IntpTN3, a unique RecA-independent homology-directed recombinase: mechanism, structure, partners and applications – IntpTN3
(1) The first approach deals with the exploration of the protein component(s) of the in vitro HR reaction. We have characterized several HR and non-HR hyperthermophilic integrases. By domain swapping and the generation of mutant enzymes, we aim at dissociating the two recombination activities and uncovering the domains involved in HR. The resolution of the tridimensional structure of IntpTN3 either alone or with DNA substrates will be instrumental in determining the accurate molecular mechanism of IntpTN3 HR. As recombinases often require secondary factor for full activity, we will employ state of the art technologies to detect and identify additional protein partners involved in IntpTN3 HR.
(2) In the second approach, we will explore the DNA component of the in vitro HR reaction. Molecular mechanisms observed in vivo involve pairing of chromosomal regions with similar or identical DNA sequence and occur in the absence of DNA breakage. An array of DNA templates with complete or interspersed homology will be constructed and assayed in vitro in the presence of IntpTN3. It has been suggested in addition that particular structures such as paranemic DNA (PX-DNA) are formed in homologous pairing but formal proofs are still lacking. By driving the HR reactions to completion, IntpTN3 will allow to investigate in vitro for the first time the topology of DNA substrates involved in these fundamental mechanisms.
(3) These approaches will contribute to better understand and also improve the in vitro IntpTN3 HR reaction. A proof of concept will demonstrate and measure the efficiency of this reaction using molecular biology techniques and bacterial genetics. This approach will pave the way to the development of new technologies in chromosome manipulation or gene therapy.
We have made substantial advances in understanding the site-specific and homologous recombination (HR) mechanisms of the IntpTN3 integrase of T. nautili. We have demonstrated and published a new evolution system allowing these integrases not only to maintain themselves but also to evolve and constitute new site specificities (Badel et al, 2020). We took stock and highlighted the particular characteristics of all Archean tyrosine recombinases in a recently published guest review article (Badel et al, 2021). Armed with all this knowledge, we developed the methodology for constructing hybrid integrases and rationalized their overproduction and purification. We were interested in identifying the domains responsible for the site-specific recombination activity of the integrases IntpTN3 and IntpTF1. These enzymes catalyze the integration of the plasmids pTN3 and pTF1 into the tRNAtRNA gene of T. nautili and the tRNASer gene of T. fumicolans, respectively. The construction of 9 hybrid integrases exhibiting a gradual exchange between the domains of these two enzymes revealed that the specificity depends on the C-terminal region (residues 150 to 450) while the N-terminal region (residues 1 to 144) is equivalent. Secondly, we were interested in the HR properties of these integrases. Using a new genomic analysis web service that we have developed, BAGET (Hepp et al, 2021) we have identified three additional integrases closely related to IntpTN3 and recognizing the same tRNALeu gene. After overproduction and purification, only one of these integrases (IntIRI33C) has, like IntpTN3, the capacity to produce HR. The very low number of substitutions and the construction of new hybrids between enzymes producing this HR or not will allow us to easily identify the domains responsible for this activity.
Spectacular preliminary results have been obtained since the onset of this project and we anticipate further essential data and important breakthroughs gaining publication in high-impact scientific journals. Selected original aspects of this research aim at generating industrial applications in gene therapy will be patent-protected by our trustees.
1: Hepp B, Da Cunha V, Lorieux F, Oberto J. BAGET 2.0: an updated web tool for the effortless retrieval of prokaryotic gene context and sequence. Bioinformatics. 2021 Feb 3:btab082. doi: 10.1093/bioinformatics/btab082. Epub ahead of print. PMID: 33532841.
2: Badel C, Da Cunha V, Oberto J. Archaeal tyrosine recombinases. FEMS Microbiol Rev. 2021 Feb 1:fuab004. doi: 10.1093/femsre/fuab004. Epub ahead of print. PMID:33524101.
3: Badel C, Da Cunha V, Forterre P, Oberto J. Pervasive Suicidal Integrases in Deep-Sea Archaea. Mol Biol Evol. 2020 Jun 1;37(6):1727-1743. doi:10.1093/molbev/msaa041. PMID: 32068866
We published recently the discovery of a new plasmid-encoded integrase (IntpTN3) from the hyperthermophilic anaerobic archaea Thermococcus nautili. In addition the canonical site-specific integration, excision and inversion properties, this tyrosine recombinase displays unprecedented activities. IntpTN3 is responsible for the generation of a number of large scale chromosomal inversion events in vivo, in its natural host T. nautili. We successfully reproduced these inversions in a purified in vitro system therefore exposing the very unusual homologous recombination (HR) properties of this enzyme. IntpTN3 able to promote HR reactions between any two segments of DNA of identical sequence as short as 100bp. Cellular HR is an essential and complex mechanism occurring in every organism; it requires energy, a number of coordinated proteins complexes and processes such as DNA synthesis. Contrarily to cellular HR, IntpTN3-promoted HR only require the enzyme and its DNA substrate: it be achieved efficiently in the test tube. The present project aims to decipher the molecular mechanisms of IntpTN3 recombination in order to improve our fundamental knowledge of HR and to use this knowledge to develop novel biotechnological applications. The project will follow three complementary approaches :
(1) The first approach deals with the exploration of the protein component(s) of the in vitro HR reaction. We have characterized several HR and non-HR hyperthermophilic integrases. By domain swapping and the generation of mutant enzymes, we aim at dissociating the two recombination activities and uncovering the domains involved in HR. The resolution of the tridimensional structure of IntpTN3 either alone or with DNA substrates will be instrumental in determining the accurate molecular mechanism of IntpTN3 HR. As recombinases often require secondary factor for full activity, we will use we will employ state of the art technologies to detect and identify additional protein partners involved in IntpTN3 HR. (2) In the second approach, we will explore the DNA component of the in vitro HR reaction. In a number of reports, molecular mechanisms observed in vivo involve pairing of chromosomal regions with similar or identical DNA sequence occurs in the absence of DNA breakage. It has been suggested that particular structures such as paranemic DNA (PX-DNA) are formed in homologous pairing but formal proofs are still lacking. By driving the HR reactions to completion, IntpTN3 will allow to investigate in vitro for the first time the topology of DNA substrates involved in these fundamental mechanisms. (3) The previous approaches will contribute to better understand and also improve the in vitro IntpTN3 HR reaction. We are proposing a proof of concept based on this knowledge which will pave the way to the development of new technologies which could be used in chromosome manipulation or gene therapy.
Spectacular preliminary results have been obtained since the onset of this project and we anticipate further essential data and important breakthroughs gaining publication in high-impact scientific journals. Selected original aspects of this research aim at generating industrial applications and will be patent-protected by our trustees.
Project coordination
Jacques OBERTO (Institut de Biologie Intégrative de la Cellule)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
MICALIS MICrobiologie de l'ALImentation au service de la Santé
LM2E LABORATOIRE DE MICROBIOLOGIE DES ENVIRONNEMENTS EXTRÊMES
Pasteur INSTITUT PASTEUR
I2BC Institut de Biologie Intégrative de la Cellule
Help of the ANR 454,811 euros
Beginning and duration of the scientific project:
September 2019
- 48 Months
