Mutationism and the Dual Causation of Evolutionary Change

The rediscovery of Mendel's laws a century ago launched the science that William Bateson called "genetics," and led to a new view of evolution combining selection, particulate inheritance, and the newly characterized phenomenon of "mutation." This "mutationist" view clashed with the earlier view of Darwin, and the later "Modern Synthesis," by allowing discontinuity, and by recognizing mutation (or more properly, mutation-and-altered-development) as a source of creativity, direction, and initiative. By the mid-20th century, the opposing Modern Synthesis view was a prevailing orthodoxy: under its influence, "evolution" was redefined as "shifting gene frequencies," that is, the sorting out of pre-existing variation without new mutations; and the notion that mutation-and-altered-development can exert a predictable influence on the course of evolutionary change was seen as heretical. Nevertheless, mutationist ideas re-surfaced: the notion of mutational determinants of directionality emerged in molecular evolution by 1962, followed in the 1980s by an interest among evolutionary developmental biologists in a shaping or creative role of developmental propensities of variation, and more recently, a recognition by theoretical evolutionary geneticists of the importance of discontinuity and of new mutations in adaptive dynamics. The synthetic challenge presented by these innovations is to integrate mutation-and-altered-development into a new understanding of the dual causation of evolutionary change--a broader and more predictive understanding that already can lay claim to important empirical and theoretical results--and to develop a research program appropriately emphasizing the emergence of variation as a cause of propensities of evolutionary change

Homology inference with specific molecular constraints

Evolutionary processes can be considered at multiple levels of biological organization. The work developed in this thesis focuses on protein molecular evolution. Although proteins are linear polymers composed from a basic set of 20 amino acids, they generate an enormous variety of form and function. Proteins that have arisen by a common descent are classified into families; they often share common properties including similarities in sequence, structure, and function. Multiple methods have been developed to infer evolutionary relationships between proteins and classify them into families. Yet, those generic methods are often inaccurate, especially when specific protein properties limit their applications. In this thesis, we analyse two protein classes that are often difficult for the evolutionary analysis: the coiled-coils – repetitive protein domains defined by a simple widespread peptide motif (chapters 2 and 3) and Rab small GTPases – a large family of closely related proteins (chapters 4 and 5). In both cases, we analyse the specific properties that determine protein structure and function and use them to improve their evolutionary inference

Compact Cas9s and Their Natural Inhibitors for Genome Editing

Recent advances with the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as genome editing tools have opened a new avenue for targeting disease-causing mutations. The programmability of the Cas9 endonuclease by RNA makes it a potentially powerful therapeutic tool to correct such mutations. The CRISPR-Cas9 system consists of a Cas9 endonuclease that is guided by RNA (sgRNA) to create double-stranded breaks in a target DNA segment complementary to the guide. This process is dependent on a 2-8 nucleotide sequence (called PAM) that is adjacent to the target and functions as a Cas9 binding signal. Each Cas9 ortholog recognizes a unique PAM. However, factors such as the size of Cas9 or the frequency of its PAM sequence in the genome have hindered its clinical use. The Cas9 from Streptococcus pyogenes (SpyCas9) is commonly used in research because its PAM (NGG, where “N” symbolizes any nucleotide) is present every ~8 bp in the genome, providing robust targeting potential. However, it is too large to fit into typical viral vectors used for in vivo delivery, namely adeno-associated vectors (AAV). While several Cas9 orthologs have been characterized, none satisfied the need for a compact, accurate Cas9 with a short PAM. In this thesis, we use two approaches to identify new compact Cas9 orthologs with small PAMs, one using anti-CRISPR proteins and one by searching through closely related Cas9s. First, we use the presence of anti-CRISPRs (naturally occurring, phage-encoded peptides that inhibit CRISPR-Cas9 described in chapter 2) in a genome as indicators of Cas9s that may be highly active. These orthologs come with the added advantage of having inhibitors that can be used as off-switches. We characterize four Cas9s that are targeted by anti-CRISPR proteins and show that they recognize diverse PAMs in vitro. One of the four Cas9’s, namely HpaCas9 from Haemophilus parainfluenzae, induces efficient genome editing in mammalian cells. However, its long N4GATTT PAM does not satisfy the short PAM criterion. For our second approach, we asked whether closely related Cas9 orthologs with drastically different PAM-interacting domains (PIDs, the domain responsible for PAM recognition) recognize different PAMs, and if so, can be used for genome editing. To this end, we exploited natural variation in the PID of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis (Nme2Cas9). Nme2Cas9 recognizes a simple dinucleotide PAM (N4CC) that provides a high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA into adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Finally, we show preliminary data on how CRISPR-Cas9 can be used for therapeutic genome editing for Amytrophic Lateral Sclerosis. Our new findings promise to accelerate the development of genome editing tools for biomedical and therapeutic applications

