Indiana University-Purdue University Indianapolis (IUPUI) / Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

The genome of mammalian cells is under constant attack from DNA-damaging agents. To maintain genomic integrity, cells activate an array of pathways primarily consisting of DNA repair and DNA damage checkpoints. Human replication protein A (RPA), a single-stranded DNA (ssDNA) binding protein, is essential for almost all DNA metabolic pathways. However, the role of RPA in nucleotide excision repair (NER), a DNA repair pathway for removing bulky DNA lesions, remains elusive. In this study, the binding of RPA to a battery of well-defined ssDNA substrates has been systematically examined using fluorescence spectroscopy. The results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA, and there was no direct contact between RPA residues and the lesion itself. These findings will help define the roles of RPA in DNA damage recognition in NER. In cells, RPA undergoes hyperphosphorylation in the N-terminus of RPA32 subunit after DNA damage. In this study, the hyperphosphorylation-induced conformational changes of RPA have been probed using mass spectrometry-based protein foot-printing, fluorescence spectroscopy and limited proteolysis. The data show that upon hyperphosphorylation RPA undergoes a subtle structural change involving its DNA-binding domain B (DBD-B), reducing its affinity for short ssDNA. These results suggest that hyperphosphorylation may modulate RPA functions by altering DBD-B-mediated RPA-DNA/protein interactions. Cellular accumulation of DNA damage has been widely implicated in premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is caused by defective maturation of lamin A and linked to accumulation of DNA double-strand breaks (DSBs). However, how lamin A dysfunction leads to genome instability and premature aging is not understood. Here evidence showed that in HGPS and RD fibroblasts DNA damage checkpoints are persistently activated and recruitment of repair factors to DSBs was impaired. Strikingly, xeroderma pigmentosum group A (XPA), a unique NER protein, formed foci and colocalized with the unrepairable DSBs in the patient cells. RNAi knockdown of XPA in HGPS cells significantly restored DSB repair. These results indicate that XPA dysfunction may play an important role in accumulating DSBs in HGPS, implicating a potential strategy for treatment of these premature aging diseases.

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The gene encoding the Dictyostelium replication protein A large subunit (DdRPA1) has been cloned by screening of an EcoR I partial genomic library and a Hind III genomic sub-library. The complete nucleotide sequence, including the promoter region of the gene has been obtained by sequencing. Though the DdRPA1 protein has a size shift during development, 62 kDa in undifferentiated cells and 81 kDa in differentiated cells; they are the products of the same gene. Northern blot analysis revealed that the expression level of the DdRPA1 was constant throughout differentiation and the size of mRNA is the same at all stages, corresponding to a 81 kDa protein. Thus, it seems that the size change between the 62 kDa and 81 kDa is probably due to posttranslational modification, most likely, proteolytic cleavage. The transcription start site for both sizes of DdRPA1 has been identified at 306 bp upstream of the coding sequence by primer extension reaction. A PCR fragment representing 27% of the gene encoding the DdRPA middle size subunit (DdRPA2) has been generated by using the degenerate primers. This PCR fragment has been cloned and sequenced. The mRNA for this subunit corresponds to a protein of about 35 kDa. A decrease of the DdRPA2 mRNA expression level during differentiation was found by comparison between undifferentiated and differentiated cells. In Dictyostelium, replication protein A is a heterotrimeric protein that can bind with specific DNA sequences in a stage-dependent pattern. These DNA sequences were identified as the cis-acting regulatory sites in differentiation-related genes, including the glycogen phosphorylase 2 gene (gp2). Therefore, it is possible that DdRPA is not only a single-stranded DNA binding protein that is used in multiple essential DNA metabolic processes, such as DNA replication, repair and recombination in undifferentiated cells, but also involved in the transcriptional regulation process during differentiation. / Master of Science

La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.

La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.

La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.

Positive-strand RNAviruses invariably assemble their viral replication complexes (VRCs) by remodeling host intracellular membranes. How viral replication proteins are targeted to specific organelle membranes to initiate VRC assembly remains elusive. Brome mosaic virus (BMV), whose replication can be recapitulated in Saccharomyces cerevisiae, assembles its VRCs by invaginating the outer perinuclear endoplasmic reticulum (ER) membrane. Remarkably, BMV replication protein 1a (BMV 1a) is the only viral protein required for such membrane remodeling. We show that ER-vesicle protein of 14 kD (Erv14), a cargo receptor of coat protein complex II (COPII), interacts with BMV 1a. Moreover, the perinuclear ER localization of BMV 1a is disrupted in cells lacking ERV14 or expressing dysfunctional COPII coat components (Sec13, Sec24 or Sec31). The requirement of Erv14 for the localization of BMV 1a is bypassed by addition of a Sec24-recognizable sorting signal to BMV 1a or by overexpressing Sec24, suggesting a coordinated effort by both Erv14 and Sec24 for the proper localization of BMV 1a. The COPII pathway is well known for being involved in protein secretion; our data suggest that a subset of COPII coat proteins have an unrecognized role in targeting proteins to the perinuclear ER membrane.

Glycogen phosphorylase 2 (gp-2) is a key enzyme during the development of Dictyostelium discoideum. The gp-2 enzyme breaks down glycogen into glucose monomers that are subsequently used to synthesize the terminal end products of cellular differentiation. This gene is an ideal candidate for studying the process of selective gene expression because its product figures so prominently in the development of this organism, implying a dependable control mechanism responsible for its developmentally regulated expression. I present in this thesis the identification of several putative cis-acting elements of gp-2 as revealed through footprint analysis. Due to the extreme AT-bias characteristic of Dictyostelium promoters, footprinting conditions required intensive optimization with respect to template, nonspecific competitor, source of protein extract and DNase I digestion. Using an endlabeled fragment containing seven repeated sequences (3 TA boxes [TAATTATA], 2 TAG boxes [TAAAAATGGT] and 2 C boxes [ACCCACT]), purified replication protein A and several developmental nuclear extracts were tested for DNA binding activity. Small footprints were observed on the TAG and C boxes of the promoter for both protein sources. However, using a more sensitive footprinting strategy involving multiple rounds of primer extension, larger footprints spanning the same promoter regions were detected. In both cases, the appearance of the footprints coincided with the documented transcriptional activity of the gene. It can be concluded from the data obtained that the TAG and C boxes are very likely cis-acting elements involved in the regulation of gp-2 expression. / Master of Science