Dans les cellules, le déroulement de l'ADN double-brin est catalysé par une famille de protéines appelées hélicases. Ces protéines sont présentes chez tous les organismes des virus jusqu'à l'homme. Parmi ces hélicases, celles de la famille RecQ jouent un rôle essentiel dans le métabolisme de l'ADN en facilitant de nombreux processus cellulaires tels que la réplication, la réparation, la recombinaison, la transcription et la maintenance des télomères. Chez l'homme, il existe cinq membres de la famille RecQ identifiés comme RECQ1, BLM, WRN, RECQ4 et RECQ5. Les mutations dans BLM, WRN et RECQ4 sont associées à une prédisposition au cancer. En plus du domaine hélicase très conservé et contenant sept motifs bien distincts, la plupart des hélicases de la famille RecQ possèdent également un domaine RecQ C-terminal (RecQ-Ct) et un domaine hélicase RNase D (HRDC). Au cours de ce travail, nous nous concentrons sur les mécanismes intrafonctionnels de certains membres de la famille RecQ des hélicases. Tout d'abord, nous avons utilisé deux isoformes naturels de l'hélicase RECQ5 humain comme modèle pour étudier la modulation fonctionnelle du domaine hélicase avec le doigt de zinc. Ici, nous montrons que la variante tronquée RECQ5α de l'hélicase RECQ5β issue d'un épissage alternatif et composée uniquement du domaine hélicase ne possède ni l'activité ATPase ni l'activité de déroulement de l'ADN. A l'inverse, et ce de matière étonnante, cette protéine est dotée d'une forte activité de réhybridation du brin déroulé. Les mesures quantitatives indiquent que l'amélioration de l'affinité de la protéine pour l'ADN que lui confère le doigt de zinc est à l'origine de ses activités ATPase et hélicase. Le plus important est que l'on constate que le doigt de zinc est capable d'agir comme un facteur moléculaire à même de supprimer l'activité de re-synthèse du brin déroulé par le domaine hélicase et de déclencher l'activité de déroulement d'ADN à travers une modulation de la fixation à l'ADN. Ensuite, nous avons analysé les propriétés biochimiques de deux isoformes de l'hélicase RecQ de Bacillus subtilis : SubL et SubS. Parmi elles, SubS ne dispose pas du domaine HRDC. Nos études montrent que le domaine HRDC est crucial pour Bacillus subtilis RecQ hélicases dans la résolution des intermédiaires de réplication et / ou de réparation de l'ADN tels que les jonctions de Holliday et la jonction de kappa. Les activités ATPase, hélicase et l'activité de rehybridation du brin déroulé sont plus importantes en présence du domaine HRDC. Ces résultats nous permettent de spéculer sur l'importance du domaine HRDC des activités de la famille de RecQ hélicase. Nous avons découvert que dans la famille RecQ, le 12 domaine HRDC peut augmenter les activités ATPases et hélicases. De manière intéressante, le domaine HRDC de Bacillus subtilis joue un rôle critique dans la résolution des intermédiaires de réplication ou de réparation de l'ADN et des jonctions de Holliday. Nous suggérons l'hypothèse que le domaine HRDC des hélicases RecQ participe à exposer leurs fonctions dans le processus de réparation de l'ADN. Dans la dernière partie, nous nous sommes intéressés à l'existence et au rôle du doigt d'arginine dans la protéine BLM. Ces études ont été menées afin de démontrer son rôle dans l'hydrolyse d'ATP et dans la conversion en mouvement mécanique permettant à la protéine de progresser le long de l'ADN. Nos études démontrent que le résidu R982, situé à proximité du γ-phosphate de l'ATP, fonctionne comme un doigt d'arginine dans la protéine BLM. Nos conclusions indiquent en outre que ce doigt d'arginine interagit avec d'autres motifs conservés situés autour du γ-phosphate des nucléotides et qu'ils effectuent ensemble les fonctions enzymatiques au sein d'un réseau complexe.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Pre-mRNA splicing and other RNA processing events occur co-transcriptionally. High resolution kinetic studies performed in our lab showed splicing-dependent RNA Pol II (RNA polymerase II) pausing near the 3’ splice site of a reporter gene. Pausing requires splicing, as mutations that block splicing lead to loss of pausing, and restoring splicing restores pausing. It was proposed that RNA Pol II pausing may occur at splicing-dependent transcriptional checkpoints. In this study, I aimed to search for splicing helicases that might couple splicing with transcription. The ts alleles prp5-1 and prp16-2 were found to cause transcription defects. These genes encode RNA helicases that were reported to act as fidelity factors during splicing. In vivo RNA labelling and RT-qPCR experiments performed with these temperature-sensitive mutants demonstrated reduced transcription coinciding with the splicing defect at restrictive temperature. Furthermore, RNA Pol II ChIP analysis showed polymerase accumulating over intron-containing genes in both mutants. ChIP analysis using antibodies specific to the phosphorylation status of the CTD (Carboxy Terminal Domain) of RNA Pol II, revealed that the apparently stalled polymerase is hyper-phosphorylated at serine 5. Intriguingly, prp8-R1753K, a ts allele of PRP8, a non-helicase splicing factor mutant also showed reduced nascent RNA synthesis but no RNA Pol II accumulation. To elucidate the reason for the observed RNA Pol II accumulation and to identify a possible splicing-dependent transcriptional checkpoint factor, prp5-1 was investigated further. RNA Pol II ChIP-Seq analysis verified that maximum enrichment genome-wide occurred on introns at restrictive conditions in prp5-1, supporting the earlier observation. Furthermore, the double mutant strain cus2Δprp5-1 abolished the RNA Pol II accumulation observed in prp5-1 at restrictive temperature and restored transcription. Recreating a stalled spliceosome in a U2 mutant strain also showed RNA Pol II accumulation in the presence of Cus2p, as observed in prp5-1. My observations suggest a link between transcription and monitoring of splicing and indicate that Cus2p, a U2 snRNP associated protein, could be a checkpoint factor in transcription prior to pre-spliceosome formation. I speculate that fidelity factors may impose transcriptional checkpoints at different stages of splicing.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-114). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 138-170).

