Genome stability has great importance in maintaining cell viability and optimal functionality of cellular processes. Loss of genome stability can lead to cell death in the simplest organisms and to deregulation of the cell proliferation machinery in higher organisms, potentially causing cancer or morbid states. The Single Stranded DNA Binding (SSB) protein of Escherichia coli is an essential protein that binds and stabilises ssDNA stretches. Its role is particularly crucial during DNA replication, recombination and repair processes and it has therefore been predicted to play a prominent role in the maintenance of genome stability. The role of SSB in genome instability was investigated using an E. coli strain in which, the expression of the ssb gene was placed under the control of the arabinose promoter. The level of SSB protein present in the cell could therefore be tuned by varying the arabinose concentration in the medium. A wide characterisation of the behaviour of the strain at low SSB level was carried out. Viability and growth tests showed that a threshold level of protein is required to allow normal growth. Microscopy analyses were carried out to follow cell division, nucleoid morphology and SOS response activation. Cells grown at low SSB level, showed a phenotype consistent with impaired cell division and altered nucleoid morphology. The SOS response was activated at low SSB levels and cell elongation was detected. Lowering the arabinose concentration in solid medium allowed the selection of suppressor strains that could form colonies under the new conditions. Sequencing of the entire genome of one such suppressor strain was carried out revealing a possible candidate for the phenotype change. The stability of a 105bp and of a 246bp DNA imperfect palindromes and the stability of CAG·CTG trinucleotide repeat arrays, inserted in the E. coli chromosome, were investigated in correlation to the SSB cellular level. Lowering the SSB level in cells grown on solid medium, increased the instability of the 105bp palindrome presumably by increasing the number of slippage events. On the other hand, SSB overexpression did not have an effect on the stability of the 246bp palindrome. The stability of a leading strand (CAG)75 repeat array was highly increased by overexpressing SSB, while the same effect was not observed for a leading strand (CTG)137 repeat array. Furthermore, excess SSB caused a change in the deletion size distribution profile for the leading strand (CAG)75 strain, lowering the bias towards big deletions. This is consistent with SSB being able to preferentially impede the formation of big DNA hairpins. Also, SbcCD nuclease was shown to have an effect on the deletion size distribution profile of the leading strand (CTG)137 strain. The lack of SbcCD led to a slight reduction of the number of big deletions.

Dda, one of three helicases encoded by bacteriophage T4, has been well- characterized biochemically but its biological role remains unclear. It is thought to be involved in origin-dependent replication, recombination-dependent replication, anti- recombination, recombination repair, as well as in replication fork progression past template-bound nucleosomes and RNA polymerase. One of the proteins that most strongly interacts with Dda, Gp32, is the only single-stranded DNA binding protein (SSB) encoded by T4, is essential for DNA replication, recombination, and repair. Previous studies have shown that Gp32 is essential for Dda stimulation of replication fork progression. Our studies show that interactions between Dda and Gp32 play a critical role in regulating replication fork restart during recombination repair. When the leading strand polymerase stalls at a site of ssDNA damage and the lagging strand machinery continues, Gp32 binds the resulting ssDNA gap ahead of the stalled leading strand polymerase. We found that a Gp32 cluster on leading strand ssDNA blocks Dda loading on the lagging strand ssDNA, blocks stimulation of fork progression by Dda, and stimulates Dda to displace the stalled polymerase and the 3' end of the daughter strand. This unwinding generates conditions necessary for polymerase template switching in order to regress the DNA damage-stalled replication fork. Helicase trafficking by Gp32 could play a role in preventing premature fork progression until the events required for error-free translesion DNA synthesis have taken place. Interestingly, we found that Dda helicase activity is strongly stimulated by the N-terminal deletion mutant Gp32-B, suggesting the N-terminal truncation to generate Gp32-B reveals a cryptic helicase stimulatory activity of Gp32 that may be revealed in the context of a moving polymerase, or through direct interactions of Gp32 with other replisome components. Additionally, our findings support a role for Dda-Gp32 interactions in double strand break (DSB) repair by homology-directed repair (HDR), which relies on homologous recombination and the formation of a displacement loop (D-loop) that can initiate DNA synthesis. We examined the D-loop unwinding activity of Dda, Gp41, and UvsW, the D-loop strand extension activity of Gp43 polymerase, and the effect of the helicases and their modulators on D-loop extension. Dda and UvsW, but not Gp41, catalyze D-loop invading strand by DNA unwinding. The relationship between Dda and Gp43 was modulated by the presence of Gp32. Dda D-loop unwinding competes with D- loop extension by Gp43 only in the presence of Gp32, resulting in a decreased frequency of invading strand extension when all three proteins are present. These data suggest Dda functions as an antirecombinase and negatively regulates the replicative extension of D- loops. Invading strand extension is observed in the presence of Dda, indicating that invading strand extension and unwinding can occur in a coordinated manner. The result is a translocating D-loop, called bubble migration synthesis, a hallmark of break-induced repair (BIR) and synthesis dependent strand annealing (SDSA). Gp41 did not unwind D- loops studied and may serve as a secondary helicase loaded subsequent to D-loop processing by Dda. Dda is proposed to be a mixed function helicase that can work both as an antirecombinase and to promote recombination-dependent DNA synthesis, consistent with the notion that Dda stimulates branch migration. These results have implications on the repair of ssDNA damage, DSB repair, and replication fork regulation, which are highly conserved processes sustained in all organisms.

