Both humans and mice display an age-related decline in immunity. Reduced generation of mature B cells may be a contributing factor due to reduced entry of mature B cells with novel B cell receptors and specificity for pathogens into the mature B cell pool. In aged mice the numbers of B cell precursors within the bone marrow are diminished; there is a severe reduction in numbers of pre-B cells and an increase in numbers of re-circulated mature B cells. Other defects in developing B cells include reduced expression of rag1 and rag2 when measured in total bone marrow precursor populations. In the pro-B cell stage of development rag expression is essential to the process of V(D)J recombination and the generation of pre-B cells. It was not known prior to this work if rag levels were lower in pro-B cells. In Chapter 2 I show that rag2 expression is reduced in pro-B cells of aged mice. The reduction in rag2 expression is correlated with a loss of V(D)J recombinase activity in pro-B cells and reduced numbers of pre-B cells. This suggests that in aged mice the reduction in rag2 expression is sufficient to result in reduced V(D)J recombinase activity and reduced generation of pre-B cells, thus contributing to fewer pre-B cells in aged mice. Furthermore, I have shown that the loss of rag2expression and recombinase activity in pro-B cells are the result of age-associated defects in the bone marrow-microenvironment as opposed to cell-intrinsic defects in developing precursors. In Chapter 3 of this thesis I examine genetic influences on age-related defects in murine B cell development and correlations between bone marrow B cell subsets and peripheral T cell subsets. It was known that longevity and age-related defects in T cell subsets are influenced by genetic differences between strains of inbred mice. The impact of genetic polymorphisms on age-related defects in B cell development had not been previously assessed. Nor was it known if these defects were correlated with age-related changes in peripheral T cell subsets. Here I present evidence that B cell subsets in the bone marrow are influenced by genetic polymorphisms between mice strains. Genetic polymorphisms on Chromosomes 15 and 19 were found to influence the frequency of re-circulated and pre-B cells in the bone marrow of aged mice. Frequencies of bone marrow B cell subsets were compared with peripheral T cell subsets. Interestingly, an association between the frequency of pre-B cells was not observed with either re-circulated B cells in the bone marrow nor peripheral T cell subsets. However the frequency of pre-B cells was inversely correlated with the frequency of B220intIgM+cells, a subset that was found to correlate with more advanced age-related T cell defects. In addition, frequencies of re-circulated B cells in the bone marrow were found to be associated with less advanced age-related defects in peripheral T cell subsets. These observations indicate that defects in B cell development, including reduced rag2 expression and V(D)J recombinase activity, are the result of changes in the aged murine bone marrow microenvironment. In addition, a genetic polymorphism located on Chromosome 19 influences the frequency of pre-B cells in aged mice. Furthermore the frequencies of B cell precursors in aged mice are not correlated with peripheral T cell subsets, but are correlated with frequencies of B220intIgM+ cells in the bone marrow. These observations advance our understanding of age-related defects in murine B cell development and may lead to identification of genes that influence B cell development in aged mice and humans as well as to help devise therapeutics aimed at restoring humoral immunity in aged individuals.

The Cre/loxP system belongs to the family of site-specific recombinases (SSR) that can precisely modify DNA that is flanked by two target sites. The reaction outcome is dependent on the structure and orientation of the target sites and includes excision, inversion and exchange of a DNA fragment. The system is established for more than 30 years and is active in vitro and in vivo in several organisms. These characteristics make the Cre/loxP system the ideal tool for genome editing. However, the strict target site preference for loxP limits its use to basic research where the loxP target sites can be introduced beforehand at the anticipated genomic locus. Directed evolution strategies have overcome this limitation and allow to generate Cre-like recombinases with altered DNA specificity. During this work, I developed the first dual-recombinase system based on evolved recombinases. Using two instead of one recombinase expands the targetability of the human genome by being more flexible in the target site search. After the identification of suitable target sites, I could show an evolved dual-recombinase system that can be used for excision and inversion of a human genomic locus. The recombinase mediated inversion reaction corrected a large genomic inversion that is frequently found in patients with severe Hemophilia A. Only two days after treating human cells with the developed dual-recombinase system, RecF8, 30% inversion could be detected in a human cell line. Applying RecF8 in patient specific endothelial cells corrected around 9% of the inversion back to the wild type sequence, which would be sufficient to drastically improve the quality of life of affected individuals. This genomic correction lead to the expression of the F8 gene, which is inactive elsewise. It remains to proof that the transcriptional reactivation of the F8 gene allows for the production of the Factor VIII protein. Before using RecF8 in a clinical setting, an in vivo study in a suitable mouse model is necessary. This study introduces a dual-recombinase system and thereby broadens the use of designer recombinases for genome editing. Moreover, in a proof on concept experiment this study shows that recombinases can be applied to correct large disease-causing genomic inversions in human cells. Altogether, the use of recombinases for scarless genome editing comes a step closer to reality.

