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PreprintCited by 6CRISPR and Genetic EngineeringBacterial Genetics and BiotechnologyBiochemical and Structural Characterization

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Large serine integrases utilise scavenged phage proteins as directionality cofactors

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Alsaleh, Abdulrazak, Pena Reyes, Tania, Baksh, Aron, Taiwo-Aiyerin, Oluwateniola T., Holland, Alexandria, Pigli, Ying, Rice, Phoebe A., Olorunniji, Femi J.·2024·Nucleic Acids Research
DOI 10.1101/2024.08.21.608926PMC PMC11795197

Preprint — published as 10.1093/nar/gkaf050

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Abstract

Recombination directionality factors (RDFs) for large serine integrases (LSIs) are cofactor proteins that control the directionality of recombination to favor excision over insertion. Although RDFs are predicted to bind their cognate LSIs in similar ways, there is no overall common structural theme across LSI RDFs, leading to the suggestion that some of them may be moonlighting proteins with other primary functions. To test this hypothesis, we searched for characterized proteins with structures similar to the predicted structures of known RDFs. Our search shows that the RDFs for two LSIs, TG1 integrase and Bxb1 integrase, show high similarities to a single stranded DNA binding (SSB) protein and an editing exonuclease, respectively. We present experimental data to show that TG1 RDF is a functional SSB protein. We used mutational analysis to validate the integrase-RDF interface predicted by AlphaFold2 multimer for TG1 integrase and its RDF, and establish that control of recombination directionality is mediated via protein-protein interaction at the junction of recombinase’s second DNA binding domain and the base of the coiled coil domain.