Monitoring viruses circulating in the human population and the environment is critical for protecting public and ecosystem health. The goal of this dissertation was to incorporate a viral metagenomic approach into virus surveillance efforts (both clinical and water quality control programs) to enhance traditional virus detection methods. Clinical surveillance programs are designed to identify and monitor etiological agents that cause disease. However, the ability to identify viruses may be compromised when novel or unsuspected viruses are causing infection since traditional virus detection methods target specific known pathogens. Here we describe the successful application of viral metagenomics in a clinical setting using samples from symptomatic patients collected through the Enterovirus Surveillance (EVS) program in the Netherlands (Appendix A). Despite extensive PCR-based testing, the viruses in a small percentage of these samples (n = 7) remained unidentified for more than 10 years after collection. Viral metagenomics allowed the identification of viruses in all seven samples within a week using minimal sequencing, thus rapidly filling the diagnostic gap. The unexplained samples contained BK polyomavirus, Herpes simplex virus, Newcastle disease virus and the recently discovered Saffold viruses (SAFV) which dominated the unexplained samples (n = 4). This study demonstrated that metagenomic analyses can be added as a routine tool to investigate unidentified viruses in clinical samples in a public-health setting. In addition, metagenomic data gathered for SAFV was used to complete four genotype 3 SAFV (SAFV-3) genomes through primer walking, doubling the number of SAFV-3 full genomic sequences in public databases. In addition to monitoring viruses in symptomatic patients, it is also important to monitor viruses in wastewater (raw and treated) to protect the environment from biological contamination and prevent further spread of pathogens. To gain a comprehensive understanding of viruses that endure wastewater treatment, viral metagenomics was used to survey the total DNA and RNA viral community in reclaimed water (the reusable end-product of wastewater treatment) (Appendix B). Phages (viruses that infect bacteria) dominated the DNA viral community while eukaryotic viruses similar to known plant and insect viruses dominated RNA metagenomic libraries suggesting that highly stable viruses may be disseminated through this alternative water supply. A plant virus, the Pepper mild mottle virus (PMMoV), was identified as a potential indicator of wastewater contamination based on metagenomic data and quantitative PCR assays (Appendix C). The metagenomic analysis also revealed a wealth of novel single-stranded DNA (ssDNA) viruses in reclaimed water. Further investigation of sequences with low-level similarities to known ssDNA viruses led to the completion of ten novel ssDNA genomes from reclaimed water and marine environments (Appendix D). Unique genome architectures and phylogenetic analysis suggest that these ssDNA viruses belong to new viral genera and/or families. To further explore the ecology of the novel ssDNA viruses, a strategy was developed to take metagenomic analysis to the next level by combining expression analysis and immunotechnology (Appendix E). This dissertation made a significant contribution to current microbiological data regarding wastewater by uncovering viruses that endure the wastewater treatment and identifying a new viral bioindicator.

