Effect of UV Irradiation on DNA as Studied by Its Thermal Denaturation Charles M. Lovetl, Jr.', Thornas N. Fdzgibbon, and Raymond Chang Williams College, Williamstown, MA 01267 Experiments dealing with the thermal denaturation of DNA are becoming increasingly prevalent in biochemistry and biophysics laboratories (1,2).Such experiments effectively introduce students to the nature of DNA secondary structure and are easily carried out with a spectrophotometer equipped with a constant temperature bath. The denaturation, or the helix to coil transition, of double-stranded DNA can be conveniently monitored by the sharp increase in absorbance at 260 nm (the absorption maximum for DNA) that accompanies the cooperative "unzippering" of the helical structure. The nucleotide bases in native DNA are stacked in such a way that the strong interactions of their s electrons suppress the absorption of incident photons. As the molecule denatures. the bases become unstacked. Consequently, there is an increase of about 40% in absorption of curve can be lirht (called the hv~erchrornicshift~.Ameltine: obtained by plotting the increase- in absorh&ce versus the increase in temperature. The temperature corresponding to the midpoint of this curve (the inflection point) is characteristic of each species of DNA and is defined as the melting temperature (T,).The value of T, reflects the stability of the native helix and usually lies hetween 60 O and 100 OC. Besides the inherent simolicitv of the ex~erimentalDrocedure, the pedagogical u k t y bf such a; experiment in a laboratory course is exceptional. It is an excellent example of cooperative phenomena and noncovalent forces, and it illustrates the ao~licabilitvof soectrosco~vin the studv of macromolecul& interactick oreo over,-the experime&al procedure can easily be modified to demonstrate interesting aspects of DNA secondary structure such as the dependence of double helix stability on ionic strength or base pair (that is, AT or GC) content. A convenient and worthwhile addition is to monitor the renaturation process by cooling. The effect of UV radiation on DNA is a phenomenon of great biological importance that can be an informative extension of the thermal denaturation experiment. A principal photoproduct in UV-irradiated DNA is the cyclohutanetype dipyrimidine, or pyrimidine dimer (Fig. 1) (3). This structure disrupts stacking interactions and is unable to form normal hydrogen bonds within a double helix. Failure of a cell to repair a non-base-pairing lesion of this type can lead to mutations in bacteria and probably cancer in higher organisms ( 4 , 5 ) .UV irradiation of DNA in the laboratory
' Corresponding author.
526
Journal of Chemical Education
can add to the value of the thermal denaturation ex~eriment by introducing students to naturally occurring phdtochemical reactions and their effect on DNA structure. It is essen-
Figure 1. Formation of cyciobutanetypeIhymine dimer by UV irradiation
Figure 2. Melting
rnand woling (m) curves ofpolyAT DNA
tially as easy to perform as the standard melting point determination and can he comnleted in a reeular lahoratorv ~ e r i od (3-4 h). The only additional equipment requirei is an inexpensive germicidal lamp. The experiment should be especially implemental in biochemistry and physical chemistry courses and is suitable for both large and small classes.
Figure 3. Schematic representing of polyAT DNA renaturatlon
Experimental
Materials
The sodium salt of polydeoxyadenylic acid-polythymidylie acid (polyAT),a double-stranded homopolymer, was obtained from Sigma Chemical Comnanv and used without further ourification. It is T for two reasons:-(1) The yield of important to use ~ O I ~ ADNA photoproducts is maximized by using DNA containinga polythymidylic acid strand, and (2) the analysis of the results is straightforward due to the uniform hydrogen bonding in this homopolymer. All other chemicals were of reagent grade. Distilled, deionized water was used for all solutions. A Gates Multi-ray lamp equipped with 8W shortwave UV Lamp bulh (available from Fisher Scientific) was used. The germicidal lamp produces UV radiation principally at 254 nm with an intensity of 17 sW/cmZat a distance of 1m. Methods
A stock DNA solution (10 Am unitslmL) was prepared by resuspending lyophilized polyAT in 10 mM Tris (pH 8.0), 1 mM EDTA, and stored at -20 OC. In a typical experiment, the stock DNA solution was diluted to a final concentration of 0.2 Am units/ML with 30 mM sodium eitrate (pH 1.01, 300 mM NaCI. A 2.0-mL sample of the DNA solution was transferred to a 50-mL beaker, immersed in an ice-water hath, and irradiated at a distance of 8 em beneath the lamp; the times of irradiation varied between 0 and 2400 s. After irradiation, the sample was equilibrated at 25 OC and its absorbance at 260 nm was measured with a Cary 219 spectrophotometer containinga water-jacketedcuvette carouselconnectedto a Lauda 2-RK circulating water hath. Absorbance values were recorded as the sample was heated to 85 OC and then cooled to 25 "C. Results and Dlscusslon
Figure 2 shows the melting and cooling curves for polyAT DNA a t 0.3 M NaC1, p H 7.5. The T,, as determined by the infection point of the melting curve, is 82.4 OC. Thermal denaturation results in a 44% increase in absorhance at 260 nm corresponding to the hyperchromic shift. The cooling curve has a n inflection point a t 67.5 OC and shows a slight decrease in sigmoidicity. The difference betweenthe melting and cooling curves is partly due t o kinetic effects on the renaturation sten: for volvAT DNA. i t is likelv t h a t the formation of the-irst few gydrogen bonds, or nuheation, is rate-limiting. Subsequent zipping of the two strands probably proceeds instantly. Upon cooling, the absorbance of the DNA sample nearly returns to the original value suggesting t h a t the denaturation process is reversible. However, the
rn
Figure 4. Menlng Irradiation for 1200 s.
