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More mutagenesis

©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison

III D. MUTATORS

Mutations that deleteriously affect the fidelity of DNA replication or repair cause an increase in the basic mutation frequency and are called mutators. There are a number of sorts of these mutations with different levels and types of effects. Two examples are mutT mutations which increase mutation frequency l03-l04 times and are specific for A/T to C/G transversion mutations, (Transversion mutations are those base substitution mutations where a purine replaces a pyrimidine or vice versa. Transition mutations are those where a purine replaces a different purine or where a pyrimidine replaces a different pyrimidine.) and mutD mutations that cause l03-l05 higher mutation frequencies and generates a range of error types (Strains with mutDmutations seem to lack the 3'-5' exonuclease activity alluded to in Section III A). These strain can be useful as a genetic tool for mutagenesis, but only when the region that you wish to mutagenize can be transiently replicated using these systems and then removed from their continuing mutagenic action.

III E. FRAMESHIFT MUTATIONS

Frameshift mutations are defined as the addition or deletion of one or two base pairs. They are termed frameshift for historical reasons, since when they occur in a region which is translated into protein, such an addition or deletion of the base will put the downstream region out of proper reading frame. Obviously, if the product is an RNA then the term frameshift is inappropriate, but used nonetheless. An example of a frameshift mutation and a plausible revertant mutation (restoring the wild-type phenotype in what is now a double mutant) that does not map at quite the same site is presented below. Whether or not such secondary mutations would be "compensatory" and generate a revertant (i.e. a strain with a pseudo wild-type phenotype) is a question of whether or not the resulting protein product functions sufficiently well.

Typically, a frameshift mutation in a translated region has three effects: (l) it puts you out of reading frame and therefore tends to destroy the function of the encoded product, (2) it often discloses nonsense signals and therefore tends to be polar to some extent, (3) it might be restored to a wild-type phenotype by a nearby compensatory frameshift mutation. The frameshift mutation is not a nonsense mutation, but rather discloses nonsense signals by shifting the reading frame. The frequency of such "disclosures" depends on the GC content of the organism, since nonsense signals are AT rich. In this latter case the region between the initial and compensatory frameshift will not be in the proper reading frame so that such "second site" revertants do not appear if this region of the encoded protein is crucial for activity. Most frameshift mutations, whether spontaneous or chemically induced, do tend to occur as the addition or deletion of a base in redundant runs of identical bases, for example, (G)GGG, (A)AAAA or (GC)GCGC; and this is thought to reflect their mechanism of generation (see below). The spontaneous frameshifts mentioned in section IIIA1, which are apparently created by a rather different mechanism, do nothave any requirement for redundant sequences.

Frameshift mutations are induced in vivo several ways. The first is by the error-prone repair system (see above) by a mechanism that is as yet unclear. A second mechanism involves chemical mutagenesis by compounds (e.g. ICR-l9l) of the structure shown in Figure 7 which seem to have the property of intercalating between base pairs of the DNA. Such a property has given rise to a model of mutagenesis which suggests that the intercalation stabilizes mispairing when the two redundant sequences on the complementary strands slide with respect to one another. These mutagens are not particularly powerful and do tend to have some specificity, in general preferring G-rich regions. It is amusing to note that ICR stands for the Institute for Cancer Research and these compounds were originally generated as anti-cancer agents. Not surprisingly, given their mutagenic effect, they are in fact cancer-causing agents. Frameshift mutations can also be produced by in vitro manipulations.

[See sample problems 1,2,3 and 3a]

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