Mastreviruses (family Geminiviridae) are plant-infecting viruses with circular single-stranded (ss) DNA genomes (~2.7kb). The genus Mastrevirus is comprised of thirty-two species which are transmitted by leafhoppers belonging to the genus Cicadulina. Mastreviruses are widely distributed and have been found in the Middle East, Europe, Asia, Australia, Africa and surrounding islands. Only one species, dragonfly-associated mastrevirus has so far been identified in the Americas, isolated from a dragonfly in Puerto Rico. Species can be group based on the host(s) they infect, those which infect monocotyledonous (monocot) plants and those which infect dicotyledonous (dicot) plants. In recent years many new mastrevirus species have been discovered. Several of these new discoveries can largely been attributed to the development of new molecular tools. The current state of sequencing platforms has made it affordable and easier to characterise mastreviruses at a genome level thus allowing scientists to delve deeper into understanding the dynamics of mastreviruses. A few mastrevirus species have been identified as important agricultural pathogens and as a result have been the focus of much of the mastrevirus research. Maize streak virus, strain A (MSV-A) has been the most extensively studied due to the devastating impact it has on maize production in Africa. Studies have shown that MSV-A likely emerged as a pathogen of maize less than 250 years following introduction of maize in Africa by early European settlers. There is compelling evidence to suggest that MSV-A is likely the result of recombination events between wild grass adapted MSV strains. It therefore is equally important to monitor viruses infecting non-cultivated plants in order to gain a greater understanding of the epidemiological dynamics of mastreviruses, which in turn is essential for implementing disease management strategies. The objective of the research undertaken as part of this PhD thesis was to investigate global mastrevirus dynamics focusing on diversity, host and geographic ranges, mechanisms of evolution, phylogeography and possible origins of these viruses. In addition to this a viral metagenomic approach was used in order to identify novel mastreviruses or mastrevirus-like present in New Zealand. The dynamics of the monocot-infecting mastreviruses are investigated in Chapter Two and Three. The work described in these two chapters focus mainly on mastreviruses which infect non-cultivated grasses in Africa and Australia, a total of 161 full mastrevirus genomes were recovered collectively in the two studies. Chapter Two reveals a high level of mastrevirus diversity present in Australia with the discovery of four new species and several new strains of previously characterised species. An extensive sampling effort in Africa undertaken in Chapter Three reveals a broader host range and geographic distribution of the African monocot-infecting mastreviruses than previously documented. Mosaic patterns of recombination are evident among both the Australian and African monocot-infecting mastreviruses. In Chapters Four, Five and Six a comprehensive investigation was undertaken focusing on the dicot-infecting mastreviruses. The study undertaken in Chapter Four entailed the recovery of 49 full mastrevirus genomes from Australia, the Middle East, Africa, Turkey and the Indian Subcontinent to investigate the diversity of dicot-infecting mastreviruses from a global context. Analyses revealed a high degree of CpCDV strain diversity and extended the known geographic range of CpCDV. For the first time phylogeographic analysis was able to investigate the origins of the dicot-infecting mastreviruses. Results revealed the likely origin of the most recent common ancestor (MRCA) of these viruses is likely closer to Australia than anywhere else that dicot-infecting mastreviruses have been sampled and illuminated a supported series of historical movements following the emergence of the MRCA. In Chapter Five two novel mastreviruses Australian-like mastreviruses were isolated from chickpea material from Pakistan. A comprehensive analysis of CpCDV isolates in the major pulse growing regions of Sudan in Chapter Six reveals that this region harbours a high degree of strain diversity. Complex patterns of intra-species recombination indicate these strains are evidently circulating in these regions and infecting the same hosts, driving the emergence of new CpCDV strains. Collectively the results discussed in Chapters Two through Six extended the current knowledge of mastrevirus diversity. The natural host range of many mastreviruses has proven to be more extensive than previously documented, with many species having overlapping host ranges and hence these hosts could be acting as ‘mixing vessels’ enabling inter-species recombination. Patterns of recombination and selection were observed in both the monocot-infecting and the dicot-infecting mastreviruses further elucidating the mechanisms these viruses employ to evolve rapidly. Extensive sampling in a wide range of geographic regions provides insights into the true geographic range of species such as MSV and CpCDV. Given that mastreviruses have been able to move globally and Australia has been identified as a major mastrevirus diversity hotspot it is conceivable that mastreviruses are also present in New Zealand. In Chapter Seven and Eight this is explored by using a viral metagenomic approach to investigate the ssDNA viral populations associated with wild grasses and sewage material in New Zealand. Although no mastreviruses were recovered, this endeavour resulted in the discovery of more than 50 novel circular Rep-encoding ssDNA (CRESS DNA) viruses associated with non-cultivated grasses and treated sewage material, many of which are similar to mastreviruses and other geminiviruses. These discoveries expand current knowledge on the diversity of ssDNA viruses present in New Zealand and further highlight this viral metagenomic approach as an effective method for ssDNA virus discovery. Overall the results discussed in this thesis provide insights into mastrevirus diversity and dynamics as well as revealing a wealth of novel CRESS DNA viruses, some of which share similarities to geminiviruses.

No description available.

