La synthèse translésionnelle (TLS) est un processus important pour franchir des lésions de l’ADN au cours de la duplication du génome dans les cellules humaines. Le modèle « d’échange de polymérases » suggère que la polymérase réplicative est transitoirement remplacée par une polymérase spécialisée, qui va franchir le dommage et permettre de continuer la synthèse d’ADN. Ces ADN polymérases spécialisées appelées Pol êta (η), iota (ι), kappa (κ), zêta (ζ), et Rev1 ont été bien caractérisées pour leur capacité à franchir différents types de lésions in vitro. Un concept en émergence est que ces enzymes pourraient également être requises pour répliquer des zones spécifiques du génome qui sont « difficiles à répliquer ». Polζ est constituée d’au moins 2 sous-unités : Rev3 qui est la sous-unité catalytique et Rev7 sous-unité augmentant l’activité de Rev3L. Jusqu'ici, la fonction la mieux caractérisée de Polζ était de sa capacité à catalyser l'extension d'un mésappariement en face d'une lésion d'ADN. Cependant, il a été montré que la sous unité catalytique Rev3 de levure et humaine interagissent avec les deux sous-unités accessoires de Polδ que sont pol31 et pol32 chez la levure et p50 et p66 chez l’humain. Il a aussi été mis en évidence que Rev3L est importante pour la réplication des sites fragiles (SFCs) dans les cellules humaines, zones connues pour être à l’origine d’une instabilité génétique et pour être répliquées de manière tardive (en G2/M). Tout ceci suggère que Polζ pourrait jouer un rôle dans la réplication du génome non endommagé, et plus spécifiquement lorsque des barrières naturelles (e.g. ADN non-B) entravent la progression normale des fourches de réplication.Chez la levure S. cerevisiae, l’inactivation du gène rev3 est viable et conduit à une diminution de la mutagenèse spontanée ou induite par des agents génotoxiques suggérant que Polζ est impliquée dans le franchissement mutagène des lésions endogènes ou induite. En revanche, l’inactivation du gène Rev3L chez la souris est embryonnaire létale alors que la plupart des autres ADN polymérases spécialisées ne sont pas vitales. Ceci suggère que Polζ a acquis des fonctions essentielles au cours de l’évolution qui restent inconnues à ce jour. Les fibroblastes embryonnaires murins (MEF) Rev3L-/- présente une grande instabilité génétique spontanée associée une forte augmentation de cassures et de translocations chromosomiques indiquant que Polζ est directement impliquée dans le maintien de la stabilité du génome. Afin de clarifier le rôle de cette polymérase spécialisée au cours de la réplication du génome, nous avons entrepris de procéder à une étude sur les relations structure/fonction/localisation de la protéine Rev3. Notre étude met en évidence que la progression en phase S des cellules Rev3L-/- est fortement perturbée, notamment en fin de phase S. Dans ces cellules invalidées pour Rev3L, on constate des changements dans le programme de réplication et plus particulièrement dans des régions de transition (TTR) répliquées à partir du milieu de la phase S. Nous montrons aussi un enrichissement global en marques épigénétiques répressives (marques associées à l’hétérochromatine et méthylation de l’ADN) suggérant qu’un ralentissement de la progression de la fourche de réplication à des loci particuliers peut promouvoir une hétérochromatinisation lorsque Rev3L est invalidé. De manière intéressante, nous constatons une diminution de l’expression de plusieurs gènes impliqués dans le développement qui pourrait peut-être expliquer la létalité embryonnaire constatée en absence de Rev3L. Enfin, nous mettons en évidence une interaction directe entre la protéine d’organisation de l’hétérochromatine HP1α et Rev3L via un motif PxVxL. Tout ceci nous suggère fortement que Polζ pourrait assister les ADN polymérases réplicatives Polδ et Polε dans la réplication des domaines compactés de la chromatine en milieu et fin de phase S. / DNA polymerase zeta (Polζ) is a key player in Translesion DNA synthesis (TLS). Polζ is unique among TLS polymerases in mammalian cells, because inactivation of the gene encoding its catalytic subunit (Rev3L) leads to embryonic lethality in the mouse. However little is known about its biological functions under normal growth conditions.Here we show that S phase progression is impaired in Rev3L-/- MEFs with a delay in mid and late S phase. Genome-wide profiling of replication timing revealed that Rev3L inactivation induces changes in the temporal replication program, mainly in particular genomic regions in which the replication machinery propagates a slower velocity. We also highlighted a global enrichment of repressive histone modifications as well as hypermethylation of major satellites DNA repeats in Rev3L-deficient cells, suggesting that fork movements can slow down or stall in specific locations, and a delay in restarting forks could promote heterochromatin formation in Rev3L-depleted cells. As a direct or indirect consequence, we found that several genes involved in growth and development are down-regulated in Rev3L-/- MEFs, which might potentially explain the embryonic lethality observed in Rev3L KO mice. Finally we discovered that HP1α directly interacts and recruits Rev3L to pericentromeric heterochromatin. We therefore propose that Polζ has been co-opted by evolution to assist DNA polymerase ε and δ in duplicating condensed chromatin domains during mid and late S phase.