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Graphical Abstract CC BY-NC · PubMed Central
Figure 1. Phage integration and excision by LSIs. (A) Phage-encoded integrase (green ovals) catalyse the integration of phage DNA at attP site (blue arrow) into the bacterial host genome at an attB site (amber). The recombination reaction results in the formation of recombinant attR and attL sites flanking the integrated prophage genome. In the reverse excision reaction, the RDF (grey ovals) is expressed from the prophage genome and binds to the integrase to promote attR × attL recombination. (B) Domain architecture of LSIs. The catalytic domain, cat (green) and the α-E helix, αE (green) mediate catalysis and provide the subunit rotation interface. The two DNA-binding domains (DBD1 and DBD2) mediate DNA binding and sequence specificity. DBD1 also known as ‘recombinase domain’ or RD is shown in pink. DBD2, also known as the zinc-binding domain or ZD is shown in yellow. The CC domain (orange) is embedded within DBD2 and mediate directionality control. The DBD2-proximal region in the CC domain is shown in pale green. (C) Schematic illustration of serine integrase domain interactions with the recombination site (attP) and the RDF based on structure of LI integrase-attP complex [53] and AlphaFold multimer-predicted interaction of the RDF with DBD2-proximal region of the CC domain [19, 31]). Colors are matched among Figs 1, 6, and Supplementary Fig. S4.
Figure 1. Phage integration and excision by LSIs. (A) Phage-encoded integrase (green ovals) catalyse the integration of phage DNA at attP site (blue arrow) into the bacterial host genome at an attB site (amber). The recombination reaction results in the formation of recombinant attR and attL sites flanking the integrated prophage genome. In the reverse excision reaction, the RDF (grey ovals) is expressed from the prophage genome and binds to the integrase to promote attR × attL recombination. (B) Domain architecture of LSIs. The catalytic domain, cat (green) and the α-E helix, αE (green) mediate catalysis and provide the subunit rotation interface. The two DNA-binding domains (DBD1 and DBD2) mediate DNA binding and sequence specificity. DBD1 also known as ‘recombinase domain’ or RD is shown in pink. DBD2, also known as the zinc-binding domain or ZD is shown in yellow. The CC domain (orange) is embedded within DBD2 and mediate directionality control. The DBD2-proximal region in the CC domain is shown in pale green. (C) Schematic illustration of serine integrase domain interactions with the recombination site (attP) and the RDF based on structure of LI integrase-attP complex [53] and AlphaFold multimer-predicted interaction of the RDF with DBD2-proximal region of the CC domain [19, 31]). Colors are matched among Figs 1, 6, and Supplementary Fig. S4.CC BY-NC · PubMed Central
Figure 2. Structural similarities between Bxb1 and TG1 RDFs and replication proteins. (A) Comparison of the predicted Bxb1 RDF structure (left) to the crystal structure of the proofreading exonuclease subunit of PolD {right; PDB: 5IHE; [37]}. The related portions of each structure are shaded from N (blue) to C (red). Zinc ions are violet and iron ions brown. One nucleotide from the predicted structure of Bxb1 RDF with dT6 is shown in gray, as is the single nucleotide seen in the active site of the exonuclease. (B) Top: comparison of the predicted TG1 RDF structure (left) to that of phage T4 SSB protein core {right; PDB: 1GPC; [46]}. Bottom: surface representations of each colored by electrostatic potential (blue = +5 kT/e, shading to red = −5 kT/e). N and C termini, or 1st and last residues within the structure shown, are labeled.
Figure 2. Structural similarities between Bxb1 and TG1 RDFs and replication proteins. (A) Comparison of the predicted Bxb1 RDF structure (left) to the crystal structure of the proofreading exonuclease subunit of PolD {right; PDB: 5IHE; [37]}. The related portions of each structure are shaded from N (blue) to C (red). Zinc ions are violet and iron ions brown. One nucleotide from the predicted structure of Bxb1 RDF with dT6 is shown in gray, as is the single nucleotide seen in the active site of the exonuclease. (B) Top: comparison of the predicted TG1 RDF structure (left) to that of phage T4 SSB protein core {right; PDB: 1GPC; [46]}. Bottom: surface representations of each colored by electrostatic potential (blue = +5 kT/e, shading to red = −5 kT/e). N and C termini, or 1st and last residues within the structure shown, are labeled.CC BY-NC · PubMed Central
Figure 3. Assay of exonuclease activities of Bxb1 RDF. (A) Design of DNA substrate for 3′-5′ exonuclease activity. The DNA substrate is a 27mer top strand labelled with a 5′ fluorescein label (5′-/56-FAM), and with four non-complementary bases at the 3′ end annealed to a 60mer unlabelled bottom strand. The region of the top and bottom strands with complementary base sequences are depicted in blue, the non-complementary bases are in red, and the single-stranded region of the 60mer are shown in grey. (B) Reactions were carried out for 30 or 60 min in the reaction buffer described in the ‘Materials and methods’ section (Bxb1 RDF, 2 and 0.5 μM DNA). Reaction products were analysed on 1.2% denaturing polyacrylamide gel electrophoresis.