A phylogenomics approach to resolving fungal evolution, and phylogenetic method development

Bien que les champignons soient régulièrement utilisés comme modèle d'étude des systèmes eucaryotes, leurs relations phylogénétiques soulèvent encore des questions controversées. Parmi celles-ci, la classification des zygomycètes reste inconsistante. Ils sont potentiellement paraphylétiques, i.e. regroupent de lignées fongiques non directement affiliées. La position phylogénétique du genre Schizosaccharomyces est aussi controversée: appartient-il aux Taphrinomycotina (précédemment connus comme archiascomycetes) comme prédit par l'analyse de gènes nucléaires, ou est-il plutôt relié aux Saccharomycotina (levures bourgeonnantes) tel que le suggère la phylogénie mitochondriale? Une autre question concerne la position phylogénétique des nucléariides, un groupe d'eucaryotes amiboïdes que l'on suppose étroitement relié aux champignons. Des analyses multi-gènes réalisées antérieurement n'ont pu conclure, étant donné le choix d'un nombre réduit de taxons et l'utilisation de six gènes nucléaires seulement. Nous avons abordé ces questions par le biais d'inférences phylogénétiques et tests statistiques appliqués à des assemblages de données phylogénomiques nucléaires et mitochondriales. D'après nos résultats, les zygomycètes sont paraphylétiques (Chapitre 2) bien que le signal phylogénétique issu du jeu de données mitochondriales disponibles est insuffisant pour résoudre l'ordre de cet embranchement avec une confiance statistique significative. Dans le Chapitre 3, nous montrons à l'aide d'un jeu de données nucléaires important (plus de cent protéines) et avec supports statistiques concluants, que le genre Schizosaccharomyces appartient aux Taphrinomycotina. De plus, nous démontrons que le regroupement conflictuel des Schizosaccharomyces avec les Saccharomycotina, venant des données mitochondriales, est le résultat d'un type d'erreur phylogénétique connu: l'attraction des longues branches (ALB), un artéfact menant au regroupement d'espèces dont le taux d'évolution rapide n'est pas représentatif de leur véritable position dans l'arbre phylogénétique. Dans le Chapitre 4, en utilisant encore un important jeu de données nucléaires, nous démontrons avec support statistique significatif que les nucleariides constituent le groupe lié de plus près aux champignons. Nous confirmons aussi la paraphylie des zygomycètes traditionnels tel que suggéré précédemment, avec support statistique significatif, bien que ne pouvant placer tous les membres du groupe avec confiance. Nos résultats remettent en cause des aspects d'une récente reclassification taxonomique des zygomycètes et de leurs voisins, les chytridiomycètes. Contrer ou minimiser les artéfacts phylogénétiques telle l'attraction des longues branches (ALB) constitue une question récurrente majeure. Dans ce sens, nous avons développé une nouvelle méthode (Chapitre 5) qui identifie et élimine dans une séquence les sites présentant une grande variation du taux d'évolution (sites fortement hétérotaches - sites HH); ces sites sont connus comme contribuant significativement au phénomène d'ALB. Notre méthode est basée sur un test de rapport de vraisemblance (likelihood ratio test, LRT). Deux jeux de données publiés précédemment sont utilisés pour démontrer que le retrait graduel des sites HH chez les espèces à évolution accélérée (sensibles à l'ALB) augmente significativement le support pour la topologie « vraie » attendue, et ce, de façon plus efficace comparée à d'autres méthodes publiées de retrait de sites de séquences. Néanmoins, et de façon générale, la manipulation de données préalable à l'analyse est loin d’être idéale. Les développements futurs devront viser l'intégration de l'identification et la pondération des sites HH au processus d'inférence phylogénétique lui-même.Despite the popularity of fungi as eukaryotic model systems, several questions on their phylogenetic relationships continue to be controversial. These include the classification of zygomycetes that are potentially paraphyletic, i.e. a combination of several not directly related fungal lineages. The phylogenetic position of Schizosaccharomyces species has also been controversial: do they belong to Taphrinomycotina (previously known as archiascomycetes) as predicted by analyses with nuclear genes, or are they instead related to Saccharomycotina (budding yeast) as in mitochondrial phylogenies? Another question concerns the precise phylogenetic position of nucleariids, a group of amoeboid eukaryotes that are believed to be close relatives of Fungi. Previously conducted multi-gene analyses have been inconclusive, because of limited taxon sampling and the use of only six nuclear genes. We have addressed these issues by assembling phylogenomic nuclear and mitochondrial datasets for phylogenetic inference and statistical testing. According to our results zygomycetes appear to be paraphyletic (Chapter 2), but the phylogenetic signal in the available mitochondrial dataset is insufficient for resolving their branching order with statistical confidence. In Chapter 3 we show with a large nuclear dataset (more than 100 proteins) and conclusive supports that Schizosaccharomyces species are part of Taphrinomycotina. We further demonstrate that the conflicting grouping of Schizosaccharomyces with budding yeasts, obtained with mitochondrial sequences, results from a phylogenetic error known as long-branch attraction (LBA, a common artifact that leads to the regrouping of species with high evolutionary rates irrespective of their true phylogenetic positions). In Chapter 4, using again a large nuclear dataset we demonstrate with significant statistical support that nucleariids are the closest known relatives of Fungi. We also confirm paraphyly of traditional zygomycetes as previously suggested, with significant support, but without placing all members of this group with confidence. Our results question aspects of a recent taxonomical reclassification of zygomycetes and their chytridiomycete neighbors (a group of zoospore-producing Fungi). Overcoming or minimizing phylogenetic artifacts such as LBA has been among our most recurring questions. We have therefore developed a new method (Chapter 5) that identifies and eliminates sequence sites with highly uneven evolutionary rates (highly heterotachous sites, or HH sites) that are known to contribute significantly to LBA. Our method is based on a likelihood ratio test (LRT). Two previously published datasets are used to demonstrate that gradual removal of HH sites in fast-evolving species (suspected for LBA) significantly increases the support for the expected ‘true’ topology, in a more effective way than comparable, published methods of sequence site removal. Yet in general, data manipulation prior to analysis is far from ideal. Future development should aim at integration of HH site identification and weighting into the phylogenetic inference process itself