O T. cruzi, é o agente causador da doença de Chagas. O estado redox NAD+/NADH intracelular é fundamental na manutenção do metabolismo celular. A GAPDH apresenta a função de proteção do telômero em mamíferos contra degradação, isto por causa de ligar se ao telômero. Aqui, mostramos que a GAPDH recombinante de T. cruzi (rTcGAPDH) interage com o DNA telomérico. A rTcGAPDH liga ao DNA de simples fita. Mostramos que a GAPDH liga ao DNA telomérico in vivo em células epimastigotas, onde a [NADH] é maior que [NAD+], mas a adição de NAD+ exógeno bloqueia esta interação. Corroborando a hipótese de que o equilíbrio NAD+/NADH determina a interação GAPDH-telômero, vimos que o tripomastigota tem maior [NAD+] intracelular que a [NADH] e a GAPDH não é capaz de ligar se ao DNA telomérico. Além disso, o NADH exógeno resgata a interação GAPDH-telómero nesta fase. É importante o equilíbrio NAD+/NADH desta interação em tripanosomas, sugerindo que a proteção do telômero do parasita pode ser regulada pelo estado metabólico das células. / The T. cruzi, is the causative agent of Chagas disease. The redox state of NAD+/NADH intracellular is critical in the maintenance of cellular metabolism. The GAPDH has the protection function of the telomere in mammals against degradation, because it is connecting to the telomere. Here we show the recombinant GAPDH of T. cruzi (rTcGAPDH) interacts with telomeric DNA. The rTcGAPDH binds to single-stranded DNA. We show GAPDH to bind to telomeric DNA in vivo epimastigotes cells, where [NADH] is greater than [NAD+], but the addition of exogenous NAD+ blocks this interaction. Corroborating the hypothesis that the NAD+/NADH balance determines the GAPDH-telomere interaction, we saw that the trypomastigote has higher [NAD+] that intracellular [NADH] and GAPDH is not able to connect to telomeric DNA. In addition, the exogenous NADH recovers the GAPDH-telomere interaction at this stage. It is important the NAD+/NADH balance this interaction in trypanosomes, suggesting that the protection of the telomere of the parasite can be regulated by the metabolic state of the cells.

The evolutionary pressure to prevent re-replication by inactivating licensing during S phase leaves higher-eukaryotes with large genomes, such as human cells, vulnerable to replication stresses. Origins licensed in G1 must be sufficient to complete replication as new origins cannot be licensed in response to irreversible replication fork stalling. Interdisciplinary approaches between cellular biology and biophysics predict that replication of the genome is routinely incomplete in G2, even in the absence of external stressors. The frequency of converging replication forks that never terminate due to irreversible stalling (double fork stall), which result in a segment of unreplicated DNA, was modelled using high quality origin-mapping data in HeLa and IMR-90 cells. From this, hypotheses were generated that related an increase in unreplicated segments of DNA to reduced functional origin number. Presented in this thesis is the confirmation of this relation by quantifying chromosome mis-segregation and DNA damage responses when origin number was reduced using RNAi against licensing factors. The number of ultrafine anaphase bridges and 53BP1 nuclear bodies are in remarkable concordance with the theoretical predictions for the number of double fork stalls, indicating that cells are able to tolerate under-replication through such post-replicative cellular responses. 53BP1 preferentially binds to chromatin associated with large replicons, and functions synergistically with dormant origins to protect the stability of the genome. Additional candidates, inspired by common fragile site research, have also been characterised as responders to spontaneous under-replication, and include FANCD2 and MiDAS, which function in early mitosis to facilitate completion of replication before cells enter anaphase. In conclusion, a series of mechanisms that sequentially function throughout the cell cycle protects the stability of the human genome against inevitable spontaneous under-replication brought about by its large size.

<p>The <i>Murine Pneumotropic Virus </i>(MPtV), in contrast to the other <i>MurinePolyomavirus</i> (MPyV), appears to be non-tumourigenic in its natural host. Instead, MPtV causes acute pneumonia and can serve as a model in studies of polyomavirus-induced disease. In initial experiments, MPtV large T-antigen (LT) was expressed in a heterologous system. LT was characterized with regard to its metabolic stability and cell immortalizing activity and, after purification, to its specific DNA binding. </p><p>The absence of permissive cell culture system for MPtV has hampered its study. We made attempts to widen the host range of the virus by modifying the regulatory and late regions of the genome. The enhancer substitution mutant (KVm1), having a transcriptional enhancer substituted with a corresponding DNA segment from MPyV, was able to replicate in mouse 3T3 cells and form virus particles that were infectious in mice. However, efficient infection of cells in vitro was not achieved with this mutant virus, possibly due to the absence of virus-specific receptors on the cells. The capsid protein substitution mutants, having capsid protein genes of MPyV, for which receptors are present on a variety of cell types, showed also no cytopathic effect, despite an enhanced viral DNA replication and assembly of virus particles. </p><p>MPtV-DNA extracted from virus in lung tissue of infected mice had a heterogeneous enhancer segment. A majority of the DNA molecules had a structure differing from the standard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was a prominent variation. Other genome variants showed complete or partial deletions of the insertion and surrounding sequences in the viral enhancer. In relation to the standard-type, all variant genomes showed differences in the activities of transcriptional promoters and the origin DNA replication. Analysis by DNA reassociation showed that a large number of nucleotide sequences related to the 220 base-pair insert in the MPtV genome were present in mouse and human DNA, but not in <i>Escherichia coli</i> DNA. Together, the data suggest that the 220 base-pair insertion is related to a transposable element of a novel type.</p>

