗ depends neither on it nor on the temperature. The difference in the free energies of denatured and native DNA at 37°C decreases from 1250 to 620 cal mole 1 if the pH increases from 7.0 to 9.7, and from 1250 to 220 cal mole 1 if the pH decreases from 5.4 to 3.2. At pH 7.0 AG decreases from 1250 to 710 cal mole 1 if the pNa increases from 0.84 to 2.04. The free energy of stabiliza-tion of the native DNA structure depends linearly on NaCl ac-tivity.
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