Mutation and mutagenesis

Chromosomes are by no means inert, stable structures, holding dead information in storage. They are constantly undergoing changes of various kinds, some of which are involved in genetic recombination. However, other changes are accidental and random in nature and, if not repaired, result in a permanent genetic mutation. In Appendices 5.2 and 5.6, some of these repair mechanisms are discussed (e.g. DNA repair), but the results of these phenomena will now be discussed further. 

DNA is subject to damage by electromagnetic radiation or reactive chemicals, many of which have been introduced into the environment as a by-product of industrial activity. Some chemicals may not be dangerous per se, but may be metabolised to products which are dangerous. There are three major classes of such chemicals07 . 

Although DNA proof-reading and repair mechanisms within cells are very effective, inevitably some errors in replication remain uncorrected, and as a result become perpetuated in the DNA of the organism. Such permanent changes are called mutations. 

Mutations caused by the replacement of a single base with an incorrect one are called substitution mutations. Such mutations will have the effect of changing one codon-triplet and, depending on the code, this may alter an amino-acid in the polypeptide chain. Examples of hypothetical mutations are shown in Table 5.15. Silent mutations are changes in the base sequence which cause no change to the 

A single-base substitution causing no change in the amino-acid sequence a silent mutation 

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If an appropriate selection can be made from the assorted mutations that block each step in enzymatic catalysis and lead to the accumulation of intermediates, such mutant enzymes can serve as direct tools to delineate a complex catalytic pathway. From both theoretical and practical standpoints, mutations and mutagenesis have significant impacts on genetic engineering. Generating mutant enzymes (or proteins) with either improved catalytic (or functional) and/or structural features or de novo specificities is a lofty goal of protein engineering. 

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

Fig. 8. Mutagenesis of the predicted ATP binding site. ATP is shown in proximity to amino acids in four loops predicted to form the ATP binding site in the nucleotide binding domain [49,134] and a fifth loop representing the phosphorylation site at Asp351 [97], Mutations and the corresponding Ca transport activity of the mutants relative to wild-type are indicated. From Clarke et al. [103).

All these techniques create genetic diversity by recombination and point mutations and are well developed. However, insertions and deletions (indels) are also important types of mutation which are probably underrepresented in many conventional mutagenesis strategies. Methods for incorporation of indels in predefined positions in a combinatorial manner have been developed.Although there are some published studies on their use in the directed evolution of biocatalysts,the full potential of these newer methods of gene mutation for enzyme improvement are yet to be demonstrated. 

Chemical clastogenesis and mutagenesis both involve a complex series of processes, including pharmacokinetic mechanisms (uptake, transport, diffusion, excretion), metabolic activation and inactivation, production of DNA lesions and their incomplete repair or misrepair, and steps leading to the subsequent expression of mutations in surviving cells or individuals (Thble 7.1). Each of the steps in these processes might conceivably involve first order kinetics at low doses (e.g., diffusion, MichaeUs-Menten enzyme kinetics) and hence be linear. In principle, therefore, the overall process edso might be linear and without threshold. 

In 1977, the NAS Safe Drinking Water Committee outlined four principles that it said should be useful in dealing with the assessment of hazards that involve chronic irreversible toxicity or the effects of longterm exposure (20). These principles (paraphrased as follows) were intended to apply primarily to cancer risks from substances whose mechanisms involve somatic mutations and may also be applicable to mutagenesis and teratogenesis ... 

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