The Murine Pneumotropic Virus (MPtV), in contrast to the other MurinePolyomavirus (MPyV), appears to be non-tumourigenic in its natural host. Instead, MPtV causes acute pneumonia and can serve as a model in studies of polyomavirus-induced disease. In initial experiments, MPtV large T-antigen (LT) was expressed in a heterologous system. LT was characterized with regard to its metabolic stability and cell immortalizing activity and, after purification, to its specific DNA binding. The absence of permissive cell culture system for MPtV has hampered its study. We made attempts to widen the host range of the virus by modifying the regulatory and late regions of the genome. The enhancer substitution mutant (KVm1), having a transcriptional enhancer substituted with a corresponding DNA segment from MPyV, was able to replicate in mouse 3T3 cells and form virus particles that were infectious in mice. However, efficient infection of cells in vitro was not achieved with this mutant virus, possibly due to the absence of virus-specific receptors on the cells. The capsid protein substitution mutants, having capsid protein genes of MPyV, for which receptors are present on a variety of cell types, showed also no cytopathic effect, despite an enhanced viral DNA replication and assembly of virus particles. MPtV-DNA extracted from virus in lung tissue of infected mice had a heterogeneous enhancer segment. A majority of the DNA molecules had a structure differing from the standard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was a prominent variation. Other genome variants showed complete or partial deletions of the insertion and surrounding sequences in the viral enhancer. In relation to the standard-type, all variant genomes showed differences in the activities of transcriptional promoters and the origin DNA replication. Analysis by DNA reassociation showed that a large number of nucleotide sequences related to the 220 base-pair insert in the MPtV genome were present in mouse and human DNA, but not in Escherichia coli DNA. Together, the data suggest that the 220 base-pair insertion is related to a transposable element of a novel type.

Homologous genetic recombination, because of its fundamental roles in the maintenance of genome stability and evolution, is an essential cellular function common to all organisms. This process also plays important roles in the repair of damaged DNA molecules, generation of genetic diversity and proper segregation of chromosomes. The genetic exchange is a highly orchestrated process that entails a plethora of control mechanisms and a large number of proteins, of which RecA and SSB are two proteins that have been chosen for further investigation(s) in the present study. In addition, we have also investigated the interaction between SSB and UvrD1, which plays an important role in DNA repair pathways, especially nucleotide excision repair (NER) and mismatch repair as well as DNA replication and recombination. Chapter 1 reviews the literature regarding various aspects of homologous recombination, with an emphasis on the biochemical and the biophysical aspects of RecA and SSB proteins. In addition, it provides an overview of the study of DNA repair and recombination in mycobacteria. RecA protein is ubiquitous and well conserved among bacterial species. Many archaeal species possess two RecA homologues (RadA and RadB) and eukarya possess multiple homologues of RecA including, Rad51, Rad51B, Rad51C, Rad51D, DMC1, XRCC2, or XRCC3. RecA or its homologues function as polymers, consisting of hundreds of monomers that cooperatively polymerize on single-stranded DNA to form a nucleoprotein filament. E. coli RecA protein participates in Trans Lesion Synthesis (TLS) of DNA and forms the minimal mutasome in association with DNA polymerase V (UmuD’2C). The fundamental mechanism underlying HR, i.e. DNA strand exchange, is one of the most fascinating examples of molecular recognition and exchange between biological macromolecules. Since the isolation of E. coli recA gene and the subsequent purification of its gene product and also from other organisms, RecA protein has been studied extensively for more than three decades. E. coli RecA protein has pivotal roles in DNA recombination and repair, and binding to DNA in the presence of ATP, is a fundamental property of RecA protein resulting in the formation of a nucleoprotein filament. This is the slow step of the HR process, and is considerably faster on ssDNA than on duplex DNA. Binding of RecA to dsDNA is slower at physiological pH, is accelerated at acidic pH, and the lag in binding at the higher pH values is due to slow nucleation. The ATP and the DNA binding functions of RecA display allosteric interaction such that ATP- binding leads to an increase in affinity to ssDNA-binding and vice-versa. X-ray structures of E. coli RecA complexed with nucleotide cofactors have implicated a highly conserved Gln196 in Mycobacterium smegmatis RecA in the coupling of ATP and the DNA binding domains. The carboxyamide group of Gln196 makes an H-bond with the γ-phosphate group of ATP and the side chain of this residue is observed to move by approximately 2Å towards the ATP, relative to the other residues involved in ATP binding. In addition, a highly conserved Arg198 has also been postulated to interact with the γ-phosphate group of bound ATP and position it for a nucleophilic attack by a conserved residue-Glu96 leading to ATP hydrolyses. To elucidate the role of Gln196 and Arg198 in the allosteric modulation of RecA functions, we generated MsRecA variant proteins, where in Gln196 was substituted with alanine, asparagine or glutamate; Arg198 was mutated to a lysine. The biochemical characterization of MsRecA and its variant proteins with the objective of defining the allosteric interaction between the ATP- and the DNA-binding sites has been described with in Chapter 2. We observed that while the mutant MsRecA proteins were proficient in ATP-binding they were deficient in ATP hydrolyses. We assayed for the ability of these proteins to bind ssDNA using either nitrocellulose filter binding or Surface Plasmon Resonance (SPR). While we did not detect any ssDNA-binding by the mutant MsRecA proteins in the filter binding assay, we