and cooling (m) curves of polyAT DNA
following W
process is not actually reversible. Because the individual strands are homopoly&ers, nucleation can occur at any region within polyA and polyl'molecules as shown in Figure 3. The duplex molecules thus formed would he long aggregates comprised of several strands. This model is supported by the fact that the averaee leneth of renatured ~olvA'l' 1)NA is considerably larger-than &at of the u n m e l & d ~ (deter~ ~ mined hv electronhoresis: data not shown). Similar behavior has been observed using hydrodynamic methods for the renaturation of polyI-polyd5BrC (6). Because the average length of continuously base-paired DNA in its renatured form is less than in its native form (compare Fig. 3c with Fig. 3a), the process would he less cooperative. The decrease in sigmoidicity observed during cooling must also reflect this decrease in cooperativity. Representative melting and cooling curves for polyAT DNA after UV irradiation for 1200 s are shown in Figure 4. PolyAT DNA irradiated for 1200 s has a T, of 76 "C, and there is a 41% increase in absorbance a t 260 nm. In eeneral. as the UV dose increases both T, and the hyperEhromic shift decrease. For examnle. followinr! 2400 s of irradiation the T, is 69 OC and the absorbance increase is 38% (data not Volume 66
Number 6
June 1969
527
perativity, it is important to realize that for polyAT DNA s (where all base airi in^ is identical) coonerativitv d e ~ e n don the length of a ~ontiniousstretchofha;epaired D N A . T ~ U ~ interru~tiono f d u ~ l e xDNA bv random dimer formation will decrease the aveiage length- of continuously base-paired TINA U A . . . .
65
I
! 0
1000
2000
3000
UV Irradiation time (s) Figure 5. Effect of UV inadlation time on T,, of polyAT DNA
shown). Figure 5 shows that over a wide range of UV fluences there is roughly a linear relationship between T, and irradiation time. In addition, as UV dose increases, a marked decrease in cooperativity can be inferred from the obvious decrease in sigmoidicity shown in melting and cooling curves. All of the effects of UV irradiation on polyAT DNA are nreciselv as expected and easily explained. The formation-of a dimer between adjacent thymines produces a strain in the helix that disrupts about four base pairs (7,8); the number of dimers formed should be . nro~ortional to irradia. tion time. Because the disruption of base-pairing decreases the stabilitv of the helix. the T, must decrease as the number of dimers increases. The decrease in hyperchromic shift with increasine UV dose is due to the increase in initial absorbance caised by the partially exposed bases in the dimer regions. In order to understand the decrease in coo-
528
Journal of Chemical Education
The results of these relatively simple experiments clearly demonstrate the cooperative nature of the helix to coil transition of DNA and the effect of UV irradiation (that is, the dimerization of adjacent thymines) on T,. Moreover, they can serve as an introduction to spectroscopy and photochemical reactions. Depending on the number of students in a laboratorv section. each erouD can be assiened two to three irradiationstudies A d t h k r results can b e pooled together for discussion. We would like to suggest the following additional experiments which can be performed by different groups of students: (1) Determination of T, in solutions of different ionic strength; T, increases with ionic strength because the secondary structure of DNA is stabilized by ions in solution (9, 10). (2) Determination of T, for naturally occurring DNA containing all four bases provides an example of the relative strength of base pairs (i.e., GC pairing involves three hydrogen bonds, whereas AT only involves two). Furthermore. the use of such DNA illustrates the irreversibility of the cooling process because natural DNA does not show a significant hypochromic shift when cooled. Literature Clted 1. Alexaoder,R.R.;Gtiffiths, J.M.: Wilhinmn,M. L.BnaicBimheminrlMethdod; Wiley: New York, 1 9 8 5 , ~89. 2. Boyer. R. F. Modern Ezperimenlol Bbehemhfry; Addison-Wesley: Reading, MA,
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