Figure 3. Assay of exonuclease activities of Bxb1 RDF. (A) Design of DNA substrate for 3′-5′ exonuclease activity. The DNA substrate is a 27mer top strand labelled with a 5′ fluorescein label (5′-/56-FAM), and with four non-complementary bases at the 3′ end annealed to a 60mer unlabelled bottom strand. The region of the top and bottom strands with complementary base sequences are depicted in blue, the non-complementary bases are in red, and the single-stranded region of the 60mer are shown in grey. (B) Reactions were carried out for 30 or 60 min in the reaction buffer described in the ‘Materials and methods’ section (Bxb1 RDF, 2 and 0.5 μM DNA). Reaction products were analysed on 1.2% denaturing polyacrylamide gel electrophoresis.CC BY-NC · PubMed Central
Figure 4. TG1 RDF is a SSB protein in vitro. Electrophoretic mobility shift assays of TG1 RDF binding to ssDNA (left) and dsDNA (right). The protein was present at 0, 200, 400, and 800 nM and the 50 nt/bp fluorescein-labeled DNA substrate was kept constant at 200 nM.
Figure 4. TG1 RDF is a SSB protein in vitro. Electrophoretic mobility shift assays of TG1 RDF binding to ssDNA (left) and dsDNA (right). The protein was present at 0, 200, 400, and 800 nM and the 50 nt/bp fluorescein-labeled DNA substrate was kept constant at 200 nM.CC BY-NC · PubMed Central
Figure 5. Single-strand DNA interferes with recombination functions of TG1 RDF in vitro. (A) Scheme depicting the in vitro intramolecular recombination reaction. Recombination of the attP × attB plasmid substrate (pTG1-PBX) gives two circular products in which the attR and attL sites are separated. Presence of the cognate RDF inhibits attP × attB recombination reaction. For the attR × attL recombination reaction, in the presence of the cognate RDF, the starting substrate plasmid (pTG1-RLX) is recombined to give attP and attB sites on separate circular plasmid products. (B) Sequences of recombination att sites for TG1 integrase. The effects of ssDNA on RDF function in in vitro recombination reactions are shown in panels (C–E). (C) attR x attL in the presence of RDF and ssDNA, (D) attP × attB in the presence of RDF and ssDNA, and (E) attR × attL in the presence of ssDNA, without RDF. In all cases, reactions were carried out for 2 h in the reaction buffer described in the ‘Materials and methods’ section (integrase, 800 nM; RDF, 800 nM), with the addition of 0, 0.5, and 1.0 μM ssDNA (polydT) as indicated. Reaction products were digested with the restriction endonuclease NruI prior to 1.2% agarose gel electrophoresis. The bands on the gel are labeled nr (non-recombinant, i.e. substrate), rec (recombination product).
Figure 5. Single-strand DNA interferes with recombination functions of TG1 RDF in vitro. (A) Scheme depicting the in vitro intramolecular recombination reaction. Recombination of the attP × attB plasmid substrate (pTG1-PBX) gives two circular products in which the attR and attL sites are separated. Presence of the cognate RDF inhibits attP × attB recombination reaction. For the attR × attL recombination reaction, in the presence of the cognate RDF, the starting substrate plasmid (pTG1-RLX) is recombined to give attP and attB sites on separate circular plasmid products. (B) Sequences of recombination att sites for TG1 integrase. The effects of ssDNA on RDF function in in vitro recombination reactions are shown in panels (C–E). (C) attR x attL in the presence of RDF and ssDNA, (D) attP × attB in the presence of RDF and ssDNA, and (E) attR × attL in the presence of ssDNA, without RDF. In all cases, reactions were carried out for 2 h in the reaction buffer described in the ‘Materials and methods’ section (integrase, 800 nM; RDF, 800 nM), with the addition of 0, 0.5, and 1.0 μM ssDNA (polydT) as indicated. Reaction products were digested with the restriction endonuclease NruI prior to 1.2% agarose gel electrophoresis. The bands on the gel are labeled nr (non-recombinant, i.e. substrate), rec (recombination product).CC BY-NC · PubMed Central
Figure 6. Integrase-RDF interface that mediates regulation of recombination. (A) Surface complementary of the predicted TG1 integrase–RDF interface. Left: the C-terminal domain of the integrase is shown as ribbons (DBD2, yellow; DBD2-proximal portion of the CC, pale green; CC pale blue), with DNA docked based on 4kis.pdb to guide the eye [53]. A surface representation of the RDF is shown, colored by electrostatic potential (blue = +5 kT/e, shading to red = −5 kT/e). Right: the RDF is shown as a gray ribbon, with the integrase surface colored by electrostatic potential (same scale). Side chains that were mutated are shown as colored sticks (see panel B). The C-terminus of the integrase (600–619) and the N-terminus [1–22] of the RDF are not shown as they were predicted to be disordered. (B) Table of mutations made. (C) The model shown in panel (A), with side chains mutated shown as sticks, color coded as in panel (B). (D, E): close-up views of integrase–RDF interactions and the side chains that were mutated.
Figure 6. Integrase-RDF interface that mediates regulation of recombination. (A) Surface complementary of the predicted TG1 integrase–RDF interface. Left: the C-terminal domain of the integrase is shown as ribbons (DBD2, yellow; DBD2-proximal portion of the CC, pale green; CC pale blue), with DNA docked based on 4kis.pdb to guide the eye [53]. A surface representation of the RDF is shown, colored by electrostatic potential (blue = +5 kT/e, shading to red = −5 kT/e). Right: the RDF is shown as a gray ribbon, with the integrase surface colored by electrostatic potential (same scale). Side chains that were mutated are shown as colored sticks (see panel B). The C-terminus of the integrase (600–619) and the N-terminus [1–22] of the RDF are not shown as they were predicted to be disordered. (B) Table of mutations made. (C) The model shown in panel (A), with side chains mutated shown as sticks, color coded as in panel (B). (D, E): close-up views of integrase–RDF interactions and the side chains that were mutated.CC BY-NC · PubMed Central
