.
Pairing between the normal (keto) forms of the bases.
 |
Spontaneous mutations arise from a variety of sources, including errors in DNA replication, spontaneous lesions, and transposable genetic elements. The first two are considered in this section; the third is examined in Chapter 20.
An error in DNA replication can occur when an illegitimate nucleotide pair (say, A–C) forms in DNA synthesis, leading to a base substitution.
Pairing between the normal (keto) forms of the bases.
Mismatched bases. (a) Mispairs resulting from rare tautomeric forms of the pyrimidines; (b) mispairs resulting from rare tautomeric forms of the purines.
), whereas the
imino and enol forms of the bases are rare. The
ability of the wrong tautomer of one of the standard bases to mispair and cause
a mutation in the course of DNA replication was first noted by Watson and Crick
when they formulated their model for the structure of DNA (Chapter 8). Figure 16-2 demonstrates some possible mispairs resulting
from the change of one tautomer into another, termed a tautomeric shift.
Mispairs can also result when one of the bases becomes ionized. This type of mispair may occur more frequently than mispairs due to imino and enol forms of bases.
Mutation by tautomeric shifts in the bases of DNA. (a) In the example diagrammed, a guanine undergoes a tautomeric shift to its rare enol form (G*) at the time of replication. (b) In its enol form, it pairs with thymine. (c and d) In the next replication, the guanine shifts back to its more stable keto form. The thymine incorporated opposite the enol form of guanine, seen in part b, directs the incorporation of adenine in the subsequent replication, shown in parts c and d. The net result is a GC → AT mutation. If a guanine undergoes a tautomeric shift from the common keto form to the rare enol form at the time of incorporation (as a nucleoside triphosphate, rather than in the template strand diagrammed here), it will be incorporated opposite thymine in the template strand and cause an AT → GC mutation. (From E. J. Gardner and D. P. Snustad, Principles of Genetics, 5th ed. Copyright © 1984 by John Wiley & Sons, New York.)
). The bacterial DNA
polymerase III (Chapter 8) has
an editing capacity that recognizes such mismatches and excises them, thus
greatly reducing the observed mutations. Another repair system (described
later in this chapter) corrects many of the mismatched bases that escape
correction by the polymerase editing function.
. With bases in the
DNA in the normal orientation, creation of a transversion by a replication
error would require, at some point in the course of replication, mispairing
of a purine with a purine or a pyrimidine with a pyrimidine. Although the
dimensions of the DNA double helix render such mispairs energetically
unfavorable, we now know from X-ray diffraction studies that G–A pairs, as
well as other purine–purine pairs, can form.Replication errors can also lead to frameshift mutations. Recall from Chapter 10 that such mutations result in greatly altered proteins.
A simplified version of the Streisinger model for frameshift formation. (a–c) In DNA synthesis, the newly synthesized strand slips, looping out one or several bases. This loop is stabilized by the pairing afforded by the repetitive-sequence unit (the A bases in this case). An addition of one base pair, A–T, will result at the next round of replication in this example. (d–f) If, instead of the newly synthesized strand, the template strand slips, then a deletion results. Here the repeating unit is a CT dinucleotide. After slippage, a deletion of two base pairs (C–G and T–A) would result at the next round of replication.
), frameshifts arise when loops in single-stranded regions
are stabilized by the “slipped mispairing” of repeated sequences.
The distribution of 140 spontaneous mutations in lacI. Each occurrence of a point mutation is indicated by a box. Red boxes designate fast-reverting mutations. Deletions (gold) are represented below. The I map is given in terms of the amino acid number in the corresponding I-encoded lac repressor. Allele numbers refer to mutations that have been analyzed at the DNA sequence level. The mutations S114 and S58 (circles) result from the insertion of transposable elements (see Chapter 20). S28 (red circle) is a duplication of 88 base pairs. (From P. J. Farabaugh, U. Schmeissner, M. Hofer, and J. H. Miller, Journal of Molecular Biology 126, 1978, 847.)
depicts the
distribution of spontaneous mutations in the lacI gene.
Compare this distribution with that in the rII genes of T4
seen by Benzer (see Figure 9-26).
Note how one or two mutational sites dominate the distribution in both
cases. In lacI, a four-base-pair sequence repeated three
times in tandem in the wild type is the cause of the hot spots (for
simplicity, only one strand of the double strand of DNA is
indicated):
The major hot spot, represented here by the mutations FS5, FS25, FS45, and FS65, results from the addition of one extra set of the four bases CTGG to one strand of the DNA. This hot spot reverts at a high rate, losing the extra set of four bases. The minor hot spot, represented here by the mutations FS2 and FS84, results from the loss of one set of the four bases CTGG. This mutant does not readily regain the lost set of four base pairs.
to the sequence shown for lacI?)
