Many mutations do not result from errors in replication. Chemical reagents can oxidize and alkylate the bases in DNA, sometimes changing their base-pairing properties. Radiation can also damage DNA. Examples of these mutagenic reactions will be discussed in this section.
Chemical Modification by Oxidation
When the amino bases, adenine and cytosine, are oxidized, they also lose an amino group. Thus the amine is replaced by a keto group in the product of this oxidative deamination reaction. For instance, oxidation of cytosine produces uracil, which base pairs with adenine (shown for deoxycytidine in Figure 7.6). Likewise, oxidation of adenine yields hypoxanthine, which base pairs with cytosine (Figure 7.7.A). Thus the products of these chemical reactions will be mutations in the DNA, if not repaired. Oxidation of guanine yields xanthine (Figure 7.7.B). In DNA, xanthine will pair with cytosine, as does the original guanine, so this particular alteration is not mutagenic.
Figure 7.6. Oxidative deamination of deoxycytidine yields deoxyuridine. The deoxyuridine in DNA would direct pairing with dA after replication.
Figure 7.7.A.Structure of hypoxanthine, the product of oxidation deamination of adenine.
Figure 7.7.B.Structure of xanthine, the product of oxidative deamination of guanine.
Both hypoxanthine and xanthine can base pair with cytosine in DNA. Why is this?
Oxidation of C to U occurs spontaneously at a high rate. The frequency is such that 1 in 1000 Cs in the human genome would become Us during a lifetime, if they were not repaired. As will be discussed later, repair mechanisms have evolved to replace a U in DNA with a T.
Methylation of C prior to its oxidative deamination will effectively mask it from the repair processes to remove U’s from DNA. This has a substantial impact on the genomes of organisms that methylate C. In many eukaryotes, including vertebrates and plants (but not yeast or Drosophila), the principal DNA methyl transferase recognizes the dinucleotide CpG in DNA as the substrate, forming 5-methyl-CpG (Figure 7.8). When 5-methyl cytosine undergoes oxidative deamination, the result is 5-methyl uracil, which is the same as thymine. The surveillance system that recognizes U’s in DNA does nothing to the T, since it is a normal component of DNA. Hence the oxidation of 5-methyl CpG to TpG, followed by a round of replication, results in a C:G to T:A transition at former CpG sites (Figure 7.8). This spontaneous deamination is quite frequent; indeed, C to T transitions at CpG dinucleotides are the most common mutations in humans. Since this transition is not repaired, over time the number of CpG dinucleotides is greatly diminished in the genomes of vertebrates and plants.
--CG-- --CG-- [O] --TG-- + NH3 --TG--
|||||| ® |||||| ® ||o||| ® |||||| +wt
--GC-- --GC-- --GC-- --AC--
Figure 7.8. Methylation of CpG dinucleotides followed by oxidative deamination results in TpG dinucleotides.
Some regions of plant and vertebrate genomes do not show the usual depletion of CpG dinucleotides. Instead, the frequency of CpG approaches that of GpC or the frequency expected from the individual frequency of G and C in the genome. One working definition of these CpG islandsis that they are segments of genomic DNA at least 100 bp long with a CpG to GpC ratio of at least 0.6. These islands can be even longer and have a CpG/GpC > 0.75. They are distinctive regions of these genomes and are often found in promoters and other regulatory regions of genes. Examination of several of these CpG islands has shown that they are not methylated in any tissue, unlike most of the other CpGs in the genome. Current areas of research include investigating how the CpG islands escape methylation and their role in regulation of gene expression.
If a CpG island were to be methylated in the germ line, what would be consequences be over many generations?
The rate of oxidation of bases in DNA can be increased by treating with appropriate reagents, such as nitrous acid (HNO2). Thus treatment with nitrous acid will increase the oxidation of C to U, and hence lead to C:G to T:A transitions in DNA. It will also increase the oxidation of adenine to hypoxanthine, leading to A:T to G:C transitions in DNA.
Chemical Modification by Alkylation
Many mutagens are alkylating agents. This means that they will add an alkyl group, such as methyl or ethyl, to a base in DNA. Examples of commonly used alkylating agents in laboratory work are N-methyl-nitrosoguanidine and N-methyl-N'-nitro-nitrosoguanidine (MNNG, Figure 7.9.A.). The chemical warfare agents sulfur mustard and nitrogen mustard are also alkylating agents.
N-methyl-nitrosoguanidine and MNNG transfer a methyl group to guanine (e.g. to the O6 position) and other bases (e.g. forming 3-methyladenine from adenine). The additional methyl (or other alkyl group) causes a distortion in the helix. The distorted helix can alter the base pairing properties. For instance, O6-methylguanine will sometimes base pair with thymine (Figure 7.9.B.).
A. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
B. 6-O-methyl-G will pair with T
Figure 7.9.A. Structure of MNNG and the base pair between O6-methyl G and T
The order of reactivity of nucleophilic centers in purines follows roughly this series:
N7-G >> N3-A > N1-A @ N3-G @ O6-G.
A common laboratory reagent for purines in DNA is dimethylsulfate, or DMS. The products of this reaction are primarily N7-guanine, but N3-adenine is also detectable. This reaction is used to identify protein-binding sites in DNA, since interaction with a protein can cause decreased reactivity to DMS of guanines within the binding site but enhanced reactivity adjacent to the site. Methylation to form N7-methyl-guanine does not cause miscoding in the DNA, since this modified purine still pairs with C.
Chemicals that Cause Deletions
Some compounds cause a loss of nucleotides from DNA. If these deletions occur in a protein-coding region of the genomic DNA, and are not an integral multiple of 3, they result in a frameshift mutation. These are commonly more severe loss-of-function mutations than are simple substitutions. Frameshift mutagens such as proflavin or ethidium bromide have flat, polycyclic ring structures (Figure 7.10.A.). They may bind to and intercalate within the DNA, i.e. they can insert between stacked base pairs. If a segment of the template DNA is the looped out, DNA polymerase can replicate past it, thereby generating a deletion. Intercalating agents can stabilize secondary structures in the loop, thereby increasing the chance that this segment stays in the loop and is not copied during replication (Figure 7.10.B.) Thus growth of cells in the presence of such intercalating agents increase the probability of generating a deletion.
Figure 7.10. Two intercalating agents (A) and their ability to stabilize loops in the template, leading to deletions in the nascent DNA strand (B). Benz(a)pyrenes are present in soot.