Figure 7. In vitro recombination activities of TG1 integrase mutants. Reactions were carried out for 2 or 16 h in the reaction buffer described in the ‘Materials and methods’ section. (A) Two hours reaction, attP × attB. Integrase was not added to reaction in lane 1. (B) Sixteen hours reaction, attP × attB. Integrase was not added to reaction in lane 1. (C) Two hours reaction, attR × attL. Integrase was not added to reaction in lane 1. (D) Sixteen hours reaction, attR × attL. Integrase was not added to reaction in lanes 1. Reaction products were digested with the restriction endonuclease NruI prior to 1.2% agarose gel electrophoresis. The bands on the gel are labeled nr (non-recombinant, i.e. substrate), rec (recombination product). The final integrase concentrations in all reactions were 800 nM. The final RDF concentrations were 800 nM (+) or 1600 nM (++). The bands on the gel are labelled nr (non-recombinant, i.e. substrate) or rec (recombination product). The sizes of the products of the recombination reactions are shown alongside each band on the gel image. MUT1: TG1 Int-3K (E537K, D541K, D545K). MUT2: TG1 Int-2K (E442K, D444K). MUT3: TG1 Int-2A (W538A, W552A).
Figure 7. In vitro recombination activities of TG1 integrase mutants. Reactions were carried out for 2 or 16 h in the reaction buffer described in the ‘Materials and methods’ section. (A) Two hours reaction, attP × attB. Integrase was not added to reaction in lane 1. (B) Sixteen hours reaction, attP × attB. Integrase was not added to reaction in lane 1. (C) Two hours reaction, attR × attL. Integrase was not added to reaction in lane 1. (D) Sixteen hours reaction, attR × attL. Integrase was not added to reaction in lanes 1. Reaction products were digested with the restriction endonuclease NruI prior to 1.2% agarose gel electrophoresis. The bands on the gel are labeled nr (non-recombinant, i.e. substrate), rec (recombination product). The final integrase concentrations in all reactions were 800 nM. The final RDF concentrations were 800 nM (+) or 1600 nM (++). The bands on the gel are labelled nr (non-recombinant, i.e. substrate) or rec (recombination product). The sizes of the products of the recombination reactions are shown alongside each band on the gel image. MUT1: TG1 Int-3K (E537K, D541K, D545K). MUT2: TG1 Int-2K (E442K, D444K). MUT3: TG1 Int-2A (W538A, W552A).CC BY-NC · PubMed Central
Figure 8. In vitro recombination activities of TG1 RDF mutants. Reactions were carried out for 2 h in the reaction buffer described in the ‘Materials and methods’ section. (A) attP × attB recombination reactions. (B) attR×attL recombination reactions. Reaction products were digested with the restriction endonuclease NruI prior to 1.2% agarose gel electrophoresis. TG1 integrase (800 nM) was used in all reactions. Integrase was not added to reactions in lane 1 of panels (A) and (B). The final RDF concentrations were 0 nM (-), 800 nM (+), or 1600 nM (++). The bands on the gel are labelled nr (non-recombinant, i.e. substrate) or rec (recombination product). The sizes of the products of the recombination reactions are shown alongside each band on the gel image. MUT4: TG1 RDF (R212D, R216D, K218D). MUT5: TG1 RDF (Δ23). MUT6: TG1 RDF (F208T, Y222D). MUT7: TG1 RDF (R28E H30D). MUT8: TG1 RDF (W107A).
Figure 8. In vitro recombination activities of TG1 RDF mutants. Reactions were carried out for 2 h in the reaction buffer described in the ‘Materials and methods’ section. (A) attP × attB recombination reactions. (B) attR×attL recombination reactions. Reaction products were digested with the restriction endonuclease NruI prior to 1.2% agarose gel electrophoresis. TG1 integrase (800 nM) was used in all reactions. Integrase was not added to reactions in lane 1 of panels (A) and (B). The final RDF concentrations were 0 nM (-), 800 nM (+), or 1600 nM (++). The bands on the gel are labelled nr (non-recombinant, i.e. substrate) or rec (recombination product). The sizes of the products of the recombination reactions are shown alongside each band on the gel image. MUT4: TG1 RDF (R212D, R216D, K218D). MUT5: TG1 RDF (Δ23). MUT6: TG1 RDF (F208T, Y222D). MUT7: TG1 RDF (R28E H30D). MUT8: TG1 RDF (W107A).CC BY-NC · PubMed Central
Figure 9. Proposed pathway for evolution of new RDFs. Recombination of two simultaneously activated prophages yields progeny with an integrase (Int) gene from prophage 1 but no cognate RDF gene (top). The chimeric phage can establish a new lysogen (middle left) but is unable to excise from the host chromosome until it evolves to use another phage protein (pink arrow) as an RDF. The mutations that allow the use of a new RDF could occur during the prophage state or during replication and could occur in either the integrase or the RDF (thus the resulting Int and new RDF genes are shown as purple and lavender rather than the original dark blue and pink).
Figure 9. Proposed pathway for evolution of new RDFs. Recombination of two simultaneously activated prophages yields progeny with an integrase (Int) gene from prophage 1 but no cognate RDF gene (top). The chimeric phage can establish a new lysogen (middle left) but is unable to excise from the host chromosome until it evolves to use another phage protein (pink arrow) as an RDF. The mutations that allow the use of a new RDF could occur during the prophage state or during replication and could occur in either the integrase or the RDF (thus the resulting Int and new RDF genes are shown as purple and lavender rather than the original dark blue and pink).CC BY-NC · PubMed Central