observed only ten-fold reduction in the affinity for ssDNA as compared to wild type MsRecA protein in MsRecAQ196A, Q196N and R198K in the SPR assay. MsRecA Q196E did not show any binding to ssDNA, in both nitrocellulose filter-binding as well as SPR assays. We assayed for the ability of the mutant RecA proteins for their ability to promote DNA-pairing as well as DNA strand exchange. While we observed limited pairing promoted by the mutant proteins relative to the wild-type MsRecA, we observed a complete abrogation of strand exchange in the case of mutant proteins. In addition, we assayed for the co-protease function of MsRecA, by monitoring the cleavage of MtLexA. We observed that only the wild-type MsRecA protein was able to cleave MtLexA, while none of the mutant RecA proteins were able to do so. In order to understand the differences observed between the wild -type and the mutant MsRecA proteins, we analyzed the conformational state of MsRecA and its variant proteins by circular dichroism spectroscopy upon ATP-binding. We observed that while MsRecA and MsRecAQ196N displayed a reduction in the absorbance at 220 nm upon ATP binding, we did not observe any such structural transitions in the other mutant MsRecA proteins that we tested. Based on our observations and the crystal structure of E. coli RecA bound to ssDNA, in Chapter 2, we propose a dual role for the Gln196 and Arg198 in modulating RecA activities. In the presynaptic filament Gln196 and Arg198 sense the presence of the nucleotide in the nucleotide binding pocket and initiate a series of conformation changes that culminate in the transition to an active RecA nucleoprotein filament. In the active RecA nucleoprotein filament these residues are repositioned such that they now form a part of the protomer-protomer interface. As such they perform two vital functions; they stabilize the protomer-protomer interface by participating in the formation of hydrogen bonds that span the interface as well transmit the wave of ATP hydrolysis across the interface leading to a coordinated hydrolyses of ATP essential for the heteroduplex extension phase of strand exchange reaction. The members of the super family of single stranded DNA binding proteins (SSB) play an important role in all aspects of DNA metabolism including DNA replication, repair, transcription and recombination. Prokaryotic SSBs bind ssDNA with high affinity and generally with positive cooperativity. Several lines of evidence suggest that prokaryotic SSBs are modularly organized into three distinct domains: the N-terminal DNA binding domain and acidic C-terminal domain are linked by a flexible spacer. Studies from our laboratory have revealed that M. smegmatis SSB plays a concerted role in recombination-like activities promoted by the cognate RecA. The C- terminal of SSB is known to be involved in its ability to interact with other proteins. We have previously reported that the C-terminal domain of M. smegmatis SSB, which is not essential for interaction with DNA, is the site for the binding of cognate RecA. The data in Chapter 3 describes the characterization of the SSB C-terminus with the objective of delineating the elements responsible for mediating protein-protein interaction, as well as to define the mechanism by which SSB is able to modulate the activities of RecA. To map the RecA interaction domain of SSB we created deletion mutants in MsSSB lacking 5, 10, 15 or 20 residues from the C-terminal. The truncated SSB proteins were expressed with a His- tag at the N- terminus and purified to homogeneity using a Ni-NTA affinity matrix. We observed unlike MsSSB, MsSSB∆C5 and MsSSB∆C10, MsSSB∆C15 and MsSSB∆C20 were unable to support three-strand exchange catalyzed by MsRecA. Based on the observation that interaction with SSB is essential for MsRecA to catalyze the strand Exchange reaction, we postulate that the RecA interacting domain of SSB is situated between the 15th and the 20th residue from the C-terminal. Further, the C-terminal of MsSSB modulates the transitions between DNA binding modes. Unlike the case with EcSSB where deletion of the last 8 residues from the C-terminal stabilizes the (SSB)35 mode of ssDNA binding, we observe that in case of MsSSB the deletion of C-terminal seems to destabilize the (SSB)35. In addition, the transition from the low density binding mode to a high density mode involves the formation of several intermediates when the C-terminal residues are deleted. With the objective of understanding the functions to the C-terminal of SSB independent of its DNA-binding domain in modulating RecA functions, we employed a peptide corresponding to the 35 residues from the C-terminal of the MsSSB. We observed that the C-terminal region alone is capable of interacting with RecA. In addition we also observed that the C-terminal domain of SSB stimulates RecA functions independent of its DNA binding domain. To address the question, whether the stimulatory effect of the C-terminal domain of SSB in the absence of its DNA-binding domain is restricted to RecA or is a generalized phenomenon associated with all SSB interacting proteins; we tested the effect of C-terminal domain of SSB on UvrD which is known to interact with SSB. UvrD participates in several pathways of DNA metabolism, which include the nucleotide excision repair (NER) and mismatch repair pathway, replication and recombination. Genetic evidence suggests that UvrD and SSB interact in vivo. We tested the effect of mycobacterial SSB on M. tuberculosis UvrD1 (MtUvrD1) functions in vitro. We observe that MtUvrd1 physically interacts with SSB. Further, presence of SSB has an inhibitory effect on the helicase activity of MtUvrD1 and that this effect is dependent on the C-terminal region as the deletion of residues from the C-terminal of SSB abrogates the inhibitory effect of SSB. However, unlike RecA, the C-terminal region of SSB alone had no effect on the helicase activity of UvrD1. We also observed that MsSSB has opposing effects on the ATPase activity of MtUvrD1. In the presence of low concentrations of SSB the ATPase activity is enhanced, while we observed an inhibition when the concentration of MsSSB is high. The precise mechanistic details of how SSB is able to act as an accessory protein to RecA, in context