Lunt, Bryce L
(2017)
Exploring the Requirements of the Mycobacterium Phage Brujita Integrase.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Temperate phages establish lysogeny and immunity to superinfection upon phage integration. Brujita Integrase (Int) is a model for a recently discovered subclass of tyrosine integrases, which lack traditional N-terminal arm-binding domains and contain additional C-terminal degradation tags. Typically, the arm-binding domain recognizes binding sites in the phage attachment DNA (attP) and serves to control the directionality in integration and excision. The
absence of this domain raises the question of how Brujita Int binds attachment site substrates to control site selectivity and directionality. In this work, we describe the DNA and protein requirements for Brujita Int function, how it implements site selection, directionality control, and higher order complex formation. We show that Brujita Int is a simple recombinase, which
resembles Cre, FLP, and XerC/D more closely than it does the tyrosine phage integrases. In contrast to Lambda Int, Brujita Int uses small DNA substrates, does not discriminate between attP and attB sites, and lacks directional control. It also shows unusual binding to its attachment sites. Binding to attB occurs at two half sites, B and B’ in a cooperative manner, such that binding at B’
is required for binding at B. Binding to attP is also unusual. The attP site is comprised of three half sites, P, P’, and an additional site to the left of P, P1. However, binding is not detected at P, although this site is required for recombination. Additionally, P1 is required for recombination, presumably to stabilize binding at P. Finally, we have solved the crystal structure of the Brujita Int and have found residues responsible for higher order complex formation. In total, our results indicate that Brujita Int may represent an evolutionary precursor to the canonical tyrosine integrases. Moreover, given its relative simplicity, Brujita Int presents a useful tool for the creation of synthetic genetic circuits as well as genome manipulation.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
28 June 2017 |
| Date Type: |
Publication |
| Defense Date: |
10 January 2017 |
| Approval Date: |
28 June 2017 |
| Submission Date: |
19 January 2017 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
162 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
phage integrase, tyrosine recombinase, site-specific recombination |
| Date Deposited: |
28 Jun 2017 20:46 |
| Last Modified: |
28 Jun 2017 20:46 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/30679 |
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