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Citations

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Metadata

Authors
Alsaleh, A., Pena Reyes, T., Baksh, A., Taiwo-Aiyerin, O. T., Holland, A., Pigli, Y., Rice, P. A., & Olorunniji, F. J.
Affiliations
  • Liverpool John Moores University (GB)
  • University of Chicago (US)
Corresponding author
  • Rice, Phoebe A.
  • Olorunniji, Femi J.
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2024-08-21
Volume
53
Issue
3
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2024.08.21.608926
PMC
PMC11795197
OpenAlex
W4407177236
Cited by
6
References
1
Citations by year
2026: 4 · 2025: 2
FWCI
1.80
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Liverpool John Moores University (acceptedVersion, cc-by)
Funders
  • National Science Foundation (2223480)
  • Directorate for Biological Sciences
  • Biotechnology and Biological Sciences Research Council (BB/X012085/1, BBSRC BB/X012085/1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
Bxb1 integrase, single-stranded DNA binding protein, exonuclease, moonlighting proteins, protein-protein interaction, coiled-coil domain, site-specific recombination
MeSH terms
Amino Acid Sequence, Bacteriophages, Coenzymes, DNA-Binding Proteins, Exonucleases, Models, Molecular, Protein Binding, Recombination, Genetic, Serine, Viral Proteins, Integrases

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