Under most conditions, meiotic recombination is essential for ensuring that organisms adapt to ever changing biotic and abiotic conditions and, as such, it shapes evolutionary change within and between species. The interplay between selection and recombination plays a role shaping levels diversity within populations. Remarkably, recombination is itself an evolving trait that varies at many levels: between distant species of eukaryotes, between closely related species and among populations (and individuals) of the same species. Recombination rates also vary across genomes. Most of the causes and mechanisms of this plasticity in recombination rates and distribution are not clearly understood. Also, our understanding of how this variability in recombination rates influences levels of diversity within populations and across genomes is incomplete. Here, I present a study combining molecular genetics with bioinformatic techniques to characterize recombination landscapes in Drosophila melanogaster. I present a model that accounts for a significant fraction of the variation in crossover rates across the genome of Drosophila melanogaster. Our predictive model suggests that crossover distribution is influenced by both meiosis-specific chromatin dynamics and very local constitutively open chromatin associated with DNA motifs that prevent nucleosome stabilization. I also present a novel method for genomic scans to identify recent events of adaptation in using nucleotide diversity data. In addition, I characterized variability in recombination rates in different populations of D. melanogaster and detected that the highest degree of variability in recombination rates across the genome is associated with intermediate genomic scales, and that this intermediate scale also plays a major role in explaining differences in recombination among populations. Our report is the first linking variation in recombination rates across genomes (genomic) and among populations (evolutionary), possibly suggesting a common mechanistic/genomic cause. Finally, I present preliminary data of the first large-scale project to study the effects of multiple environmental conditions in recombination rates at genome-wide level. In conclusion, these studies provide a new framework to investigate variation in recombination rates and to understand the genomic causes and evolutionary consequences.

The fate of any lineage is contingent on the rate at which its genome changes over time. Genome dynamics are influenced by patterns of mutation and recombination. Mutations as the raw force of variation can be acted on independently during exchanges of homologous genetic regions via meiotic recombination. While molecular evolution in sexual lineages is impacted by both mutation and recombination, asexual lineage fate is primarily influenced by the mutation rate; recombination is often altered or absent in asexuals. Although multiple studies show accelerated mutation accumulation in asexual lineages that have lost recombination, virtually nothing is known about rate patterns when meiosis is retained. Here, I use parasitic wasps in genus Diachasma to investigate genome evolution in a recently-derived asexual lineage. I provide evidence that asexual Diachasma possess a canonical set of meiosis genes as well as high levels of genomic homozygosity. Taken together, these observations support an active, albeit modified, form of meiosis in this asexual lineage. In addition, I present the first documentation of accelerated mutation accumulation in the nuclear genome of a naturally-occurring, meiotically- reproducing organism. If harmful, these mutations could impede asexual lineage persistence and contribute strong support for the long-term benefits of sex.

Les extrémités des chromosomes portent des structures nommées télomères, nécessaires à la survie des cellules. La sénescence réplicative induite par l’altération des télomères bloque les proliférations cellulaires excessives. Cependant, certaines cellules échappent à ce contrôle et deviennent immortelles. Je me suis intéressée aux mécanismes permettant de reconstituer des télomères fonctionnels par recombinaison chez la levure. Le noyau est divisé en sous-compartiments plus ou moins permissifs pour la recombinaison. J’ai étudié comment la localisation des télomères dysfonctionnels aux pores nucléaires (NPC) conditionne leur réparation et la prolifération des cellules. J’ai montré que les lésions réplicatives au télomères sont relocalisées aux NPC ce qui favorise leur réparation par un mécanisme conservatif. J’ai aussi montré la présence aux NPC de cercles d’ADN télomérique qui pourraient servir de matrice pour allonger les télomères et sortir de la sénescence réplicative. / Chromosome ends are formed by structures called telomeres, which are necessary for genome integrity and cell survival. Upon aberrant proliferation, as in precancerous cells, telomere alterations blocks cell proliferation, a mechanism called replicative senescence. However, some cells escape this control and become immortals. Using budding yeast as model, my work aimed to understand the mechanisms that allow cells to reconstitute functional telomeres using homologous recombination. The nucleus is divided in sub-compartments that are differently permissive for recombination. I investigated how the localization of dysfunctional telomeres to the nuclear pore complex (NPC) determines their repair and favors cell proliferation. I showed that replicative lesions at telomeres relocate to the NPC allowing conservative repair by recombination. I further shed light on the presence at NPC of telomeric DNA circles that may serve as template for telomere elongation and thus escape from senescence.

