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Bisulfite has similar effects.Both agents are used as preservatives in processed foods to prevent thegrowth of toxic bacteria. They do not appear to significantly increasecancer risks when used in this way, perhaps because they are used insmall amounts and their contribution to the overall levels of DNA damage is minor. (The potential health risk from food spoilage if thesepreservatives were not used is much greater.)Alkylating agents can alter certain bases of DNA.
For example, thehighly reactive chemical dimethylsulfate (Fig. 12-34b) can methylatea guanine residue to yield O6-methylguanine, which is unable to basepair with cytosine. Many similar reactions are brought about by alkylating agents normally present in cells, such as S-adenosylmethionine(see Fig. 17-20) and other compounds.Possibly the most important source of mutagenic alterations inDNA is oxidative damage. Excited-oxygen species such as hydrogenperoxide, hydroxyl radicals, and superoxide radicals arise during irradiation or as a byproduct of aerobic metabolism. Cells possess an elaborate defense system to destroy these reactive species, including enzymes such as catalase and superoxide dismutase.
A fraction of theseoxidants inevitably escapes cellular defenses, however, and damage toDNA involves a large, complex group of reactions ranging from oxidation of sugar and base moieties to breaking strands. Accurate estimates for the extent of this damage are not yet available, but it is clearthat each day the DNA in each human cell is subject to thousands ofdamaging oxidative reactions.This is merely a sampling of the best-understood reactions. Manycarcinogenic compounds present in food, water, or air exert theircancer-causing effects by modifying bases in DNA. In the cell, the integrity of DNA as a polymer is nevertheless maintained better thanthat of either RNA or protein, because DNA is the only macromoleculehaving biochemical repair systems.
These repair processes (describedin Chapter 24) greatly lessen the impact of damage to DNA.Chapter 12 Nucleotides and Nucleic Acids347DNA Is Often MethylatedCertain nucleotide bases in DNA molecules are often enzymaticallymethylated. Adenine and cytosine are methylated more often thanguanine and thymine.
Methylation of these bases is not random but isgenerally confined to certain sequences or regions of a DNA molecule.In some cases the function of methylation is well understood; in othersthe function is still unclear. All known DNA methylases use S-adenosylmethionine as a methyl group donor. In E. coli there are two prominent methylation systems. One serves as part of a cellular defensemechanism that helps to distinguish the cell's own DNA from foreignDNA (restriction modification, described in Chapter 28). The other system methylates adenine to A^-methyladenine (see Fig. 12-5a) withinthe sequence (5')GATC(3').
This is mediated by an enzyme called theDam methylase, which functions as part of a system that repairs mismatched base pairs formed occasionally during DNA replication(Chapter 24).In eukaryotic cells, about 5% of cytosine residues are methylated toform 5-methylcytosine (see Fig. 12-5a). Methylation is most commonat CpG sequences, producing methyl-CpG symmetrically on bothstrands of the DNA. The extent of methylation of CpG sequences variesin different regions of large eukaryotic DNA molecules, and is ofteninversely related to the degree of gene expression.
These methylationshave structural as well as regulatory significance. The presence of5-methylcytosine in an alternating CpG sequence markedly increasesthe tendency for that sequence to take up the Z conformation.GLong DNA Sequences Can Be DeterminedIn its capacity as a repository of information, the most important property of a DNA molecule is its nucleotide sequence. Until the late 1970s,obtaining the sequence of a nucleic acid containing even five or tennucleotides was difficult and very laborious. The development of twonew techniques in 1977, one by Alan Maxam and Walter Gilbert andthe other by Frederick Sanger, has made it possible to sequence everlarger DNA molecules with an ease unimagined just a few decades ago.The techniques depend upon an improved understanding of nucleotidechemistry and DNA metabolism, and on electrophoretic methods thatallow the separation of DNA strands differing in size by only one nucleotide.
Electrophoresis of DNA is similar to the electrophoresis of proteins (see Fig. 6-4). Polyacrylamide is often used as the gel matrix forshort DNAs (up to a few hundred nucleotides). Agarose is generallyused as the gel matrix for separating longer DNAs.In both Sanger (dideoxy) and Maxam-Gilbert sequencing, the general principle is to reduce the DNA to be sequenced to four sets oflabeled fragments. The reaction producing each set is base-specific, sothat the lengths of the fragments correspond to positions in the DNAsequence where a certain base occurs.
For example, for an oligonucleotide with the sequence pAATCGACT, a reaction that produces onlyfragments ending in C will generate fragments four and seven nucleotides long, whereas a reaction producing fragments ending in G willproduce only a five-nucleotide fragment.
The fragment sizes correspond to the relative positions of C and G residues in the sequence.When the sets of fragments corresponding to each of the four bases areelectrophoretically separated side by side, they produce a ladder ofbands from which the sequence can be read directly (Figs. 12-35, 12-Figure 12-35 Section of an autoradiogram produced by the method developed by Sanger and colleagues. Side-by-side electrophoresis of the DNAfragments generated by each dideoxynucleotide generates a ladder of bands. Each band on the filmcorresponds to a population of DNA fragments of aspecific length produced in the sequencing reactions(see Fig.
12-36). The identity of the base at eachposition in the sequence is determined from thelane in which a band is observed; the order of thebands read from the bottom of the gel correspondsto the DNA sequence.348Part II Structure and CatalysisFigure 12—36 DNA sequencing by the Sanger(dideoxy) method. This method makes use of themechanism of DNA synthesis by DNA polymerases(Chapter 24). DNA polymerases require both aprimer, to which nucleotides are added, and a template strand to guide selection of each new nucleotide (a).
The 3'-hydroxyl group of the primer reactswith the incoming deoxynucleoside triphosphate(dNTP), forming a new phosphodiester bond. TheSanger sequencing procedure usesdideoxynucleoside triphosphate (ddNTP) analogs(b) to interrupt DNA synthesis. When the dNTP isreplaced by the ddNTP, strand elongation is haltedafter the analog is added because it lacks the 3'hydroxyl group needed for the next step. The DNAto be sequenced is used as the template strand, anda short primer (usually radioactively labeled) isannealed to it (c). By adding small amounts of asingle ddNTP, for example ddCTP, to an otherwisenormal reaction system, the synthesized strandswill be prematurely terminated at locations wheredC normally occurs. Because there is much moredCTP than ddCTP, there is only a small chancethat the analog will be incorporated whenever adC is to be added, but there is generally enoughddCTP that each new strand has a high probabilityof acquiring one ddC at some point during synthesis.
The result is a solution containing fragmentsrepresenting each C residue in the sequence. Thesize of the fragments, separated by electrophoresis,reveals the location of C residues in the sequence.This procedure is repeated separately for each ofthe four ddNTPs, and the sequence can be readdirectly from an autoradiogram of the gel (c). Because shorter DNA fragments migrate faster, thefragments near the bottom represent the nucleotidepositions closest to the primer (the 5' end), and thesequence is read from bottom to top. Note that thesequence obtained is that of the strand complementary to the strand being analyzed.
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