DNA mutagenesis

Miller, B.M., Zitzelsberger, H.F., Weier, H.-U.G. Adler, I.-D. (1991) Classification of micronuclei in murine erythrocytes immunofluorescent staining using CREST antibodies compared to in situ hybridization with biotinylated gamma satellite DNA. Mutagenesis, 6, 297-302... 

Table 11.2 Selected DNA mutagenesis and recombination methods (Kurtzman, 2001).

The mixtures of extremozymes produced by the expression process are then tested to see if they have catalytic activity for the industrial processes of interest. If a given mixture shows catalytic activity, it is then usually subjected to random DNA mutagenesis or molecule breeding to see whether random evolution of the enzymes will lead to improved activities. 

The answer is e. (Murray, pp 412-434. Scriver, pp 677-704. Sack, pp 3-29. Wilson, pp 99-121.) Xeroderma pigmentosum (278700) appears to be due to the inability of an excision-repair system to remove thymine dimers, which are formed on exposure of DNA to ultraviolet radiation. This results in a deficiency in the ability to repair the damaged DNA. Mutagenesis by this mechanism is presumably the basis for the multiple neoplasms that occur in patients who have this disease. 

Ahmed MS, Ramesh V, Nagaraja V, Parish JH, Hadi SM (1994) Mode of binding of quercetin to DNA. Mutagenesis 9 193—197... 

Linker-Scanning Mutagenesis. Using linker-scanning mutagenesis (24) small sequences of DNA are removed and replaced with a synthetic restriction fragment or linker. This technique is commonly used in analysis of promoters and other control sequences in DNA, while preserving the spatial relationship between the sequences. 

Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

How is the binding specificity of the heterodimer achieved compared with the specificity of Mat a2 alone The crystal structure rules out the simple model that the contacts made between the Mat a2 homeodomain and DNA are altered as a result of heterodimerization. The contacts between the Mat o2 homeodomain and DNA in the heterodimer complex are virtually indistinguishable from those seen in the structure of the Mat o2 monomer bound to DNA. However, there are at least two significant factors that may account for the increased specificity of the heterodimer. First, the Mat al homeodomain makes significant contacts with the DNA, and the heterodimeric complex will therefore bind more tightly to sites that provide the contacts required by both partners. Second, site-directed mutagenesis experiments have shown that the protein-protein interactions involving the... 

The specific role of each amino acid residue for the function of the protein can be tested by making specific mutations of the residue in question and examining the properties of the mutant protein. By combining in this way functional studies in solution, site-directed mutagenesis by recombinant DNA techniques, and three-dimensional structure determination, we are now in a position to gain fresh insights into the way protein molecules work. 

Figure 2.5 Scheme for DNA shuffling illustrated for the case in which the parent genes originate from the WT by some form of mutagenesis [7e],... 

---