of homologous recombination and stimulates its biochemical activities have been a subject of debate. Whereas research from some groups has shown that the stimulatory effect SSB is mediated through its ability to melt DNA secondary structure, thereby allowing RecA to overcome the kinetic barrier imposed by the presence of secondary structure in ssDNA, others postulate that SSB plays a direct role in the stabilization of RecA nucleoprotein filament and prevents its dissociation. Chapter 3 discusses the experimental evidence in favor of the aforesaid models and based on the results of our experiments; we propose that the accessory functions of SSB may be mediated by a mechanism that involves elements of both models. While interaction with SSB can bring about a conformational change in RecA that is reflected in the enhanced levels of strand exchange and co-protease activity, the helix destabilizing function of SSB is essential during heteroduplex extension and to sequester the displaced strand such that it does not participate in any further pairing reactions. The novel finding that we present in Chapter 3 is that the interaction of SSB C-terminal alone has a stimulatory effect upon RecA activities. Furthermore, we observed that M. tuberculosis UvrD1 is a weak interaction partner of SSB. The physical and functional interactions between MsSSB with RecA on the one hand, and MsSSB and UvrD1 on the other highlight different types of cross-talk between the components of HR and DNA repair pathways. In contrast to the results of earlier studies, our results indicate that protein-protein interactions alone between SSB and RecA may modulate the RecA mediated processes of presynapsis, homologous pairing and strand exchange between homologous DNA molecules as well as modulate its co-protease activity. In addition, our studies indicate that a direct protein-protein interaction is responsible for the modulation of UvrD1 activities by SSB.

Homologous recombination (HR) is a highly conserved cellular process involved in the maintenance of chromosomal integrity and generation of genetic diversity. Biochemical and genetic studies have suggested that HR is crucial for repair of damaged DNA arising from various endogenous or exogenous assaults on the genome of any organism. Further, HR is vital to repair fatal DNA damage during DNA replication. An instructive example of cross-talk between the processes of DNA recombination and replication can be construed in the processing of replication/recombination/repair intermediates. The impediment(s) to the progression of DNA replication fork is one of the underlying causes for increased genome instability and consequently this might compromise the survival of organism. Various processes manifest at stalled replication forks before they can be rendered competent for the replication-restart. One of the mechanisms of replication-restart involves replication fork reversal (RFR), which envisage unwinding of the blocked forks with simultaneous annealing of the parental and daughter strands o generate a Holliday junction intermediate adjacent to DNA double strand end. Genetic evidence shows that in E. coli dnaEts mutant, holD mutant and in helicase defective rep mutant, RFR is catalyzed by RuvAB complex. Classically, HJ intermediates are generated during the terminal stages of the HR pathway. In E. coli, branch migration and resolution of HJ intermediates is promoted by RuvA, RuvB and RuvC proteins, which participate at the late stages of HR. Structural, biochemical and mutational analysis suggest that E. coli RuvA binds Holliday junction DNA with high affinity and specificity. RuvB, a member of the AAA+ (ATPase associated with various cellular activities) family, is recruited to the RuvA-Holliday junction complex and functions as a motor protein. Together, RuvA and RuvB catalyze ATP dependent branch migration of HJ. The resolution of HJ is catalyzed by the RuvC endonuclease, which introduces coordinated cuts at two symmetrical sites across the junction. RuvAB complex, the Holliday junction branch migration apparatus, is ubiquitous in bacteria. Genetic, biochemical and structural studies have not only established the in vivo role of E. coli RuvAB, in context of HR pathway, but have also provided valuable insights into the mechanism of HJ processing by RuvAB complex. However, the paucity of extensive studies examining the biochemical properties of each member of the RuvABC protein complex restricts models in deciphering the functions of the individual components of this tripartite protein complex. Our current understanding of the biochemical function of E. coli RuvA is within the context of its interacting cellular partner, RuvB. Consequently, the inherent activities of RuvA in the context of DNA repair and HR are poorly understood. Moreover, it remains to be ascertained if RuvABC protein complex, its different sub-complexes, or the individual subunits can function differently in the processing of HJ intermediates generated during DNA repair and HR. The information from these studies would be helpful in understanding the mechanistic details of HR pathway in mycobacteria. Additionally, a number of important questions regarding the molecular basis of RuvAB catalyzed fork reversal remain unanswered. Therefore, exploration of biochemical details of the RuvAB mediated RFR would provide mechanistic insights into the dynamics of fork reversal process. Moreover, analysis of RuvAB catalyzed RFR might be helpful in validating the different assumptions of the RFR model that has been proposed on the basis of genetic analysis of certain E. coli replication mutants. Another interesting question that remains to be answered is, how under in vivo conditions, RuvABC protein complex or its individual subunits are regulated to function differently in the context of HR and DNA repair? Mycobacterium tuberculosis is an important intracellular pathogen which is likely to experience substantial DNA damage inside the host and thus may require an efficient DNA recombination and repair machinery for its survival. Our knowledge about the mechanistic aspects of genetic exchange in mycobacteria is rather limited. Therefore, understanding of the processes catalyzed by the components of HR pathway may help in