Biology subjects

Olorunniji, F. J.

Publications and source records attributed to Olorunniji, F. J..

5 recordsLinked to original sources

bioRxiv2025

Structural basis of directionality control in large serine integrases

Large serine integrases (LSIs) catalyze unidirectional site-specific insertion of large DNA payloads, and in the presence of a cognate recombination directionality factor (RDF), catalyze unidirectional excision. Because neither reaction changes the net number of covalent bonds, the preferred direction must be controlled by the energetics of the changing protein-DNA complexes along these reaction pathways. However, a detailed understanding has been hampered by a lack of structural information. Here, we report 8 structures of SP{beta} integrase-DNA complexes along the integrative (-RDF) and excisive (+RDF) reaction pathways, at resolutions extending to 3.15 [A]. These complexes include tetrameric intermediates before and after strand exchange and product-bound dimers for both pathways. Our findings reveal that both recombination-induced conformational changes and RDF-mediated repositioning of the integrases coiled-coil subdomain (1) dictate which pairs of DNA sites can be assembled into a synaptic complex to initiate recombination and (2) dictate which product complexes will be conformationally locked, preventing back reactions. Critically, we find that the synaptic complex in which excision occurs is fundamentally different from that in which integration occurs. These mechanistic insights provide a conceptual framework for engineering efficient and versatile genome editing tools.

biochemistry↗

bioRxiv2024

Large serine integrases utilise scavenged phage proteins as directionality cofactors

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 recombinases second DNA binding domain and the base of the coiled coil domain.

molecular biology↗

bioRxiv2024

Identification of cognate recombination directionality factors for large serine recombinases by virtual pulldown

Integrases from the "large serine" family are simple, highly directional site-specific DNA recombinases that have great promise as synthetic biology and genome editing tools. Integrative recombination (mimicking phage or mobile element insertion) requires only integrase and two short ([~]40 - 50) DNA sites. The reverse reaction, excisive recombination, does not occur until it is triggered by the presence of a second protein termed a Recombination Directionality Factor (RDF), which binds specifically to its cognate integrase. Identification of RDFs has been hampered due to their lack of sequence conservation and lack of synteny with the phage integrase gene. Here we use Alphafold2-multimer to identify putative RDFs for more than half of a test set of 98 large serine recombinases, and experimental methods to verify predicted RDFs for 4 of 5 integrases chosen as test cases. We find no universally conserved structural motifs among known and predicted RDFs, yet they are all predicted to bind a similar location on their cognate integrase, suggesting convergent evolution of function. Our methodology greatly expands the available genetic toolkit of cognate integrase - RDF pairs.

synthetic biology↗

bioRxiv2024

Variable orthogonality of RDF-large serine integrase interactions within the phiC31 family

Large serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome (attP) and a corresponding host genome sequence (attB), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of attP and attB sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse, excisive reaction, generating new recombination sites, attR and attL. In addition to promoting attR x attL reaction, the RDF inhibits attP x attB recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases ({phi}C31, {phi}BT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both attP x attB and attR x attL reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases - RDF pairs: {phi}C31 integrase was the least selective, with all three RDFs activating it for attR x attL recombination. Similarly, {phi}C31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate {phi}C31 integrase. {phi}BT1 and TG1 RDFs were noticeably more effective than {phi}C31 RDF at inhibiting attP x attB recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase - RDF pair. The binding surface on RDF is much more conserved than the binding surface on integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase - RDF pairs. Additionally, the ability of a particular integrases preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches.

molecular biology↗

bioRxiv2019

Control of ΦC31 integrase-mediated site-specific recombination by protein trans splicing

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system for rapid regulation of site-specific recombination events in vivo. The{varphi} C31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in E. coli generated a fully functional{varphi} C31 integrase. Protein splicing is necessary for recombination activity; no activity was observed when the{varphi} C31 integrase N-and C-terminal extein domains without the intein sequences were co-expressed, nor when a key intein catalytic residue was mutated. As a proof of principle, we used a bistable switch based on an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system. We used araC and tet inducible promoters to regulate the expression of the two parts of the split recombinase. Inversion of a DNA segment containing a constitutive promoter, catalyzed by trans-spliced integrase, switches between RFP and GFP expression only when both inducible promoters are ON. We used the same split inteins to regulate the reconstitution of a split integrase-RDF fusion that efficiently catalyzed the reverse attR x attL recombination, demonstrating that our split-intein regulated recombination system can function as a reversible AND gate in which the forward reaction is catalyzed by the integrase, and the reverse reaction by the integrase-RDF fusion. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

synthetic biology↗