Chez les bactéries à chromosomes circulaires, le crossing-over introduit par la recombinaison homologue peut conduire à des échanges de chromatides soeurs. Des nombres impairs de ces échanges aboutissent à la dimérisation des deux chromatides nouvellement répliquées compromettant ainsi leur ségrégation. Par conséquent, la plupart des bactéries utilisent le système de recombinaison spécifique de site Xer pour convertir les dimères de chromosomes et de plasmides en monomères stables. Ce système comporte deux recombinases de la famille Tyrosine recombinase, XerC et XerD, agissant sur le site dif. Cependant, quelques ε-protéobactéries n’ont besoin que d'une seule recombinase XerH agissant sur un site difH. Il parait intéressant d’étudier le système de recombinaison XerH de Campylobacter jejuni, surtout que l'augmentation spectaculaire de l'incidence de campylobactériose est alarmante. Cette étude vise à mieux comprendre comment la protéine XerH catalyse la réaction de recombinaison au niveau du site difH en mettant en évidence les séquences indispensables pour la liaison et le clivage. Grâce à ces expériences, nous avons pu confirmer que XerH est capable de se lier à la séquence entière difH; XerH est capable de cliver les deux brins supérieurs et inférieurs de difH avec une réaction plus efficace au niveau du brin inférieur; les nucléotides conservés du site de liaison sont indispensables pour la réaction de liaison; la modification de la longueur de l’espaceur améliore la réaction de liaison et de clivage et les modifications apportées au site de clivage prédit ont aboli la réaction de liaison et affecté la réaction de clivage au niveau du brin supérieur et inférieur du site difH. Ces expériences aideront à comprendre comment la recombinase XerH/difH contrôle la résolution des dimères chromosomiques chez Campylobacter jejuni en identifiant les séquences et les facteurs indispensables pour qu’un certain système soit fiable. Notre étude représente un pas vers l’avant pour comprendre un mécanisme important chez un agent pathogène ayant un grand impact sur la santé publique. / In bacteria with circular chromosomes, cross-over induced by homologous recombination can lead to sister chromatid exchanges, odd numbers of these exchanges result in dimerization of the two newly replicated chromatids compromising their segregation. Therefore, most bacteria use the Xer site-specific recombination system to convert chromosomal and plasmid dimers into stable monomers. This system involves two recombinases of the Tyrosine recombinase family, XerC and XerD, acting at the dif site. However, some ε-proteobacteria require only one XerH recombinase acting on a difH site. It seems interesting to study the XerH recombination system of Campylobacter jejuni, especially since the dramatic increase in the incidence of campylobacteriosis is alarming. This study aims to better understand how the XerH protein catalyzes the recombination reaction at the difH site by identifying the sequences required for binding as well as the factors regulating this reaction. As a result of these experiments, we were able to confirm that XerH is able to bind to the entire difH sequence; it is able to cleave both the top and bottom strands of difH with a more efficient reaction at the bottom strand; The conserved nucleotides in the binding site are essential for the binding reaction, modification of the spacer length improves the binding and cleavage reaction, and modifications in the predicted cleavage site abolished the binding reaction and affected the cleavage reaction at both the top and bottom strands of the difH site.. These experiments will help to understand how the XerH/difH recombinase controls the resolution of chromosomal dimers in Campylobacter jejuni by identifying the essential sequences and factors required for a certain system to be reliable. Our study represents a step forward in understanding an important mechanism in a pathogen with great impact on public health.

ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA. Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament. The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes. In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.

L'état circulaire du chromosome bactérien pose un problème particulier lors de la réplication. Un nombre impair d'événements de recombinaison homologue donne des chromosomes dimères concaténés qui ne peuvent pas être divisés en cellules filles. Pour résoudre ce problème, les bactéries ont mis au point un mécanisme de résolution des dimères basé sur un système de recombinaison spécifique au site. Ceci est effectué par le système Xer/dif. Dans ce système, les protéines Xer effectuent une réaction de recombinaison dans le site dif au niveau du septum cellulaire immédiatement avant la division cellulaire. Dans la plupart des bactéries, cette réaction est effectuée par deux recombinases, XerC et XerD. Cependant, Streptococcus suis, un agent pathogène zoonotique important utilise un système de recombinaison différent, constitué d'une seule enzyme recombinase appelée XerS, qui catalyse la réaction de recombinaison dans un site dif non conventionnel. Pour caractériser le mode de clivage de XerS, des expériences EMSA ont été réalisées en utilisant des fragments de PCR marqués par HEX et des "suicide substrates". Nos données suggèrent que 1.) XerS est capable de lier la séquence entière de difSL; 2.) XerS lie plus efficacement le côté gauche des mutants difSL incomplets que le côté droit; 3.) XerS coupe les brins supérieur et inférieur du site difSL, avec une réaction plus efficace au bas. 4.) Modifications des nucléotides de la région la plus externe ou de la région centrale changent les préférences de clivage. 5.) XerS n'a montré aucune activité spécifique sur un autre site dif non conventionnel des Firmicutes, 6.) XerS interagit avec la sous-unité FtsK-y. L'ensemble des résultats présentés permet de mieux comprendre le fonctionnement de la recombinaison XerS dans le système de recombinase unique de Streptococcus et comment cette recombinaison est régulée par des facteurs de l'hôte. / The circular state of the bacterial chromosome presents a specific problem during replication. An odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. To solve this problem, bacteria have developed a mechanism of dimer resolution based on site-specific recombination system. This is performed by the Xer/dif system. In this system, the Xer proteins perform a recombination reaction in the dif site at the cell septum immediately prior to cell division. In most bacteria this reaction is performed by two recombinases, XerC and XerD. However, an important zoonotic pathogen; Streptococcus suis harbors a different recombination system, composed by a single recombinase enzyme called XerS, that catalyzes the recombination reaction in an unconventional dif site; difSL. A region characterized by two imperfect inverted repeat regions that flank a central region of 11 bp.To characterize the mode of cleavage of XerS, EMSA experiments were performed by using HEX-labelled PCR fragments and “nicked suicide substrates”. Our data suggests that; 1.) XerS is able to bind the entire difSL sequence; 2.) XerS binds more efficiently the left half side on incomplete difSL mutants than the right half side; 3.) XerS cleaves both the top and bottom strands of the difSL site, with a more efficient reaction at the bottom strand; 4.) Nucleotides at the outermost region of a T rich region seem to be determinant for binding selectivity and modifications of the extra spacing between the inverted repeat arms as well as length modifications of the central region change cleavage preference. 5.) XerS did not show any specific activity on another unconventional dif site in Firmicutes, as tested on difH. 6.) XerS interacts with FtsK-y subunit. This research aims to understand how XerS recombination works in the single recombinase system of Streptococcus and how this recombination is regulated by host factors. Exploration of these recombinases will provide a better understanding of the mechanisms of DNA exchange and genome stability in bacteria. It can also increase our knowledge of the evolution and speciation of recombinogenic bacteria.

