Vaccinia virus (VV), the prototype member of the orthopoxvirus family, is a large virus of complex morphology which contains a 191 Kbp double-stranded DNA genome whose expression is tightly regulated in a temporal fashion during viral replication. The regulation of gene expression can be exerted at various of levels, including transcriptional, translational, and post-translational points of control. In addition to transcriptional regulatory mechanisms, the occurrence of a variety of post-translational modifications in VV has been demonstrated. In an effort to better understand the role played by post-translational modifications during the viral replication cycle, we chose to focus on one specific modification event, ADP-ribosylation. Experiments were designed to determine whether any VV proteins might be subject to ADP-ribosylation. The ability to metabolically label a subset of viral proteins by growth of the virus in the presence of [3H]adenosine, in addition to the effects of the ADP-ribosylation inhibitor nicotinamide on viral core protein precursor processing and replication, provided evidence that this or some similar modification is an obligatory event during VV replication. Immunological reagents were used to identify several of the modified proteins. Biochemical evidence obtained via labeling with various precursor compounds, boronate affinity chromatography, and reverse phase HPLC analysis confirmed that the proteins were modified by ADP-ribose or a closely related compound. Additional ADP-ribosylation inhibitor studies provided further support for the initial finding that the viral proteins are subject to ADP-ribosylation or some related modification, and the evidence obtained from these experiments supports a model where this modification event might serve a function in either the proteolytic processing of the core protein precursors, or in localization of the mature core proteins to sites of VV replication within infected cells. / Graduation date: 1992

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Les poly(ADP-ribose) polymérases (PARPs/ARTDs) sont des senseurs des cassures de l’ADN et utilisent la nicotinamide adénine dinucléotide (NAD+) pour synthétiser un polymère de poly(ADP-ribose) (PAR) long et branché attaché aux résidus accepteurs des protéines nucléaires. Le travail présent montre que les protéines des mammifères PARP-1, PARP-2 et PARP-3 sont capables d’ADPribosyler les extrémités des duplex d’oligonucléotides in vitro. PARP-1 modifie préférentiellement les substrats resequés, alors que PARP-2 et PARP-3 modifient préférentiellement les substrats avec un nick, et sont capables d’ADP-ribosyler les groupements phosphate en 5’ ou 3’ au niveau des extrémités des cassures double brin des duplex d’ADN contenant à proximité un nick ou gap phosphorylé en 5’. Cet étude présente une caractérisationdétaillée des préférences de substrat d’ADN pour les enzymes structuralement proches PARP-2 et PARP-3, et propose un modèle putatif du mécanisme de l’ADP-ribosylation des extrémités de l’ADN catalysée par PARP-3 et PARP-2. L’ADPribosylationefficiente d’un fragment d’ADN d’environ 3kb par PARP-3 et PARP-2, l’ADPribosylationmédiée par les PARPs dans les extrait cellulaires ainsi qu’un signal persistant générée par l’anticorps anti-PAR sur de l’ADN génomique purifié en série à partir des cellules HeLa déplétées de PARG et traitées à la bléomycine suggèrent que certains types de cassures complexes de l’ADN peuvent être efficacement PARylés par les PARPs comme réponse cellulaire aux dommages de l’ADN. Cet nouveau type de modification postréplicative de l’ADN médiée par les PARPs in vitro apporte des nouveaux éléments sur les mécanismes moléculaires sous-jacents aux enzymes qui catalysent l’ADP-ribosylation. / Poly(ADP-ribose) polymerases (PARPs/ARTDs) act as DNA break sensors and use nicotinamide adenine dinucleotide (NAD+) to catalyze the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor residues of nuclear proteins. The present work shows that mammalian PARP-1, PARP-2 and PARP-3 can ADP-ribosylate strand break termini in DNA oligonucleotide duplexes in vitro. PARP-1 preferentially modifies recessed substrates, while PARP2 and PARP3 preferentially modify nicked DNA and can ADPribosylate 5′- and 3′-terminal phosphate residues at double-strand break termini of a DNA duplex containing a proximal 5′-phosphorylated nick or a gap. This work provides detailed characterization of DNA substrate preferences for structurally

The activity of Adenosine diphosphoribosyl transferase (ADPRT), the chromatin-bound enzyme which specifically catalyses the cleavage of oxidized NAD⁺ with the concomitant covalent attachment of the ADP-ribose moiety to acceptor proteins, was investigated in Polyoma Virus-Transformed (PyY) and Untransformed BHK21/C13 (BHK) cells. It was shown that ADPRT activity was consistently 2-4 fold higher in PyY cells than in BHK cells. The spectrum of (ADP-ribose)_n residues synthesised by the two cell types was very similar when analysed by hydroxyapatite column chromatography. Poly (ADP-ribose) Glycohydrolase activity in the two cell types was identical with 25-30% degradation of the poly(ADP-ribose) over a period of 90 minutes. DNA damage resulting from incubation with Deoxyribonuclease was reflected by an immediate increase in ADPRT activity and an increase in (ADP-ribose)_n chain length by both cell types. Polyamines which are present at high concentrations in rapidly dividing tissues were able to stimulate ADPRT activity both <i>in vitro</i> and <i>in vivo</i> in BHK and PyY cells. In general the average chain length of ADP-ribose residues synthesised remained unaltered. No significant increase in the level of DNA-strand breakage could be detected in the polyamine-treated cells. Depletion of the cellular polyamine levels resulted in stimulation of ADPRT activity, but there was no significant difference in the spectrum of (ADP-ribose)_n residues synthesised. Again no significant increase in the level of DNA-strand breaks could be detected in the polyamine-depleted cells. These results suggest that DNA-damage may not be the only means of regulating ADPRT activity and that polyamines may have a role to play in this regard <i>in vivo</i>.

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 183-207). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

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A method for the high resolution fractionation of ADP-ribose polymers has been developed that allows isolation of highly purified polymers of defined size and branching fequency. The key features of the method are purification using dihydroxyboronyl-BioRex 70 and fractionation by anion exchange HPLC.

In this study, ADP-ribosylation reactions in the murine myelomonocytic cell line WEHI-3BD+ were investigated.

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