molecular genetic analysis of mycobacteria. Sequence analysis of M. tuberculosis genome, followed by various comparative genomic studies, has revealed the presence of putative homologs of E. coli rec genes but it is not known whether these gene products are able to catalyze the reactions similar to their E. coli counterparts. In M. tuberculosis, the genes encoding for the enzymatic machinery required for branch migration and resolution of HJ intermediates are present. The ruvA, ruvB and ruvC genes form an operon, and are probably translationally coupled. Further, these ruv genes are DNA damage inducible. The transcript level of ruvC is regulated by both RecA dependent and independent mechanisms whereas ruvA and ruvB are induced only through RecA dependent SOS response. During M. tuberculosis infection of host cells, expression of ruvA and ruvB genes is upregulated. We therefore surmise that their gene product might be required for DNA replication, recombination or repair, and would be physiologically relevant under in vivo conditions. However, the details of reactions involved in the processing of HR intermediates and rescue of stalled replication forks in M. tuberculosis remains unknown. In the initial part of this study, we have investigated the function of M. tuberculosis RuvA protein using Holliday junctions containing either homologous or heterologous core. In the later part, we have explored the ability of M. tuberculosis RuvA and RuvB proteins to catalyze in vitro replication fork reversal. M. tuberculosis ruvA gene was isolated by PCR amplification and cloned in an expression vector to generate the pMTRA construct. Genetic complementation assays, using the pMTRA construct transformed into E. coli ΔruvA mutant, indicated that M. tuberculosis ruvA is functional in E. coli and suggested that it can substitute for E. coli RuvA in conferring resistance to MMS and survival following UV irradiation. Having established the functionality of M.tuberculosis ruvA, a method was developed for heterologous over-expression and purification of M. tuberculosis RuvA protein (MtRuvA). MtRuvA was purified to homogeneity and the identity of purified protein was verified using western blot analysis using the anti-MtRuvA antibodies. Purified MtRuvA was free of any contaminating endo- or exo-nuclease activity. Biochemical functions of MtRuvA were defined by performing detailed investigations of DNA-binding and Holliday junction processing activities. Substrate specificity of purified MtRuvA was examined,through DNA binding assays, by using oligonucleotide substrates mimicking differentintermediates involved in the pathway of recombinational DNA repair. Purified M. tuberculosis RuvA exhibited high affinity for HJ substrate but also formed stable complex with replication fork and flap substrate. DNase I footprinting of MtRuvA-homologous Holliday junction complex confirmed that MtRuvA bound at the junction center. The DNase I protection conferred by MtRuvA, on homologous HJ, was two-fold symmetric; the continuous footprint was 10 bp longon one pair of symmetrical arms and 7 bp on the opposite pair of arms. In parallel, DNase footprinting of MtRuvA-heterologous Holliday junction complex generated a footprint that encompassed 16 nucleotide residues on each strand of the Holliday junction. Different crystallographic studies have envisaged an important role for RuvA in base pair rearrangement atthe center of the junction. Also, in crystal structure of tetramer of EcRuvA-HJ complex twobases at the junction center were unpaired. To explore if RuvA binding leads to helical distortionof Holliday junction, MtRuvA-HJ complexes were subjected to chemical probing with KMnO4.In case of heterologous HJ, binding of MtRuvA resulted in appearance of sensitive T residues at the junction crossover. By contrast, binding of MtRuvA to homologous HJ rendered the T residues at the junction center and within the homologous core sensitive to oxidation by KMnO4.Taken together, these observations suggested that binding of MtRuvA distorts two base pairs at the junction crossover in heterologous HJ, whereas in case of homologous HJ base pairs distortion extends into the arms of the junction. These observations with KMnO4 probing were independently validated, in real time, by using sensitive to 2-aminopurine fluorescence spectroscopy measurements of MtRuvA-HJ complexes. To follow structural distortions upon interaction with MtRuvA, HJ variants carrying 2-AP substitution were generated for both homologous and heterologous HJ substrate. In each junction species, the 2-AP residue was uniquely present either at the junction center, adjacent to the center or away from the center. Incase of heterologous HJ, binding of MtRuvA resulted in increase of fluorescence emission of2-AP residues located at the junction crossover but not those of 2-AP residues that were present1-2 base pairs away from the junction center. Binding of MtRuvA to homologous HJ resulted in increase of fluorescence emission of 2-AP residues located at the junction crossover. Further, increase in fluorescence emission was also observed for 2-AP residues present within the homologous core or adjacent to the homologous core in a pair of symmetrically related arms. Thus, 2-AP fluorescence results suggested that binding of MtRuvA to homologous HJ causes base pair distortion within and adjacent to the homologous core whereas in case of heterologous HJ the base pair distortion is restricted to the junction center. Together, these results suggest thatMtRuvA causes two distinct types of base pair distortions between homologous and heterologous HJ substrates. To explore the relationship between binding of MtRuvA and alterations in global structure of the junction DNA, we employed the established technique of comparative gel electrophoresis. Analysis of data from comparative gel electrophoresis revealed that MtRuvA, upon binding to the Holliday junctions, converts the stacked-X structure of HJ to square-planar form and stabilizes the same for loading of RuvB rings and subsequent branch migration by RuvAB complex. Our results underline the possible existence of distinct pathways for RuvA function, which presumably depend on the structure and the nature of the DNA repair or HR intermediates. In