Rationalizing in vitro evolution by structural principles- Implications for target specificity of site-specific DNA recombinases.

• Applicant: Professorin Dr. María Teresa Pisabarro
• Subject Area: Bioinformatics and Theoretical Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 408106607

Recent advances in genome editing technology have improved our ability to manipulate DNA. The unique properties of site-specific recombinases (SSRs), which cut and re-join DNA with nucleotide precision, can be exploited for precise genome manipulation. An important step in broadening the use of SSRs in sophisticated genome engineering resides on their recognition of pre-defined target sites. However, customizing target site specificity of SSRs is not straightforward.Innovative substrate-linked in vitro evolution methods applied to SSRs have recently ensued in important clinical implications. These techniques have the advantage of not relying on pre-existing knowledge, but they are arduous and do not provide straightforward understanding of selectivity. Designing tailored high bio-specificity requires a good comprehension of the structure-function relationships ruling the system of interest. Directed molecular evolution of SSRs alters specificity by modifying protein-DNA interactions, and it is the inherent complexity of protein-DNA recognition what controls SSRs selectivity. Thus, structural knowledge can be used to rationalize and predict SSRs specificity. We have already demonstrated that structure-based in silico approaches combined with in vitro evolution provide a first step toward understanding how SSRs achieve binding specificity.In this project, we will apply structural bioinformatics approaches to investigate key molecular aspects of SSRs specificity using the evolved Brec1 recombinase as model system. Brec1 specifically recognizes a target site (loxBTR) present in the long terminal repeats of the majority of clinically relevant HIV-1 strains and subtypes, and it has been shown to excise the integrated provirus from human infected cells without measurable off-target effects (Karpinski et al. Nat. Biotech. 2016). First analysis of Brec1 molecular evolution data in comparison to other evolved SSRs has provided valuable information about mutational hot spots, which will be particularly investigated. We will use molecular modeling and simulation to establish atom-detailed protein-DNA models and study their structural and dynamic properties including interfacial hydration. In an in-depth systematic and comparative analysis including Brec/loxBTR wild type, mutational variants and also other natural and evolved SSRs, we will set up profiles for their natural/altered DNA recognition. This comprehensive analysis is expected to provide advanced understanding of the bases for SSRs specificity. The final goal is to establish a rationale for SSRs specificity, which should facilitate the rapid generation of enzymes with customized functional profiles. Rationally designed SSRs will be experimentally tested and used to enhance the predictive capabilities of our theoretical models. The outcome of our work will be applicable to the engineering of other proteins of interest, and therefore significantly advance the fields of biotechnology and biomedicine.

• DFG Programme: Research Grants
• Cooperation Partner: Professor Dr. Frank Buchholz