Sexual reproduction is expected to facilitate the removal of deleterious mutations from populations because biparental inheritance (i.e., segregation) and recombination during meiosis break down linkage disequilibria (LD), allowing mutations to be selected independently from their genetic background. Accordingly, the absence of recombination and segregation is expected to increase selective interference between loci, translating into reduced efficacy of natural selection. While there now exist multiple lines of evidence demonstrating that asexual lineages do experience accelerated accumulation of putatively harmful mutations, whether these mutations influence phenotype in a manner that could contribute to the maintenance of sex remains almost entirely unevaluated. Here, I use the New Zealand freshwater snail, Potamopyrgus antipodarum, to address these questions. In particular, I take advantage of the fact that the mitochondrial genome is expected to suffer from these mutational effects and interacts extensively with the nuclear genome to evaluate potential harmful effects of mutation accumulation in asexuals on a genome-wide scale. I present evidence that harmful mutations remain extant longer in asexual populations than in sexual populations, that the degree of functional constraint determines the extent of mutation accumulation in asexuals, that there is genetic variation for mitochondrial function in asexual lineages of P. antipodarum, and that phenotypic variation for mitochondrial function is mediated by both genetic and environmental variation. Together, these analyses provide strong evidence that asexual lineages are accumulating deleterious mutations, and that there is genetic variation, structured by lake, for mitochondrial function.

DNA double strand break (DSB) repair is critical to maintain genomic integrity and cell viability. DSBs can occur during the course of cell cycle during replication or transcription, or by exogenous agents such as chemicals or ionizing radiation. For my thesis, I studied homologous recombination (HR), which has two sub-pathways: Homology Directed Repair (HDR) and Single Strand Annealing (SSA). HDR involves strand invasion of a homologous template to prime DNA synthesis; SSA involves annealing of homologous segments flanking a DSB. Background data showed that depletion of XAB2 protein by RNA interference reduced both HDR and SSA events. XAB2 protein contains 15 tetratricopeptide repeat (TPR) motifs, which likely enable protein-protein interactions. While XAB2 is speculated to have a role in transcription coupled repair and pre-mRNA splicing, its role in HR pathway is uncertain. The overall hypothesis for my thesis is that XAB2 mediates a specific step of HR (5’-3’ end resection), and the TPR motifs present in XAB2 enable the protein to function in a complex during HR. By using an end resection assay and cell biology analysis, I found that XAB2 is essential for 5’ – 3’ end resection, an intermediate step common to both HDR and SSA pathways. With a functional complementation assay I developed, I have shown that specific TPR regions are critical for XAB2 functions in HR. Overall, my research demonstrates that XAB2 protein has a key role in the 5’-3’ end resection step of HR, and its function in HR requires specific sets of its TPR regions.

Accurate knowledge of heat transfer to materials in recombining plasmas is needed to improve heat shield designs. A lack of understanding of the chemical component of surface heating motivates the use of conservative assumptions with regards to surface catalysis in the design of thermal protection systems (TPS) that detrimentally impact payload capability. Chemical heating is the release of potential energy from recombining reactive species on the surface to form molecules. For a stable surface interacting with partially-dissociated air, the chemical heating component is due to surface-catalyzed recombination reactions of atomic O and N to produce molecular O2, N2, and NO. Unfortunately, heat flux measurements provide no fundamental information about the surface recombination pathways involved, or how the energy reaches the surface. Rather, they give a total heating rate. This work has taken steps to advance the current poor understanding about the chemical energy transport to and from material surfaces in high-temperature, recombining plasmas. A combination of spatially resolved laser-based diagnostics and emission spectroscopy was used to measure the number densities and gradients of the reactants (N, O), the products (NO, N2) and the energy distribution of recombined molecules (NO, N2) in the boundary layer adjacent to a plasma heated material. Laser excitation can probe individual species by electronic state (atoms) and by electronic, vibrational and rotational states (molecules). Emission can probe the radiative emission for a range of species and electronic, vibrational and rotational states of both atoms and molecules. These measurements of spatial variations in species concentrations through the boundary layer are directly related to near-surface gas-phase chemistry and energy exchange and have provided experimental information that was not currently available. Results provide the initial steps to determine recombination rates and the energy deposited on the surface due to surface catalyzed recombination of atomic nitrogen and oxygen in air plasma.

