Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Biochemistry

Supervisor

Dr. David Edgell

Abstract

A systematic approach to reprogram protein-DNA interactions has yet to be discovered. This study investigates the ability of co-variation analyses to identify potential protein-DNA contacts that regulate specificity. Here, 27 LAGLIDADG Homing Endonucleases (LHEs) and their 22-basepair DNA targets were collated into a Multiple Sequence Alignment (MSA) that was subjected to pairwise co-variation calculations. Using the LHE I-OnuI as a reference, an amino acid-DNA pair, lysine (K) 231 and adenine +3, generated the highest score. To test if the K231/A3 score was biologically relevant we tested protein mutants for altered nuclease specificity at +3 DNA point mutants. Randomizing the 231st amino acid did not alone restore cleavage activity on substrate mutants but randomization in conjunction with, aspartic acid (D) 240, restored cleavage activity on A3T and A3G substrates. In conclusion, co-variation analyses identified in part, amino acids that could be mutated to alter DNA specificity. Future work should focus on mapping more LHE-DNA target sequences to increase MSA diversity.

LHE_DNA_alignment.fa (18 kB)
MSA

CountTable.txt (66 kB)
count table from Miseq run


Included in

Biochemistry Commons

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