Whether a deletion or an addition is generated depends on whether the
slippage occurs on the template or on the newly synthesized strand,
respectively. In a similar fashion, the addition mutant can slip out one
CTGG sequence and stabilize a structure with 13 base pairs (verify this for
the FS5 sequence shown for lacI), which
explains the rapid reversion of mutations such as FS5.
However, there are only five base pairs available to stabilize a slipped-out
CTGG in the deletion mutant, accounting for the infrequent reversion of
mutations such as FS2 in the sequence shown for
lacI.
Deletions in lacI. Deletions occurring in S74 and S112 are shown at the top of the figure. As indicated by the gold bars, one of the sequence repeats (aqua) and all the intervening DNA are deleted, leaving one copy of the repeated sequence. All mutations were analyzed by direct DNA sequence determination. (From P. J. Farabaugh, U. Schmeissner, M. Hofer, and J. H. Miller, Journal of Molecular Biology 126, 1978, 847.)
. The majority, although not all, of the
deletions occur at repeated sequences. Figure 16-6 shows the results for the first 12 deletions
analyzed at the DNA sequence level, presented by Miller and his co-workers
in 1978. Further studies showed that hot spots for deletions are in the
longest repeated sequences. Duplications of segments of DNA
have been observed in many organisms. Like deletions, they often occur at
sequence repeats.
) could explain why
deletions predominate at short repeated sequences. Alternatively, deletions
and duplications could be generated by recombinational mechanisms (to be
described in Chapter 19).In addition to replication errors, spontaneous lesions, naturally occurring damage to the DNA, can generate mutations. Two of the most frequent spontaneous lesions result from depurination and deamination.
The loss of a purine residue (guanine) from a single strand of DNA. The sugar-phosphate backbone is left intact.
). A mammalian cell
spontaneously loses about 10,000 purines from its DNA in a 20-hour
cell-generation period at 37°C. If these lesions were to persist, they would
result in significant genetic damage because, in replication, the resulting
apurinic sites cannot specify a base complementary to the
original purine. However, as we shall see later in the chapter, efficient repair
systems remove apurinic sites. Under certain conditions (to be described later),
a base can be inserted across from an apurinic site; this insertion will
frequently result in a mutation.
Deamination of (a) cytosine and (b) 5-methylcytosine.
5-Methylcytosine hot spots in E. coli. Nonsense mutations occurring at 15 different sites in lacI were scored. All result from the GC → AT transition. The asterisks (*) mark the positions of 5-methylcytosines. Open bars depict sites at which the GC → AT change could be detected but at which no mutations occurred in this particular collection. It can be seen that 5-methylcytosine residues are hot spots for the GC → AT transition. Of 50 independently occurring mutations, 44 were at the 4 methylated cytosine sites and only 6 were at the 11 unmethylated cytosines. (From C. Coulondre et al., Nature 274, 1978, 775.).
). Unrepaired uracil residues will pair with
adenine in replication, resulting in the conversion of a G–C pair into an A–T
pair (a GC → AT transition). In 1978, deaminations at
certain cytosine residues were found to be the cause of one type of mutational
hot spot. DNA sequence analysis of GC → AT transition hot spots in the
lacI gene showed that 5-methylcytosine residues are
pres-ent at each hot spot. (Certain bases in prokaryotes and eukaryotes are
methylated.) Some of the data from this lacI study are shown in
Figure 16-9. The height of each bar
on the graph represents the frequency of mutations at each of a number of sites.
It can be seen that the positions of 5-methylcytosine residues correlate nicely
with the most mutable sites.
) generates thymine (5-methyluracil), which is not recognized by
the enzyme uracil-DNA glycosylase and thus is not repaired. Therefore, C → T
transitions generated by deamination are seen more frequently at
5-methylcytosine sites, because they escape this repair system.
A consequence of the frequent mutation of 5-methylcytosine to thymine is the underrepresentation of CpG dinucleotides in higher cells, because this sequence is methylated to give 5-methyl-CpG, which is gradually converted into TpG.