The RecQ family of helicases has been termed the “Caretakers of the Genome,” and rightfully so. These proteins are highly conserved from bacteria to humans and have been implicated in functions from homologous recombinatorial repair to damage checkpoint response to telomere maintenance and more. Mutant genes of three of the human RecQ helicases lead to syndromes characterized by a high incidence of cancer, premature aging and early death. Despite their implications in several biological functions and importance to the integrity of the human genome and suppression of cancer, many aspects of the RecQ family structure and function remain unknown. To date, much is known about the catalytic function of the helicase domain and accompanying domains, but considerably less is known about the non-catalytic N-terminus in these proteins, which, in many cases, including those human orthologs involved in disease, can make up about half of the total protein length. While experiments have been able to identify protein partners that interact with the N-terminal region, few are able to narrow the binding sites to minimally functional parts and fewer still describe any detail regarding the structural features of these binding areas. In fact, some reviews have generally described the N-terminus as “featureless,” a concept we challenge in our studies. Many of the N-termini of these RecQs have long been known to contain large stretches of acidic residues, a feature of intrinsically disordered regions. These regions/proteins are rich in charged and polar residues, lack compactness that makes crystallography possible, and have flexible and dynamic conformations that are prevalent in “high specificity, low affinity” interactions. Disordered proteins are well-known to be hot spots for protein/protein interactions and post-translational modifications, amongst other functions. Considering these facts, and recognizing the ties between these and what we know about the N-termini of the RecQs, we hypothesized that these proteins likely have long disordered termini. In Chapter 3, we confirm the presence of disorder at the Top3/Rmi1 binding site on Sgs1, the Saccharomyces cerevisiae RecQ helicase. We show that even in a disordered state, this binding region is not “featureless,” but in fact contains a transient alpha-helical molecular recognition element that is necessary to facilitate complex formation between Sgs1, Top3 and Rmi1. Loss of helical structure at this site leads to increased genomic instability and sensitivity to DNA damaging agents. Based on these results, we suggest that there are likely many more such elements in the N-terminus that that are important for other Sgs1 protein/protein interactions and provide an estimate for the number of interactions in this region. In Chapter 4, we evaluate the prevalence of disorder in a set of Chromatin Processes proteins in an effort to establish a role for disorder with regards to maintaining chromatin integrity. In our bioinformatics study, we found that disorder is overrepresented in the Chromatin Processes proteins, and that a major driving force for disorder in these proteins is protein/protein interaction and post-translational modification. We also show a biological connection to disorder and increased protein/protein interaction by investigating these parameters in the context of the DNA damage checkpoint response and in complex formations. Mediators between highly structured kinases in the checkpoint were the most interactive proteins and over half of all predicted interaction sites occurred in disordered areas. Complexed proteins often contained one protein with a high number of disordered sites and a high number of predicted interactions, while the rest were considerably more ordered. Chapter 5 explores a Sgs1 interaction partner, Rmi1 and uses bioinformatics to design structurally-based point mutations in an effort to further elucidate Rmi1 function in yeast, which remains largely unknown outside of its enhancement of Top3/Sgs1 catalytic function. Using AGADIR, which predicts alpha-helical structure and is particularly useful in our hands for guided-mutagenesis in disordered regions, we identified several point mutations that lead to Δrmi1 phenotypes or intermediate growth on hydroxyurea. We hypothesize that these mutants are important in maintaining Rmi1 stability. Together, these studies suggest an important change in how the field approaches further studies into the RecQ helicases; traditional methods of primary sequence comparisons and crystal structures limit the study of disordered regions that are still functionally important. Future care should be given to consider the conservation of structure or structural elements in the RecQs over strict alignments when comparing functional regions between orthologs. Our studies also suggest that it is highly likely that structural motifs for important protein interactions in RecQs are being overlooked because they are not readily obvious using traditional methods. By understanding these motifs and the interactions they facilitate, we may be able to more easily identify polymorphisms in patients with genomically unstable conditions like cancer and, having better understood the biological process these structures facilitate, design drugs to counteract detrimental effects.

