GSBS Dissertations and Theses

Approval Date

5-24-2013

Document Type

Doctoral Dissertation

Academic Program

Biochemistry and Molecular Pharmacology

Department

Molecular, Cell and Cancer Biology Department

First Thesis Advisor

Scot Wolfe, PhD

Keywords

Homeodomain Proteins, DNA-Binding Proteins

Subjects

Dissertations, UMMS; Homeodomain Proteins; DNA-Binding Proteins

Abstract

Homeodomains (HDs) are a large family of DNA-binding domains contained in transcription factors that are most notable for regulating body development and patterning in metazoans. HDs consist of three alpha helices preceded by an N- terminal arm, where the third helix (the recognition helix) and the N-terminal arm are responsible for defining DNA-binding specificity. Here we attempted to engineer the HDs by fully randomizing positions in the recognition helix to specify each of the 64 possible 3’ triplet sites (i.e. TAANNN). We recovered HD variants that preferentially recognize or are compatible with 44 of the possible sites, a dramatic increase from the previously observed range of specificities. Many of these HD variants contain combinations of novel specificity determinants that are uncommon or absent in extant HDs, where these determinants can be grafted into alternate HD backbones with an accompanying alteration in their specificity. The identified determinates expand our understanding of HD recognition, allowing for the creation of more explicit recognition models for this family. Additionally, we demonstrate that HDs can recognize a broader range of DNA sequences than anticipated, thus raising questions about the fitness barrier that restricts the evolution HD-DNA recognition in nature. Finally, these new HD variants have utility as DNA-binding domains to direct targeting of customizable sequence-specific nuclease as demonstrated by site-specific lesions created in zebrafish. Thus HDs can guide sequence-specific enzymatic function precisely and predictably within a complex genome when used in engineered artificial enzymes.

DOI

10.13028/M2DW3Z

Rights and Permissions

Copyright is held by the author, with all rights reserved.

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