DNA damage products formed after attack by oxygen radicals. dR = deoxyribose.
shows two products of oxidative damage. The 8-oxo-7-hydrodeoxyguanosine
(8-oxodG, or GO) product frequently mispairs with A, resulting in a high level
of G → T transversions. Thymidine glycol blocks DNA replication if unrepaired
but has not yet been implicated in mutagenesis.
Spontaneous mutations can be generated by different processes. Replication errors and spontaneous lesions generate most of the base-substitution and frameshift mutations. Replication errors may also cause some deletions that occur in the absence of mutagenic treatment.
DNA sequence analysis has revealed the mutations responsible for a number of human hereditary diseases. The previously discussed studies of bacterial mutations allow us to suggest mechanisms that cause these human disorders.
Sequences of wild-type (WT) mitochondrial DNA and deleted DNA (KS) from a patient with Kearns-Sayre syndrome. The 13-base boxed sequence is identical in both WT and KS and serves as a breakpoint for the DNA deletion. A single base (boldface type) is altered in KS, aside from the deleted segment.
depicts one of these deletions. Note how
similar it is in form to the spontaneous E. coli deletions
shown in Figure 16-6.
Expansion of the CGG triplet in the FMR-1 gene seen in the fragile X syndrome. Normal persons have from 6 to 54 copies of the CGG repeat, whereas members of susceptible families display an increase (premutation) in the number of repeats: normally transmitting males (NTMs) and their daughters are phenotypically normal but display from 50 to 200 copies of the CGG triplet; the number of repeats expands to some 200 to 1300 in those showing full symptoms of the disease.
). This syndrome is the
most common form of inherited mental retardation, occurring in close to 1 of
1500 males and 1 of 2500 females. It is evidenced cytologically by a fragile
site in the X chromosome that results in a break in vitro.
The inheritance of fragile X syndrome is unusual in that 20 percent of the males with a fragile X chromosome are phenotypically normal but transmit the affected chromosome to their daughters, who also appear normal. These males are said to be normally transmitting males (NTMs). However, the sons of the daughters of the NTMs frequently display symptoms. The fragile X syndrome results from mutations in a (CGG)n repeat in the coding sequence of the FMR-1 gene. Patients with the disease show specific methylation, induced by the mutation, at a nearby CpG cluster, resulting in reduced FMR-1 expression.
Why do symptoms develop in some persons with a fragile X chromosome and not in others? The answer seems to lie in the number of CGG repeats in the FMR-1 gene. Humans normally show a considerable variation in the number of CGG repeats in the FMR-1 gene, ranging from 6 to 54, with 29 repeats in the most frequent allele. [The variation in CGG repeats produces a corresponding variation in the number of arginine residues (CGG is an arginine codon) in the FMR-1-encoded protein.] Both NTMs and their daughters have a much larger number of repeats, ranging from 50 to 200. These increased repeats have been termed premutations. All premutation alleles are unstable. The males and females with symptoms of the disease, as well as many carrier females, have additional insertions of DNA, suggesting repeat numbers of 200 to 1300. The frequency of expansion has been shown to increase with the size of the DNA insertion (and thus, presumably, with the number of repeats). Apparently, the number of repeats in the premutation alleles found in NTMs and their daughters is above a certain threshold and thus is much more likely to expand to a full mutation than is the case for normal persons.
) involving a
one-step expansion of the four-base-pair sequence CTGG. However, the
extraordinarily high frequency of mutation at the three-base-pair repeats in the
fragile X syndrome suggests that in human cells, after a threshold level of
about 50 repeats, the replication machinery cannot faithfully replicate the
correct sequence, and large variations in repeat numbers result.
A second inherited disease, X-linked spinal and bulbar muscular atrophy (known as Kennedy disease), also results from the amplification of a three-base-pair repeat, in this case a repeat of the CAG triplet. Kennedy disease, which is characterized by progressive muscle weakness and atrophy, results from mutations in the gene that encodes the androgen receptor. Normal persons have an average of 21 CAG repeats in this gene, whereas affected patients have repeats ranging from 40 to 52.
Myotonic dystrophy, the most common form of adult muscular dystrophy, is yet another example of sequence expansion causing a human disease. Susceptible families display an increase in severity of the disease in successive generations; this increase is caused by the progressive amplification of a CTG triplet at the 3′ end of a transcript. Normal people possess, on average, five copies of the CTG repeat; mildly affected people have approximately 50 copies, and severely affected people have more than 1000 repeats of the CTG triplet. Additional examples of triplet expansion are still appearing—for instance, Huntington disease.