Au cours de la réplication de l’ADN, les cellules rencontrent régulièrement des obstacles d’origine endogène et exogène qui peuvent mettre en péril la réplication des génomes et menacer la duplication et ségrégation des chromosomes en mitose. La Recombinaison Homologue (RH) a un rôle bien caractérisé dans la réparation des cassures double-brin. Par contre, son rôle dans la protection et le redémarrage des fourches de réplication est moins bien caractérisé. Il a été montré par l’équipe que le redémarrage des fourches bloquées par la RH dépend de la formation d’ADN simple-brin et pas d’une cassure double-brin.Afin d’étudier les mécanismes par lesquels la RH contribue au sauvetage des fourches de réplication bloquées, un système permettant de bloquer localement la progression d’une seule fourche de réplication a été utilisé. Cet essai génétique a permis de montrer que le redémarrage de fourches bloquées par la RH est associé à une synthèse d’ADN fautive suite à des événements de glissement de la polymérase au niveau de micro-homologies. Un marqueur génétique a été associé à la barrière de réplication afin de mesurer l’efficacité de redémarrage des fourches bloquées et d’étudier l’étape de résection (i.e formation de l’ADN simple brin) dans différents fonds génétiques.Dans ce travail, le rôle de facteurs impliqués dans la résection a été étudié dans le contexte d’un blocage de fourche de réplication. Comme pour la réparation de cassures double-brin, la résection des fourches bloquées se fait en deux étapes : résection initiale et extensive. La résection initiale, de faible portée, dépend du complexe MRN (Mre11/Rad50/Nbs1) et Ctp1. A cette étape, la dégradation de l’ADN néosynthétisé se fait sur une distance de 110 bp. Cette résection est suffisante pour permettre de recruter les facteurs de la RH, mais est aussi nécessaire pour que les fourches continuent à être résectées. L’absence de MRN et/ou Ctp1 conduit à un défaut de redémarrage. La résection extensive, qui expose de l’ADN simple brin sur une distance de 0,8 à 1Kb, est largement dépendante de la nucléase Exo1. Contrairement à la résection initiale, la résection extensive n’est pas critique pour le redémarrage des fourches par la RH.De façon intéressante, le facteur Ku, connu pour être impliqué dans la jonction d’extrémités non-homologue, a un rôle dans le contrôle de la résection initiale et extensive et dans l’optimisation du redémarrage des fourches bloquées. Plus précisément, en absence de Ku, de l’ADN simple-brin s’accumule en amont des fourches bloquées, et la dynamique de redémarrage est affaiblie, mais pas abolie. Globalement, ces résultats clarifient une étape cruciale dans le redémarrage des fourches par la RH : la résection. / On a regular basis, cells encounter endogenous and exogenous replication stresses that jeopardize the progression of replication forks, thus threatening both the accuracy of chromosome duplication and their segregation during mitosis. Homologous recombination (HR) has a well-known role in repairing DNA double strand breaks (DSB). Other less acknowledged functions of HR are to protect and restart impeded forks. As it was previously reported by the team, restarting replication forks by HR requires the exposure of a single-stranded gap through fork resection, and not a DSB, to allow the recruitment of recombination factors.To study the effects of HR in blocked replication forks, a conditional fork barrier (RFB) was used to terminally-arrest replication at a specific locus. This construct allowed to determine that replication restart by HR is error-prone, leading to replication forks liable to slippage at micro-homology. A genetic reporter assay was placed in the vicinity of the RFB to allow the efficiency of replication restart and the step of resection to be quantified.In here, we explored factors involved in the formation of ssDNA gaps at halted replication forks. Similarly to DSB repair, resection in fork restart occurs in two steps. The initial resection is performed by MRN (Mre11/Rad50/NBS1) and Ctp1. This small degradation of approximately 110 bp of newly synthetized strands is sufficient to recruit HR factors and is required to promote the subsequent resection. The absence of either MRN or Ctp1 leads to defective replication restart by HR. The extensive resection (about 0.8-1Kb in size) is largely dependent on the nuclease Exo1, and it is not required for efficient fork restart.Interestingly, the non-homologous end-joining factor Ku was found to have a role in orchestrating initial and extensive resection and fine-tuning fork restart. Specifically, in the absence of Ku, ssDNA accumulates at the terminally-arrested replication forks, and fork restart dynamics is decreased, but not abolished. Overall, these results shed light on a delicate step of replication fork recovery by homologous recombination: resection.

