Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

PCNA and RPA are required for DNA mismatch repair (MMR), but their rolesin the pathway are not fully understood. Using an affinity pull-down approach, weshow that (1) increased PCNA binding to DNA heteroduplexes is associated withthe appearance and accumulation of excision products; and (2) RPAphosphorylation occurs when DNA polymerase ?? binds to the DNA substrate. Wetherefore hypothesize that PCNA plays an important role in mismatch-provokedexcision and that RPA phosphorylation plays an important role in DNA resynthesis.To determine the role of PCNA in MMR, mismatch-provoked and nick-directedexcision was assayed in a cell-free system in the presence of the PCNA inhibitor,p21CIP1/WAF. We show that whereas PCNA is essential for 3' directed excision, it isdispensable for the 5' directed reaction, suggesting a differential role for PCNA inMMR. We further find that the PCNA-dependent pathway is the only pathway for3' directed excision, but there are at least two pathways for 5' directed excision,one of which is a PCNA-independent 5' excision pathway. To determine if RPAphosphorylation facilitates DNA resynthesis, a gap-filling assay was developedusing both a cell-free system and a purified system, and we demonstrate that RPAphosphorylation stimulates DNA polymerase ??-catalyzed resynthesis in bothsystems. Kinetic studies indicate that phosphorylated RPA has a lower affinity forDNA compared with un-phosphorylated RPA. Therefore, the stimulation ofresynthesis by phosphorylated RPA is likely due to the fact that phosphorylationpromotes the release of RPA from DNA, thereby making DNA template availablefor resynthesis.

<p>DNA sequences from the <i>rps16</i> intron and the <i>psbE-petL</i> spacer from the chloroplast genome, the ribosomal nuclear ITS region, and introns from the low copy nuclear genes <i>RPA2</i>, <i>RPB2</i>, <i>RPD2a</i> and <i>RPD2b</i>, are in different combinations used to infer phylogenetic relationships in <i>Sileneae</i> (<i>Caryophyllaceae</i>). Used in concert, the biparentally inherited nuclear regions are useful to distinguish between paralogy due to allopolyploidy and single gene duplications, respectively, because the latter are not expected to give rise to repeated phylogenetic patterns in potentially unlinked sequence regions. In addition, the sequences resolve previously poorly known relationships in the tribe <i>Sileneae</i>. Several independent losses and incomplete concerted evolution are inferred between the two <i>RPD2</i> paralogues in a subgroup of <i>Silene</i>.</p><p>An allopolyploid origin is suggested for the tetraploid <i>S. aegaea</i>, with the maternal ancestor from the diploid <i>S. pentelica</i> lineage, and the paternal contributor from the diploid <i>S. sedoides</i> lineage.</p><p><i>Silene involucrata</i> originated as an allotetraploid with the diploid lineage of Arctic <i>S. uralensis</i> as cytoplasmic donor and the diploid Siberian/Northeast Asian <i>S. ajanensis</i> lineage as pollen donor. A subsequent allopolyploidization with the <i>S. ajanensis</i> lineage as pollen donor and the tetraploid <i>S. involucrata</i> lineage as cytoplasmic donor resulted in the hexaploid lineage of <i>S. sorensenis sensu lato</i>.</p><p>A monophyletic origin of the North American polyploids is rejected. One lineage consists of tetraploid <i>S. menziesii</i> and its diploid allies. A separate lineage leads to a clade consisting of both diploid and polyploid Arctic, European and Asian taxa in addition to the majority of the North American polyploids. The tetraploid <i>S. californica</i> and the hexaploid <i>S. hookeri</i> are derived from separate allopolyploidization events between these two lineages.</p>

