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Thecloser the evolutionary relationship between the species, the more extensively will their DNAs hybridize. For example, human DNA hybridizes much more extensively with mouse DNA than with DNA fromyeast.The hybridization of DNA strands from different sources forms thebasis of a powerful set of techniques essential to the modern practice ofmolecular genetics. It is possible to detect a specific DNA sequence orgene in the presence of many other sequences if one already has anappropriate complementary DNA strand (usually labeled in some way)to hybridize with it (Chapter 28).
The complementary DNA can be froma different species or from the same species; in some cases it is synthesized in the laboratory, using techniques described later in this chapter. Hybridization techniques can be varied to detect a specific RNArather than DNA. The isolation and identification of specific genes andRNAs relies on these techniques, and new applications of this technology are making it possible to accurately identify an individual on thebasis of a single hair left at the scene of a crime or predict the onset ofsome diseases in an individual decades before symptoms appear (seeBox 28-1).Duplex ofspecies 1HybridduplexDuplex ofspecies 2Figure 12-31 Principle of the hybridization test.Two DNAs from different species are completelydenatured by heating. When mixed and slowlycooled, complementary DNA strands of each specieswill associate and anneal to form normal duplexes.If the different DNAs have significant sequencehomology, they will tend to form partial duplexesor hybrids with each other: the greater the sequence homology between the two DNAs, thegreater the number of hybrids formed.
Hybrid formation can be measured by different procedures,such as chromatography or isopycnic centrifugation.Usually one of the DNAs is labeled with a radioactive isotope to simplify the measurements.Part II Structure and Catalysis344Nucleotides and Nucleic Acids UndergoNonenzymatic TransformationsDeaminationCH3HypoxanthineO(a)Purines and pyrimidines, along with the nucleotides of which they area part, undergo a number of reactions involving spontaneous alteration of their covalent structure.
These reactions are generally veryslow, but they are physiologically significant because of the cell's verylow tolerance for alterations in genetic information. Alterations inDNA structure that lead to permanent changes in the genetic information encoded therein are called mutations, and much evidence suggests an intimate link between the accumulation of mutations and theprocesses of aging and cancer.Several bases undergo spontaneous loss of their exocyclic aminogroups (deamination) (Fig.
12-32a). For example, under conditionsfound in a typical cell, deamination of cytosine (in DNA) to uracil willoccur in about one of every 107 cytosines in 24 h. This corresponds toabout 100 spontaneous events per day in an average mammalian cell.Deamination of adenine and guanine is about 100 times slower.The slow cytosine deamination reaction seems innocuous enough,but it is almost certainly the reason why DNA contains thymine ratherthan uracil. The product of cytosine deamination (uracil) is readilyrecognized as foreign in DNA and is removed by a repair system (Chapter 24).
If DNA normally contained uracil, recognition of uracils resulting from cytosine deamination would be more difficult, and unrepaireduracils would lead to permanent sequence changes as they were pairedwith adenines during replication. Cytosine deamination would gradually lead to a decrease in G=C base pairs and an increase in A=U basepairs in the DNA of all cells. Over the millennia, the cytosine deamination reaction could eliminate G=C base pairs and the genetic code thatdepends on them. Establishing thymine as one of the four bases inDNA may well have been one of the key turning points in evolution,making the long-term storage of genetic information possible.DepurinationOHH2NO"-O—P—O—CH 2 . 0OHApurinicresidueGuanosine residue(in DNA)(b)Figure 12—32 Some well-characterized reactions ofnucleotides.
(a) Deamination reactions. Only thebase is shown, (b) Depurination, in which a purineis lost by hydrolysis of the iV-glycosyl bond. Thedeoxyribose remaining after depurination is readilyconverted from the /3-furanose to the aldehyde form(see Fig.
12-3).Another important reaction in deoxynucleotides is the hydrolysisof the glycosyl bond between the base and the pentose (Fig. 12-32b).This occurs much faster for purines than for pyrimidines. In DNA asmany as one in 105 purines (10,000 per mammalian cell) are lost every24 h under typical cellular conditions. Depurination of ribonucleotidesand RNA is much slower and generally is not considered physiologically significant. In the test tube, loss of purines can be accelerated bydilute acid.
Incubation of DNA at pH 3 causes selective removal of thepurine bases, resulting in a derivative called apurinic acid.Chapter 12 Nucleotides and Nucleic Acids345AdjacentthyminesKink11(b)HCH 36-4 PhotoproductCyclobutane thymine dimer(a)Other reactions are promoted by certain types of radiation. In thelaboratory, UV light will induce the condensation of two ethylenegroups to form a cyclobutane ring. In the cell, the same reaction occursbetween adjacent pyrimidine bases in nucleic acids to form cyclobutane pyrimidine dimers. This happens most frequently between adjacent thymine residues on the same DNA strand (Fig.
12-33). A secondtype of pyrimidine dimer formed during UV irradiation, called a 6-4photoproduct, is also shown in Figure 12-33. Ionizing radiation (x raysand gamma rays) can cause ring opening and fragmentation of basesas well as breaks in the covalent backbone of nucleic acids.Virtually all forms of life are exposed to energy-rich radiation capable of causing chemical changes in DNA. UV radiation (having wavelengths of 200 to 400 nm), which makes up a significant portion of thesolar spectrum, can cause pyrimidine dimer formation and other chemical changes in the DNA of bacteria and of human skin cells. There is aconstant field of ionizing radiation around us in the form of cosmicrays, which can penetrate deep into the earth, as well as radiationemitted from radioactive elements, such as radium, plutonium, uranium, radon, 14C, and 3 H.
X rays used in medical or dental examinations and in radiation therapy of cancer and other diseases are anotherform of ionizing radiation. It is estimated that UV and ionizing radiations are responsible for about 10% of all DNA damage caused by nonbiological agents.DNA also may be damaged by reactive chemicals introduced intothe environment as products of industrial activity. Such products maynot be injurious per se, but may be metabolized by cells into forms thatare.
There are three major classes of such reactive chemical agentsFigure 12-33 Formation of thymine dimers induced by UV light, (a) The reaction on the left results in the formation of a cyclobutyl ring involvingC-5 and C-6 of each thymine residue, just as UVlight will induce the formation of cyclobutane fromtwo molecules of ethylene. An alternative lightinduced reaction between adjacent thymines resultsin a linkage between C-6 of one residue and C-4 ofits neighbor, as shown on the right.
A bend or kinkis introduced into the DNA on formation of a cyclobutane thymine dimer (b).346Part II Structure and CatalysisNaNO 2Sodium nitriteCH 3N—N=OCH 3DimethylnitrosamineNaNO 3Sodium nitrateH5-BromouracilCH3oVR1N—N=00oOCH3DimethylsulfateNitrosaminesHNCH2 —CH2 —ClNitrogen mustard(b)(a)NNCH2 —CH2 —ClH2NN2-Aminopurine(c)Figure 12-34 Chemical agents that cause sometypes of DNA damage, (a) Nitrous acid precursors.(b) Alkylating agents, (c) Base analogs.HNH 2 N' TNT"NrlGuaninetautomersH 2 N' ^N" JjO 6 -MethylguanineH 2 N ' "1S(Fig. 12-34): (1) deaminating agents, particularly nitrous acid (HNO2)or compounds that can be metabolized to nitrous acid or nitrites, (2) alkylating agents, and (3) compounds that can simulate or mimic thenormal bases present in DNA.Nitrous acid, formed from organic precursors such as nitrosaminesand from nitrite and nitrate salts, is a potent reagent that acceleratesthe deamination of bases described above.
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