Human RPA (replication protein A), a single-stranded DNA-binding protein, is required for many cellular pathways including DNA repair, recombination and replication. However, the role of RPA in nucleotide excision repair remains elusive. In the present study, we have systematically examined the binding of RPA to a battery of well-defined ssDNA (single-stranded DNA) substrates using fluorescence spectroscopy. These substrates contain adducts of (6-4) photoproducts, N-acetyl-2-aminofluorene-, 1-amino-pyrene-, BPDE (benzo[a]pyrene diol epoxide)- and fluorescein that are different in many aspects such as molecular structure and size, DNA disruption mode (e.g. base stacking or non-stacking), as well as chemical properties. Our results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA and that the affinity of RPA for damaged ssDNA depends on the type of adduct. Interestingly, the bulkier lesions have a greater effect. With a fluorescent base-stacking bulky adduct, (+)-cis-anti-BPDE-dG, we demonstrated that, on binding of RPA. the fluorescence of BPDE-ssDNA was significantly enhanced by up to 8-9-fold. This indicated that the stacking between the BPDE adduct and its neighbouring ssDNA bases had been disrupted and there was a lack of substantial direct contacts between the protein residues and the lesion itself. For RPA interaction with short damaged ssDNA, we propose that, on RPA binding, the modified base of ssDNA is looped out from the surface of the protein, permitting proper contacts of RPA with the remaining unmodified bases.

<p>Bacteria of the genus <em>Deinococcus</em> are perhaps the most resilient life forms ever discovered, demonstrating extreme resistance to ionizing radiation, ultraviolet radiation, desiccation, and a variety of mutagenic chemical agents. The most studied member of this genus, <em>D. radiodurans</em>, has been observed to rapidly reassemble its genome following severe fragmentation by hundreds of γ-radiation induced double-strand DNA breaks. Amongst the numerous factors contributing to DNA repair, a single-stranded annealing protein, DdrB, is believed to play an important role during the initial phases of recovery. The work described in this thesis represents the first structural characterization of DdrB, revealing a novel fold for single-stranded DNA binding. Together with biochemical data delineating the DNA-binding interface, two crystal structures of the DdrB/ssDNA complex were also solved, providing a comprehensive illustration of this interaction. Quaternary assemblies observed in these crystal structures also informed on the potential contribution of higher-order nucleoprotein complexes to the function of DdrB in single-stranded annealing. Most significantly, a face-to-face assembly of DdrB/ssDNA complexes provided insight into the mechanism by which DdrB mediates annealing of DNA, which may represent a common mechanism shared by other single-stranded annealing proteins.<strong></strong></p> / Doctor of Philosophy (PhD)

Mesozooplankton are of critical importance to marine food webs by transferring energy from the microbial food web to higher trophic levels and depositing energy to the deeper ocean layers through fecal deposition. While decades of research have shown that viruses have significant impacts in the oceans, and infect a wide range of organisms from bacteria to whales, there is still little known about the impacts of viruses on the mesozooplankton community. As copepods are the most abundant mesozooplankton group, this study sought to characterize the viruses present in natural populations of the calanoid copepods Acartia tonsa and Labidocera aestiva in Tampa Bay, Florida. Viral metagenomics revealed two virus genomes, named Acartia tonsa copepod circovirus (AcCopCV) and Labidocera aestiva copepod circovirus (LaCopCV), which were discovered in their respective copepod species. Both viruses show amino-acid similarities to known circoviruses, and phylogenetic and genomic analyses suggest they may be divergent members of the Circoviridae family. LaCopCV was found to be extremely prevalent in the L. aestiva population, with up to 100% of individuals infected. High viral loads for LaCopCV were observed by quantitative PCR, with an average viral load of 1.3x105 copies per individual. In addition, transcription of the LaCopCV replication gene was detected in L. aestiva, demonstrating active viral replication. AcCopCV could be detected sporadically in A. tonsa populations throughout the year. The circoviruses were specific to their respective hosts, and were not detected in the other copepod species or surrounding seawater. Virus-like particles were observed in A. tonsa and L. aestiva under transmission electron microscopy, demonstrating that viruses were actively proliferating in copepod connective tissue, as opposed to gut tissue, parasites, or symbionts. Preliminary results from in-situ hybridization show that the AcCopCV genome can be detected in A. tonsa tissue, linking the discovered genomes to virus propagation in copepod tissue. This is the first study describing viruses in copepods, as well as the first discovery of circoviruses infecting marine organisms. These results suggest that viruses impact marine copepod populations, necessitating further studies to determine the ecological impacts of viruses on the mesozooplankton community.

Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.