Insight into the light driven assembly of the oxygen evolving complex of photosystem II

Photosystem II (PSII) of plants, algae, and cyanobacteria utilize solar energy to catalyze one of the most important and most thermodynamically demanding reactions in nature: the oxidation of water into protons and molecular oxygen. Oxygen produced by PSII is toxic byproduct, however it is essential for respiration, the ozone layer and the extracted electrons drive the fixation of atmospheric CO2 to create biomass. The mechanism of water splitting driven by the light-induced charge separation is relatively well studied and high-resolution crystal structures are available to reveal the molecular aspects of PSII complex, however considerably less is known about how the inorganic Mn4O5Ca cluster is assembled de novo.The photosynthetic apparatus continuously experiences damage due to high light intensity and this results in the loss of photosynthetic activity. The primary photodamage occurs within main functional PSII unit, the D1 protein. To perform a highly efficient and sustained photosynthetic activity, damaged D1 protein should be replaced, with consequent reassembly of PSII. The key step in obtaining functional PSII de novo is the assembly of Mn4CaO5 core, driven by series of photo-oxidative reactions with incorporation of Mn and Ca ions into the coordination environment of PSII. The initial rate-limiting steps of the assembly of the PSII Mn4CaO5 core requires at least two quanta of light with the rate-limiting dark rearrangement step between them. A sensitive polarographic technique was used to track the assembly process under flash illumination as a function of the constituent Mn2+ and Ca2+ ions in genetically engineered membranes of the cyanobacterium Synechocystis sp. PCC6803 to elucidate the action of Ca2+ and peripheral proteins. We show that the protein scaffolding that organizes this process is allosterically modulated by the assembly protein Psb27, which together with Ca2+, stabilizes photoactivation intermediates.Photoactivation experiments with site-directed mutants D1-E189K and D1-E189R identified the role of D1-E189 in the formation the high affinity site of PSII. We have concluded that D1-E189 ligand is crucial during initial steps of photoactivation since it supports photoactivation intermediates by coordinating Ca2+ at its effectors site, which prevents the formation of inappropriately bound high-valency Mn at the oxygen evolving complex site