Recent years have witnessed the integration of theoretical advances in population genetics with large-scale analyses of complete genomes. As a result, a growing number of studies suggest the frequent occurrence of deleterious as well as adaptive mutations. Given the evidence for the widespread occurrence of selection, the finite sizes of natural populations, and the limited recombination in every genome, mutations under selection are expected to alter the fate of genetically linked mutations. The consequences of this non-independent behavior of mutations can be described by the Hill-Robertson effect in terms of the reduction in the effective population size (Ne). Reduction in the effective population size has two effects: 1) a reduction in levels of genetic variation and 2) a reduction in the effectiveness of selection that is manifested in an increased probability of fixation of deleterious mutations and a reduced probability of fixation of advantageous mutations. Changes in Ne that have previously been frequently associated with changes in recombination rate can also occur locally, in association with changes in the number of sites under selection even when the recombination rate remains uniform. The main objective of the work presented in this thesis is to investigate these local effects of the non-independent behavior of mutations on patterns of polymorphism and divergence in Drosophila using computer simulation and experimental approaches. A computer simulation approach is developed to investigate the local consequences of linked selection on estimates of selection and the proportion of adaptive substitutions using the McDonald-Kreitman framework. The results suggest that even a high level of recombination is unlikely to remove all the effects of linked selection. Ignoring these local linkage effects leads to misleading estimates of the intensity of selection and the proportion of adaptive substitutions. Two predictions of the Hill-Robertson effect were tested empirically by examining patterns of polymorphism and divergence combined with codon bias estimates in genes with and without introns: 1) the effectiveness of selection and polymorphism levels are expected to be reduced in the center of the long coding sequence of genes without introns (the intragenic Hill-Robertson effect), and 2) introns are expected to function as modifiers of recombination thereby increasing the effectiveness of selection in the central region of the coding sequence of genes containing centrally located introns. The evidence from divergence and codon bias patterns in genes with a long coding sequence supports the presence of the intragenic Hill-Robertson effect. However, polymorphism levels do not show the expected decrease in the center of the coding sequence. With regard to the second prediction, results indicate that intron presence does not increase the effectiveness of selection at synonymous sites in the set of investigated genes. Rather, intron presence is associated with increased levels of adaptation at nonsynonymous sites. Further investigations are necessary to clarify the role of introns in mediating the increase in adaptation.

Homologous DNA recombination and rearrangement has been modeled with a class of four-regular rigid vertex graphs called assembly graphs which can also be represented by double occurrence words. Various invariants have been suggested for these graphs, some based on the structure of the graphs, and some biologically motivated. In this thesis we use a novel method of data analysis based on a technique known as partial-clustering analysis and an algorithm known as Mapper to examine the relationships between these invariants. We introduce some of the basic machinery of topological data analysis, including the construction of simplicial complexes on a data set, clustering analysis, and the workings of the Mapper algorithm. We define assembly graphs and three specific invariants of these graphs: assembly number, nesting index, and genus range. We apply Mapper to the set of all assembly graphs up to 6 vertices and compare relationships between these three properties. We make several observations based upon the results of the analysis we obtained. We conclude with some suggestions for further research based upon our findings.

<p>Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c.</p><p>The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are investigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and thereafter lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regulated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro-tein has been shown to repress the Pc promoter and thereby making tran-scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes.</p><p>In this work the importance of the native size for biologically active integrase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein-protein interfaces. In addition it is shown that P2 Cox and the P2 relative Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed.</p><p>The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.</p>

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.