7. Mutations are concentrated at hotspots

1.7 Mutations are concentrated at hotspots

Key terms defined in this section
Back mutation reverses the effect of a mutation that had inactivated a gene; thus it restores wild type.
Forward mutations inactivate a wild-type gene.
Hotspot is a site at which the frequency of mutation (or recombination) is very much increased.
Modified bases are all those except the usual four from which DNA (T, C, A, G) or RNA (U, C, A, G) are synthesized; they result from postsynthetic changes in the nucleic acid.
Neutral substitutions in a protein are those changes of amino acids that do not affect activity.
Silent mutations do not change the product of a gene.

So far we have dealt with mutations in terms of individual changes in the sequence of DNA that influence the activity of the genetic unit in which they occur. When we consider mutations in terms of the inactivation of the gene, most genes within a species show more or less similar rates of mutation relative to their size. This suggests that the gene can be regarded as a target for mutation, and that damage to any part of it can abolish its function. As a result, susceptibility to mutation is roughly proportional to the size of the gene. But consider the sites of mutation within the sequence of DNA; are all base pairs in a gene equally susceptible or are some more likely to be mutated than others?


What happens when we isolate a large number of independent mutations in the same gene? Many mutants are obtained. Each is the result of an individual mutational event. Then the site of each mutation is determined. Most mutations will lie at different sites, but some will lie at the same position. Two independently isolated mutations at the same site may constitute exactly the same change in DNA (in which case the same mutational event has happened on more than one occasion), or they may constitute different changes (three different point mutations are possible at each base pair).




Figure 1.17 Spontaneous mutations occur throughout the lacI gene of E. coli, but are concentrated at a hotspot.

The histogram of Figure 1.17 shows the frequency with which mutations are found at each base pair in the lacI gene of E. coli. The statistical probability that more than one mutation occurs at a particular site is given by random-hit kinetics (as seen in the Poisson distribution). So some sites will gain one, two, or three mutations, while others will not gain any. But some sites gain far more than the number of mutations expected from a random distribution; they may have 10 or even 100 more mutations than predicted by random hits. These sites are called hotspots. Spontaneous mutations may occur at hotspots; and different mutagens may have different hotspots.


A major cause of spontaneous mutation in E. coli results from the presence of an unusual base in the DNA. In addition to the four bases that are inserted into DNA when it is synthesized, modified bases are sometimes found. The name reflects their origin; they are produced by chemically modifying one of the four bases already present in DNA. The most common modified base is 5-methylcytosine, generated by a methylase enzyme that adds a methyl group to a small proportion of the cytosine residues (at specific sites in the DNA).


Sites containing 5-methylcytosine provide hotspots for spontaneous point mutation. In each case, the mutation takes the form of a G PC to A PT transition. The hotspots are not found in strains of E. coli that cannot methylate cytosine.




Figure 1.18 The deamination of 5-methylcytosine produces thymine (causing C-G to T-A transitions), while the deamination of cytosine produces uracil (which usually is removed and then replaced by cytosine).

The reason for the existence of the hotspots is that 5-methylcytosine suffers spontaneous deamination at an appreciable frequency. Replacement of the amino group by a keto group converts 5-methylcytosine to thymine. Figure 1.18 shows why deaminating the (rare) 5-methylcytosine causes a mutation, whereas deamination of the more common cytosine does not have this effect (Coulondre et al., 1978).




Figure 1.15 Mutations can be induced by chemical modification of a base.

The deamination of cytosine generates uracil. However, E. coli contains an enzyme, uracil-DNA-glycosidase, that removes uracil residues from DNA (see 14.13 Base flipping is used by methylases and glycosylases). This action leaves an unpaired G residue, and a "repair system" then inserts a C base to partner it. The net result of these reactions is to restore the original sequence of the DNA. Presumably this system serves to protect DNA against the consequences of spontaneous deamination of cytosine (although it is not active enough to prevent the effects of nitrous acid; see Figure 1.15).


But the deamination of 5-methylcytosine leaves thymine. Because this base is a respectable constituent of DNA in its own right, the system does not recognize the change, and a mutation results. The conversion creates a mispaired G PT partnership, whose separation at the subsequent replication produces one wild-type G PC pair and one mutant A PT pair.


The operation of this system casts an interesting light on the use of T in DNA compared with U in RNA. Perhaps it relates to the need of DNA for stability of sequence; the use of T means that any deaminations of C are immediately recognized, because they generate a base (U) not usually present in the DNA.


Spontaneous mutations that inactivate gene function occur in bacteria at a rate of ~10 V5 V10 V6 events per locus per generation. This mutation rate corresponds to changes at individual nucleotides of 10 V9 V10 V10 per generation. We have no accurate measurement of the rate of mutation in eukaryotes, although usually it is thought to be somewhat similar to that of bacteria on a per-locus per-generation basis. We do not know what proportion of the spontaneous events results from point mutations.


Not all mutations in DNA lead to a detectable change in the phenotype. Mutations without apparent effect are called silent mutations. They fall into two types. Some involve base changes in DNA that do not cause any change in the amino acid present in the corresponding protein. Others change the amino acid, but the replacement in the protein does not affect its activity; these are called neutral substitutions.


Mutations that inactivate a gene are called forward mutations. Their effects are reversed by back mutations, which are of two types.


An exact reversal of the original mutation is called true reversion. So if an A PT pair has been replaced by a G PC pair, another mutation to restore the A PT pair will exactly regenerate the wild-type sequence.


Alternatively, another mutation may occur elsewhere in the gene, and its effects compensate for the first mutation. This is called second-site reversion. For example, one amino acid change in a protein may abolish gene function, but a second alteration may compensate for the first and restore protein activity.


A forward mutation results from any change that inactivates a gene, whereas a back mutation must restore function to a protein damaged by a particular forward mutation. So the demands for back mutation are much more specific than those for forward mutation. The rate of back mutation is correspondingly lower than that of forward mutation, typically by a factor of ~10.



Research
Coulondre, C. et al. (1978). Molecular basis of base substitution hotspots in E. coli. Nature 274, 775-780.



Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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