Functional Mapping of Mu Transposition Machinery : MuA Protein Modification and Engineering for Hyperactivity
Year: 2013
Type: dissertation
Abstract: Transposable DNA elements constitute a class of discrete genome segments, which use fundamentally similar reactions for their movement within and between genomes without the need for sequence homology to the target site. Transposons are ubiquitous and abundant residents scattered throughout the genomes of virtually all living organisms. The ability of transposons to integrate into various DNA sequences is utilized as tools in molecular biology. The similarity of the transposition reaction mechanisms is reflected by their enzymes, the transposases. Many DNA transposases share a conserved RNase H-like fold that contains a catalytic DDE motif. The bacteriophage Mu encodes MuA transposase, which is a well characterized member of the DDE transposases. Mu transposition proceeds within a nucleoprotein complex known as a transpososome, the minimal core of which contains four MuA molecules and two synapsed transposon ends. It forms the basis of Mu transposon tools that are widely used in genetics/genomics applications. The study reveals important structure-function relationships of MuA in the context of the transpososome. In addition, this study critically evaluates different random DNA mutagenesis methods and describes a universal assay to quantitatively analyse DNA transposition events. Initially, we characterized mutagenic properties of several most commonly used random mutagenesis methods and used them for MuA mutant library generation. Then, we established the quantitative transposition assay for mutant screening purposes. We analysed MuA structure-function relationships by scanning pentapeptide insertion library to dissect insertion tolerant against insertion intolerant regions in MuA structure. The comprehensive structure-function analysis was complemented with data from the sequence alignment of protein homologues. We identified potential sites for protein modifications. In addition, the different random MuA mutant libraries were screened for increased transposition frequency. We show that MuA mutant variants with enhanced activity could be generated. By the mapping of hyperactivity-inducing substitutions to the Mu transpososome structure, we could characterize the factors behind the activity enhancement. Furthermore, several MuA mutants were discovered to be valuable for Mu in vitro transposition applications. The results of this study provide a comprehensive structure-function map of MuA transposase. Moreover, the methods described in this study and the activity enhancing substitutions discovered in RNase H-like fold can be used for mechanistic analyses of other DDE transposases or used for the analyses of their close relatives such as HIV integrase and the RAG recombinase of the V(D)J recombination.
Source: Työväentutkimus Vuosikirja
Author: Tiina Rasila
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2188396331