summary, our results show that binding of MtRuvA to the HJ induced changes in the local conformation of junction, which might augment RuvB catalyzed branch migration. An unexpected finding is the observation that MtRuvA causes two distinct types of structural distortions, depending on whether the Holliday junction contains homologous or heterologous core. These observations support models wherein RuvA facilitates, in a manner independent of RuvB, base pair rearrangements at the crossover point of both homologous and heterologous Holliday junctions. Although the genetic basis of ruvA ruvB catalyzed RFR in E. coli has been understood in some detail but less is known about the genetic and molecular mechanism of fork reversal in mycobacteria or other organisms. Specifically, to examine if the E. coli paradigm can be generalized to other RuvAB orthologs, we explored the RFR activity of M. tuberculosis RuvAB using a series of oligonucleotides and plasmid-based substrates that mimic stalled replication fork intermediates. This approach might be useful in genetic analysis of factors involved in processing of stalled forks in M. tuberculosis wherein technical difficulties associated with the isolation and characterization of appropriate mutants have limited our understanding of DNA metabolism. Importantly, we have asked the questions as to how the structure at fork junction, extent of reversal and presence of sequence heterology might determine the outcome of RuvAB mediated RFR. The results from this study will be helpful in consolidating the proposed in vivo role for RuvAB complex in fork reversal. The open reading frame corresponding to M. tuberculosis ruvB gene was PCR amplified and cloned in an expression vector to generate the pMTRB construct. Genetic complementation assays were performed to assess the functionality of M. tuberculosis ruvB in E. coli ΔruvB mutant. The data from these assays suggested that M. tuberculosis ruvB is active in E. coli and it is able to make functional contacts with E. coli RuvA. Moreover, the efficient alleviation of MMS toxicity in E. coli ΔruvB mutant suggested that M. tuberculosis ruvB might have a role in relieving replication stress generated under specific in vivo conditions. For biochemical analysis, M. tuberculosis RuvB protein (MtRuvB) was over-expressed in a heterologous system and purified to homogeneity. The identity of purified MtRuvB was verified using western blot analysis using the anti-MtRuvB antibodies. Purified MtRuvB was free of any contaminating endo- or exo- nuclease activity. The DNA-binding properties of MtRuvB were analyzed, in conjunction with its cognate RuvA, by using different substrates that are most likely to occur as intermediates during the processes of DNA replication and/or recombination. MtRuvAB bound HJ, three-way junction and heterologous replication fork with high affinity but with relatively weaker affinity to flap and flayed duplex substrates. MtRuvB displayed very weak affinity for linear duplex and failed to bind linear single-stranded DNA. The high affinity of MtRuvB for HJ substrate, in presence of its cognate RuvA, is indicative of direct and functional interaction between RuvA and RuvB. To further test this idea, the catalytic activity of MtRuvB was assayed in the in vitro HJ branch migration assay. In this assay,MtRuvB, in association with its cognate RuvA, promoted efficient branch migration of homologous HJ over heterologous HJ. To decipher the role of MtRuvAB in processing of stalled replication fork we performed in vitro replication fork reversal (RFR) assay using both oligonucleotide and plasmid based model replication fork substrates. Initially, binding of MtRuvAB to different homologous fork (HomFork) substrates was analyzed using the electrophoretic mobility shift assays. MtRuvAB exhibited similar binding affinity towards different HomFork substrates bearing different spatial orientation of nascent leading and lagging strands. To gain insight into the role of MtRuvAB in processing of replication forks, in vitro RFR reactions were carried out using an array of synthetic homologous fork substrates. In all these reactions, MtRuvAB catalyzed efficient fork reversal leading to generation of both parental duplex and daughter duplex. In the kinetics of fork reversal reaction, for all the fork substrates,the accumulation of daughter duplex increased with time whereas the increase in parental or nascent strand DNA was negligible. Taken together, our results suggest that MtRuvAB can efficiently catalyze in vitro replication fork reversal reaction to generate a Holliday junction intermediate thus implicating that RuvAB mediated fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. Equally important, we demonstrate the reversal of forks carrying hemi-replicated DNA, thus indicating that MtRuvAB mediated fork reversal is independent of symmetry at the fork junction. For understanding the role of RuvAB mediated processing of stalled forks at chromosome level, the fork reversal assays were performed using plasmid derived model “RF” substrate. Fork reversal was monitored by restriction enzyme digestion mediated release of 5’ end labeled fragments of specific size from the fourth arm extruded at the branch point of fork junction. In these reactions MtRuvAB complex was proficient at generating the reversed arm de novo from the RF substrate. Further, MtRuvAB complex catalyzed extensive fork reversal as analyzed by release of linear duplex of2.9 kb from a JM substrate. Use of non hydrolysable analogs of ATP and analysis of restriction digestion mediated release of duplex fragments from the reversed arm suggested that MtRuvAB catalyzed RFR reaction is ATP hydrolysis dependent progressive and processive reaction. MtRuvAB complex catalyzed fork reversal on plasmid substrate that had been linearized thus indicating that MtRuvAB mediated RFR is uncoupled from DNA supercoils in the substrate. Notably, MtRuvAB promoted reversal of forks in a substrate containing short stretch of heterologous sequences, indicating that sequence heterology failed to impede fork reversal activity of MtRuvAB complex. These results are discussed in the context of recognition and processing of varied types of replication fork structures by RuvAB enzyme complex.