PARP/XRCC1 surveillance of the human genome

DNA single strand breaks (SSBs) are one of the most common lesions to genomic DNA, arising from various endogenous and exogenous sources. Single strand break repair (SSBR) constitutes a biochemical pathway whereby SSBs are detected, enzymatically processed and ligated. Whilst the general mechanisms of SSBR are relatively well described in vitro, there are remaining questions concerning how the pathway operates in vivo. For example, an early step in SSBR is thought to be the poly(ADP-ribose) polymerase (PARP)-dependent modification of SSB-proximal proteins with ADP-ribose, which is a signal for the recruitment of downstream repair factors, including the central scaffold XRCC1. Yet, the presence of multiple DNA-dependent PARP genes (PARP1, PARP2 and PARP3) has caused confusion regarding their specific roles in SSBR. This thesis potentially clarifies some contentious aspects of PARP function in the repair of SSBs induced by reactive oxygen species (ROS) and by Topoisomerase 1 (Top1). By employing PARP1-/-/PARP2-/- cells generated herein using CRISPR-Cas9 technology, in combination with preextraction immunofluorescence imaging and high-content analysis, I demonstrate that both PARP1 and PARP2 contribute towards ROS-induced ADP-ribosylation and XRCC1 chromatin-localization, but that in response to Top1-SSBs, these functions are specifically supported by PARP1 alone. Furthermore, using TDP1-/- and XRCC1-/-/TDP1-/- cells also generated herein, I characterize a striking hyper-ADP-ribosylation phenotype in response to Top1-SSBs. The clinical significance of this was confirmed by co-workers, who observed a similar phenotype in an XRCC1-deficient patient, where mutations in XRCC1 underlie a novel cerebellar neurodegenerative disease. This phenotype could be utilized in future to screen for genes with novel functions in SSBR. Finally, I investigate the functional implications of disrupted SSBR genes for rates of repair and cellular viability using alkaline single-cell electrophoresis and clonogenic survival assay. In doing so, I unexpectedly discovered that deletion of PARP1 suppresses CPT-induced comet tail moments of WT and XRCC1-/- cells

Untangling the Effects of Codon Mutation and Amino Acid Exchangeability

Determining the relative contributions of mutation and selection to evolutionary change is a matter of great practical and theoretical significance. In this paper, we examine relative contributions of codon mutation rates and amino acid exchangeability on the frequencies of each type of amino acid difference in alignments of distantly related proteins, alignments of closely related proteins, and among human SNPs, using a model that incorporates prior estimates of mutation and exchangeability parameters. For the operational exchangeability of amino acids in proteins, we use EX, a measure of protein- level effects from a recent statistical meta-analysis of nearly 10,000 experimental amino acid exchanges. EX is both free of mutational effects and more powerful than commonly used biochemical distance measures (7). For distaflt protein relationships, mutational effects (genetic code, transition/transversion bias) and operational exchangeability (EX) account for roughly equal portions of variance in off-diagonal values, the complete model accounting for R2 - 0.35 of the variance. For human/chimpanzee alignments representing closely related proteins relationships, mutational effects (including CpG bias) account for 0.52 of the variance; adding EX to the model increases this to 0.67. For natural variation in human proteins, the variance explained by mutational effects alone, and by mutational effects and operational exchangeability are, respectively, 0.66 and 0.70 for SNPs in HGVBase, and 0.56 and 0.60 for disease-causing missense variants in HGMD. Thus, exchangeability has a stronger relative effect for distant protein evolution than for the cases of closely related proteins or of population variation. A more detailed model for the hominid data suggests that 1) there is a threshold in EX below which substitutions are highly unlikely to be accepted, corresponding to roughly 30 % relative protein activity; 2) selection against missense mutants is a slightly convex function of protein activity, not changing much as long as protein activity is low; and 3) the probability of disease-causing effects decreases nearly linearly with EX