Two diverse works regarding DNA-Drug Interaction are presented here. The first portion deals with covalent interactions between compounds that are derivatives of heterocyclic aza-enediynes and DNA (conventional Watson-Crick base paired double stranded DNA) and the second is related to non-covalent interactions of these compounds with G-quadruplex DNA. The aza-enediynes have been studied for their ability to undergo aza-variants of the Bergman and Myers cyclizations, and the potential role of the ensuing diradicals in DNA cleavage chemistry. The aza-Myers-Saito cyclization of aza-enyne allenes that are derived from base-promoted isomerization of skipped aza-enediynes has been recently reported. In the first part of the dissertation, the synthesis and DNA cleavage chemistry of a series of pyridinium skipped aza-enediynes (2-alkynyl-Npropargyl pyridine salts) are reported. Efficient DNA cleavage requires the presence of the skipped aza-enediyne functionality, and optimal DNA cleavage occurs at basic pH. An optimized analog containing a p-methoxyphenyl substituent was prepared. Studies with radiolabeled DNA duplexes reveal that this analog generates nonselective frank DNA strand breaks, via deoxyribosyl 4'-hydrogen atom abstraction, and also leads to oxidation of DNA guanine bases. This is the first report of enediynelike radical-based DNA cleavage by an agent designed to undergo an alternative diradical-generating cyclization. The second part is based upon the growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for replication. A prototypical replicative helicase - SV40 large T-antigen (T-ag), a multifunctional protein with duplex DNA helicase activity is shown to also unwind G-quadruplex DNA structures. A series of G-quadruplex-interactive agents, particularly perylene diimide derivatives, is explored for inhibition of T-ag duplex and G-quadruplex DNA unwinding activities, and it is revealed that certain perylene diimides are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic Gquadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.

Pre-mRNA splicing by the spliceosome requires the precise and regulated efforts of the five snRNAs (U1, U2, U4, U5, and U6) and numerous associated proteins. Following assembly and activation of the spliceosome, two consecutive reactions result in intron removal and exon ligation from pre-mRNA substrates. It has been established that several members of the DExH/D-box family of helicases act transiently on the spliceosome prior to the chemical steps to authorize the successive reactions by hydrolyzing ATP and consequently inducing structural rearrangements. While it has been suggested that these changes produced in the structure of the spliceosome result in optimal positioning of the reactive species, the mechanisms and products of these reorganizations remain uncharacterized. The work presented here describes the genetic strategy for accumulating and purifying spliceosomes arrested in vivo, during the catalytic steps of the splicing cycle. Using these complexes, we have defined the components required to proceed through the first and second steps of splicing, in addition to the factors required for the release of the spliced message. Analysis of these functional, synchronized particles has also allowed us to define a function for Prp2p in initiating the first step of pre-mRNA splicing. Our data suggest that Prp2p may act in an ATP-independent manner to remodel the spliceosome prior to using its ATPase function to displace the SF3 complex. We propose that the SF3 complex, in addition to its role in identification of the branchpoint, also acts to sequester the reactive 2’OH of the branchpoint adenosine to prevent premature reactivity. Following the two catalytic steps of the splicing cycle, the spliceosome must disassemble and recycle its snRNPs for further rounds of splicing. The essential U6 snRNP component Prp24p, mediates one of the early assembly events - the annealing between the U4 and U6 snRNAs. We have discovered that although Prp24p is essential for viability, its function(s) can be bypassed by overexpressing the U6 snRNA. Additionally, biochemical characterizations of various forms of the U4/U6 snRNP provide evidence that Prp24p must be released before other components of the U4/U6 snRNP are permitted to interact and facilitate tri-snRNP formation. / text

No description available.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.