Random mutations, protein mutability, and DNA repair: understanding protein tolerance to random amino acid changes through directed evolution
Guo, Haiwei H., 1974-
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In nature, evolution has given rise to the astonishing and wonderful diversity of organisms on this planet. In the laboratory, directed evolution can be a powerful technique to generate variants of a given protein with novel characteristics. The end products of the selection for desired traits from large combinatorial sets of mutants can also yield insight into protein structure and function. In both laboratory and nature, new mutations may be beneficial, but are often neutral or deleterious to overall protein function. One salient question that arises is the degree of tolerance of a protein to random amino acid change.A major part of the corpus of this dissertation addresses this question of protein tolerance to random change. This basic question is quantitatively defined as calculating the probability that a random amino acid replacement will lead to a protein's functional inactivation. Using the human DNA repair enzyme 3-methyladenine DNA glycosylase (AAG), we develop a method to calculate inactivation probabilities from libraries of AAG mutants harboring random mutations throughout the gene. This analytical method is then applied to a range of diverse proteins. Remarkably, inactivation probabilities were observed to be similar among many proteins. To delineate the nature of tolerated mutations, 244 surviving AAG mutants were sequenced. Over 920 tolerated mutations were assembled into "substitutability indices" of each amino acid position across the entire AAG gene and mapped onto secondary and tertiary structures. I discuss the general factors determining the tolerability of amino acid substitutions within the same chapter.The next section of this dissertation describes the use of targeted random oligonucleotide cassette mutagenesis to study the AAG enzyme active site. Selection for altered properties yield novel human DNA glycosylases with altered active sites. This study also reveals the degree of plasticity of the human AAG substrate recognition pocket and highlights the essential residues for substrate recognition and catalysis.This dissertation demonstrates a general approach to understanding proteins' tolerance to random mutations, and to creating novel DNA glycosylases. This work can serve as a stepping stone toward new enzymes remove select altered DNA bases for potential therapeutic and biotechnological applications.
- Pathology