DNA sequences from the rps16 intron and the psbE-petL spacer from the chloroplast genome, the ribosomal nuclear ITS region, and introns from the low copy nuclear genes RPA2, RPB2, RPD2a and RPD2b, are in different combinations used to infer phylogenetic relationships in Sileneae (Caryophyllaceae). Used in concert, the biparentally inherited nuclear regions are useful to distinguish between paralogy due to allopolyploidy and single gene duplications, respectively, because the latter are not expected to give rise to repeated phylogenetic patterns in potentially unlinked sequence regions. In addition, the sequences resolve previously poorly known relationships in the tribe Sileneae. Several independent losses and incomplete concerted evolution are inferred between the two RPD2 paralogues in a subgroup of Silene. An allopolyploid origin is suggested for the tetraploid S. aegaea, with the maternal ancestor from the diploid S. pentelica lineage, and the paternal contributor from the diploid S. sedoides lineage. Silene involucrata originated as an allotetraploid with the diploid lineage of Arctic S. uralensis as cytoplasmic donor and the diploid Siberian/Northeast Asian S. ajanensis lineage as pollen donor. A subsequent allopolyploidization with the S. ajanensis lineage as pollen donor and the tetraploid S. involucrata lineage as cytoplasmic donor resulted in the hexaploid lineage of S. sorensenis sensu lato. A monophyletic origin of the North American polyploids is rejected. One lineage consists of tetraploid S. menziesii and its diploid allies. A separate lineage leads to a clade consisting of both diploid and polyploid Arctic, European and Asian taxa in addition to the majority of the North American polyploids. The tetraploid S. californica and the hexaploid S. hookeri are derived from separate allopolyploidization events between these two lineages.

This thesis focuses on phylogenetic relationships among some of the major lineages in Silene subgenus Behenantha (Caryophyllaceae) using DNA sequences from multiple, potentially unlinked gene regions from a large taxonomic and geographic sample. Both traditional phylogenetic analyses and a strategy to infer species trees and gene trees in a joint approach are used. A new strategy to optimize species classifications, based on the likelihoods of the observed gene trees, is presented. Silene latifolia, S. dioica and the other dioecious species previously classified in section Elisanthe are not closely related to the type of the section (S. noctiflora). The correct name for the group of dioecious species is section Melandrium. The chloroplast DNA data presented indicate a geographic, rather than a taxonomic, structure in section Melandrium. The nuclear genes investigated correlate more to the current taxonomy, although hybridization has likely been influencing the relationships within section Melandrium. Incongruence between different parts of the gene SlXY1 in two Silene lineages is investigated, using phylogenetic methods and a novel probabilistic, multiple primer-pair PCR approach. The incongruence is best explained by ancient hybridization and recombination events. A survey of mitochondrial substitution rate variation in Sileneae is presented. Silene section Conoimorpha, S. noctiflora and the closely related S. turkestanica have elevated synonymous substitution rates in the mitochondrial genes investigated. Morphological and phylogenetic data reject that the Californian S. multinervia should be treated as a synonym to the Asian S. coniflora, as has previously been suggested. Furthermore, none of the genes investigated, or a chromosome count, support the inclusion of S. multinervia in section Conoimorpha. Data from multiple genes suggest that S. noctiflora and S. turkestanica form a sister group to section Conoimorpha. The calyx nervature, which is a potential synapomorphy for S. multinervia and section Conoimorpha, may be explained either by parallelism or by sorting effects.

Heliosperma (nom. cons prop.) comprises 15—20 taxa, most of them endemic to the Balkan Peninsula. DNA sequences from the chloroplast (rps16 intron, psbE-petG spacer) and the nuclear genome (ITS and four putatively unlinked RNA polymerase genes) are used to elucidate phylogenetic relationships within Heliosperma, and its position within Sileneae. Three main lineages are found within Heliosperma: Heliosperma alpestre, H. macranthum and the H. pusillum-clade. The relationships among the lineages differ between the plastid and the nuclear trees. Relative dates are used to discriminate among inter- and intralineage processes causing such incongruences, and ancient homoploid hybridisation is the most likely explanation. The chloroplast data strongly support two, geographically correlated clades in the H. pusillum-group, whereas the relationships appear poorly resolved by the ITS data, when analysed under a phylogenetic tree model. However, a network analysis finds a geographic structuring similar to that in the chloroplast data. Ancient vicariant divergence followed by hybridisation events best explains the observed pattern. The morphological and taxonomical diversity in the H. pusillum-group is possibly ecology-induced, and is not correlated with the molecular data. Phylogenetic patterns regarding the origin of Heliosperma are complicated, probably influenced by reticulate and sorting events. At least two ancient lineages have been involved in its evolution, one most closely related to Viscaria/Atocion and the other to Eudianthe/Petrocoptis. Atocion and Viscaria are sister genera, most species-rich on the Balkans, and including six/three species. Phylogenies do not support their traditional classification, and provide a framework for a taxonomic revision. Atocion compactum is found in three different positions in the chloroplast tree, and in a single clade in the nuclear gene trees. Using relative dates we demonstrate that hybridisation with subsequent chloroplast capture is a feasible explanation for the pattern observed. This, and other observed reticulate patterns, highlights the importance of hybridisation in plant evolution.