Examination of role and function of the Coiled-Coil domain of φC31 integrase

Abramiuk, Jakub Filip (2026) Examination of role and function of the Coiled-Coil domain of φC31 integrase. MSc(R) thesis, University of Glasgow.

Abstract

φC31 Integrase (Int) is a recombinase enzyme used by the φC31 bacteriophage to integrate its DNA into the chromosome of a host bacterium at initiation of the lysogenic cycle. The enzyme has several useful properties, such as strong recombination site specificity, high reaction efficiency in optimal conditions and unidirectionality of the recombination, which can be reversed in the presence of a Recombination Directionality Factor (RDF) - a secondary protein factor. For those reasons, it (and other related integrases from the Large Serine Recombinase (LSR) family) have been used in biotechnological and genetic applications, such as targeted insertion/excision of large DNA fragments.

Despite this interest in the applications of the enzyme, the biochemistry underlying properties of its activity is still not fully understood and is under ongoing examination, especially with regard to directionality control. During the forward reaction φC31 Int targets and forms a synapse on a heterologous pair of sites called attP and attB, forming hybrid sites attL and attR as products of the reaction. The presence of RDF allows φC31 Int to form a synapse on attL and attR sites and inhibits synapse formation at attP and attB, resulting in a reverse reaction. Several structural studies in closely related LSRs have provided evidence of involvement of the Coiled-Coil (CC) domain (located near the C-terminus of the φC31 Int, between residues G445 and A531) in the formation of a tetrameric complex during synapse formation. This could mean that CC domain interactions are part of the control mechanism for φC31 Int reaction directionality.

This project aims to expand the knowledge on φC31 Int reaction specificity and directionality by examining several point mutations in the CC domain. Residues of interest were chosen based on sequence alignments with other LSRs, and are thought to be analogous to residues responsible for CC-CC interactions during formation of the synaptic complex in those LSRs. Information on their role in φC31 Int activity and specificity could be important for understanding the biochemistry of the enzyme, and subsequently could help unlock the ability to create designer versions with altered properties for genetic engineering and synthetic biology applications.

Mutants of L474 and Y475 residues, as well as a complete CC deletion (ΔCC) variant were produced. Other identified residues of interest included L468, L471, E472, V487, H491, F492, R493, Q496, L499, T500 and E507. In vitro assays for reaction efficiency (including tests with att sites associated with different LSRs to check for site specificity disruptions) as well as synaptic binding (including experiments with unorthodox att site pairings), were conducted for the produced mutants. The alanine mutant of L474 showed some activity, albeit reduced in comparison with wild-type. Y475A and ΔCC mutants were unable to facilitate the recombination reaction under any conditions, cementing the view that the CC domain is vital during the synapsis, as well as confirming the suspicion that Y475-mediated interactions are important to CC domain function. Interestingly, the Y475H mutant showed activity in a forward reaction, which may imply that the interactions this residue takes part in are at least partially dependent on the shape and size of the tyrosine sidechain. However, it was almost completely inactive on attL x attR substrates, suggesting that its polarity might be important for interactions enabling the reverse reaction. All active mutants retained high site specificity.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH345 Biochemistry; Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Stark, Professor Marshall and Colloms, Dr. Sean
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-85742
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 04 Feb 2026 14:49
• Last Modified: 04 Feb 2026 14:53
• Thesis DOI: 10.5525/gla.thesis.85742
• URI: https://theses.gla.ac.uk/id/eprint/85742