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming. To accelerate the development of designer-recombinases I evaluated two sequencing approaches and gathered the sequence information of over two million Cre-like recombinase sequences evolved for 89 different target sites. With this information I first investigated the sequence compositions and residue changes of the recombinases to further our understanding of their target site selectivity. The complexity of the data led me to a generative deep learning approach. Using the sequence data I trained a conditional variational autoencoder called RecGen (Recombinase Generator) that is capable of generating novel recombinases for a given target site. With computational evaluation of the sequences I revealed that known recombinases functional on the desired target site are generally more similar to the RecGen predicted recombinases than other recombinase libraries. Additionally, I could experimentally show that predicted recombinases for known target sites are at least as active as the evolved recombinases. Finally, I also experimentally show that 4 out of 10 recombinases predicted for novel target sites are capable of excising their respective target sites. As a bonus to RecGen I also developed a new method capable of accurate sequencing of recombinases with nanopore sequencing while simultaneously counting DNA editing events. The data of this method should enable the next development iteration of RecGen.

Genome engineering, the deliberate alteration of an organism's genetic material, has revolutionized biotechnology and biomedical research, enabling precise modifications to DNA sequences. Among the tools developed for this purpose, site-specific recombinases (SSRs) stand out for their ability to catalyze targeted DNA rearrangements between defined target sites. The Cre/loxP system, in particular, has been widely used for conditional gene inactivation and recombinase-mediated cassette exchange, facilitating targeted DNA excision, inversion, or integration through the recognition and recombination of loxP target sites. While the inherent specificity of Cre towards the loxP target sequence has been invaluable, it also limits its application to other genomic loci of therapeutic interest. Understanding the factors that govern the enzyme’s DNA specificity opens the possibility to engineer and retarget the complex to non-native sequences, significantly broadening the range of targetable genomic loci. To address this challenge, I describe the development of a high-throughput method to quantify Cre recombination efficiency across a library of loxP-like spacer variants. This method systematically analyzes the impact of spacer sequence alterations to reveal DNA specificity determinants. Through comprehensive screening, the study identified spacer sequences that exhibit inefficient recombination by Cre, despite both full lox sites having matching spacer sequences. Directed evolution was used to enhance Cre activity on these previously 'inert' spacer sequences, generating variants with altered spacer specificity. Detailed molecular analyses, including mutational studies and molecular dynamics simulations, elucidated the structural basis for altered spacer selectivity in evolved Cre variants. The study provides mechanistic insights into the role of specific amino acid residues in determining spacer specificity and highlights the potential for the rational design of recombinases with tailored spacer preferences. Building upon this foundation, I describe the engineering of heterospecific Cre-type SSRs capable of recombining asymmetric DNA target sites. By combining two evolved Cre variants with unique half-site specificities, a functional heterotetrameric complex forms, capable of excising DNA fragments flanked by asymmetric target sequences naturally occurring in the human genome. This approach expands the applicability of SSRs and holds promise for correcting chromosomal inversions underlying genetic disorders, as demonstrated in the correction of the int1h inversion associated with hemophilia A. However, harnessing the full potential of heterospecific SSRs presents challenges, particularly concerning off-target effects resulting from the formation of undesired functional homotetrameric complexes. To mitigate these risks, I investigated strategies to render SSR monomers functionally active in heterotetrameric, but not homotetrameric complexes. Through substrate-linked directed evolution, I identified mutations that confer obligate heterospecificity, leading to safer and more precise genome engineering applications. Together, these studies highlight the transformative potential of engineered SSRs in genome editing and underscore the importance of ongoing research efforts to enhance their specificity, efficacy, and safety for therapeutic interventions and biotechnological applications. By manipulating the highly specific Cre/loxP complex to retarget different lox sequences and analyzing evolved or naturally occurring recombinase recombination specificity, we can better understand how these enzymes can be optimized for therapeutic applications. Furthermore, the ability to confer obligate heterospecificity increases the overall safety of these engineered SSRs, expanding their potential applications in genome engineering, particularly for therapeutic targets that require editing asymmetric (non-palindromic) target sites.