Les papillomavirus sont des virus à ADN qui infectent la peau et les muqueuses. Ils causent des verrues et peuvent aussi mener au développement de cancers, dont le cancer du col de l’utérus. La réplication de leur génome nécessite deux protéines virales : l’hélicase E1 et le facteur de transcription E2, qui recrute E1 à l’origine de réplication virale. Pour faciliter l’étude de la réplication du génome viral, un essai quantitatif et à haut débit basé sur l’expression de la luciférase a été développé. Parallèlement, un domaine de transactivation a été identifié dans la région régulatrice N-terminale de la protéine E1. La caractérisation de ce domaine a montré que son intégrité est importante pour la réplication de l’ADN. Cette étude suggère que le domaine de transactivation de E1 est une région protéique intrinsèquement désordonnée qui permet la régulation de la réplication du génome viral par son interaction avec diverses protéines. / Papillomaviruses are small DNA viruses that infect skin and mucosa. They cause warts and can also lead to the development of cancers, including cervical cancer. Replication of their genome requires two viral proteins: the E1 helicase and the E2 transcription factor, which recruits E1 to the viral origin of replication. To facilitate the study of viral genome replication, a quantitative and high-throughput assay based on luciferase expression has been developed. In parallel, a transactivation domain has been identified in the N-terminal regulatory region of the E1 protein. Characterization of this domain showed that its integrity is important for DNA replication. This study suggests that the E1 transactivation domain is an intrinsically unstructured protein region that allows regulation of viral genome replication by its interaction with diverse proteins.

La leucémie lymphoïde représente environ 30% des cas de cancer chez l’enfant. Elle est souvent causée par des réarrangements chromosomiques impliquant des gènes encodant des facteurs de transcription, qui contrôlent des programmes génétiques complexes. Par exemple, LMO2 (LIM-only 2) est un facteur de transcription oncogénique fréquemment exprimé de façon aberrante dans les leucémies lymphoblastiques aigues des cellules T (T-ALL). Dans l’hématopoïèse normale, LMO2 est essentiel à la génération des cellules souches hématopoïétiques à l’origine de toutes les cellules sanguines. D’ailleurs, certaines cellules leucémiques possèdent des propriétés normalement réservées aux cellules souches hématopoïétiques. Ainsi, l’étude de la fonction de LMO2 dans les cellules souches hématopoïétiques peut être pertinente autant dans le contexte hématopoïétique normal que leucémique. Afin de mettre en évidence de nouvelles fonctions moléculaires pour LMO2, j’ai choisi d’identifier les protéines qui s’y associent. En plus de ses partenaires connus, j’ai identifié plusieurs protéines de transcription/remodelage de la chromatine, en accord avec son rôle transcriptionnel. Plusieurs nouvelles fonctions potentielles ont été révélées, indiquant que cette protéine adaptatrice pourrait faire partie de complexes non transcriptionnels, régulant d’autres processus cellulaires. Les oncogènes comme LMO2 pourraient être des régulateurs à large spectre. Particulièrement, j’ai identifié des interactions entre LMO2 et des protéines de réplication de l’ADN. J’ai montré que LMO2 contrôle la réplication de l’ADN dans les cellules hématopoïétiques, et possiblement durant la leucémogenèse, indépendamment de son rôle transcriptionnel. Ensemble, ces études ont donc permis de révéler de nouvelles fonctions pour LMO2, et pourraient servir de paradigme pour d’autres facteurs de transcription oncogéniques, particulièrement aux autres protéines de la famille LMO, qui sont aussi des oncogènes puissants. / Lymphoid leukemia represents about 30% of childhood cancer cases. It is often caused by chromosomal rearrangements involving genes coding for transcription factors, controlling complex genetic programs. As an example, the oncogenic transcription factor LMO2 (LIM-only 2) is often aberrantly expressed in T cell acute lymphoblastic leukemia (T-ALL). In normal hematopoiesis, LMO2 is essential for the generation of hematopoietic stem cells that give rise to all blood cells. Moreover, some leukemic cells possess properties normally reserved to hematopoietic stem cells. Thus, studying the role of LMO2 in hematopoietic stem cells could be relevant to the contexts of normal hematopoiesis and leukemogenesis. To reveal new molecular functions for LMO2, I chose to identify its associated proteins. In addition to its known protein partners, I identified many proteins involved in transcription/chromatin remodeling, in agreement with its transcriptional role. In addition, several new potential functions have been revealed, indicating that this scaffold protein could be part of non-transcriptional protein complexes, regulating different cell processes. Oncogenes like LMO2 could be master regulators in normal hematopoietic and leukemic cells. Particularly, I identified protein-protein interactions between LMO2 and DNA replication proteins. I demonstrated that LMO2 controls S phase progression in hematopoietic cells, independently of its association in transcriptional complexes. LMO2 overexpression in mice induces T-ALL and affects specifically the cell cycle status of thymocyte progenitors, which are targets of transformation by LMO2. Thus, LMO2 promotes DNA replication in hematopoietic cells, and possibly in leukemogenesis. Together, these studies allowed to reveal new functions for LMO2, and could serve as a paradigm for other oncogenic transcription factors, especially for other LMO proteins which are all potent oncogenes.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.