Functional Divergence of Photolyase and Cryptochrome-DASH: The Role of Loop Dynamics

Date created: 
2015-09-11
Identifier: 
etd9264
Keywords: 
Photolyase
Cryptochrome-Dash
Cyclobutane pyrimidine dimer
Conformational dynamics
Directed evolution
DNA repair
Abstract: 

Photolyase (PL) is an enzyme that repairs thymine dimers, a form of DNA damage caused by UV light. To do so, the damaged bases of the cyclobutane pyrimidine dimer (CPD) are displaced from the duplex into an extrahelical position. A “recognition loop” in PL must be displaced to allow it to bind substrate. Cryptochrome-DASH (CRYD) is a structurally homologous protein to PL, with a high sequence identity, which cannot perform the same physiological function as PL. Here I ask how PL is able to bind to its substrate and how CRYD functionally diverged from PL. I hypothesize that there exist optimized conformational dynamics of these recognition loop regions that allow PL to bind CPD. Limited proteolysis experiments determined that the recognition loops of PL and CRYD are the most dynamic regions of each of these proteins. Furthermore, the conformational dynamics of the CRYD recognition loop are greater than that of PL. A difference between the dynamics is consistent with my hypothesis, but does not definitively prove it. The differences in recognition loop conformational dynamics could be due to primary, secondary or tertiary structures. To determine the impact sequence has on these conformational dynamics, end-to-end contact was measured in isolated recognition loop peptides using fluorescence quenching. It was found that the CRYD recognition loop peptide is more dynamic than that of PL. This indicates that sequence, at least in part, is responsible for conformational dynamic differences in these protein loops. To definitively determine the role the recognition loop plays in how PL and CRYD functionally diverged, I outlined a series of directed evolution experiments. To this end random mutagenesis was planned to be carried out only in the PL recognition loop region in search for partially functional PLs. If a correlation exists between conformational dynamics and function, then the recognition loop would be implicated in the functional divergence of these proteins. Technical development, including random mutagenic PCR, a mutation rate detection assay, in vivo functional assays and in vitro functional assay was undertaken to test this hypothesis.

Document type: 
Thesis
Rights: 
This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
File(s): 
Supervisor(s): 
Edgar Young
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.
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