Positive strand RNA viruses, inculding tombusviruses, are known to utilize cellular membranes to assemble their replicase complexes (VRCs). Two tombusviruses , Tomato bushy stunt virus (TBSV) and Carnation Italian ringspot virus (CIRV), replicate on different organellar membranes, peroxisomes or endoplasmic reticulum (ER) for TBSV and mitochodria outer membranes in case of CIRV. I showed that both TBSV and CIRV replicase proteins could assemble VRCs and replicate viral RNA on purified microsomes (ER) and mitochondria. Different efficiencies of assembly was shown determined by multiple domains on TBSV or CIRV replication proteins. To study why VRC assembly could occur on an alternative organellar membranes, I focused on the phospholipids, key lipid components in ER or mitochondria membranes. Phospholipids directly interact with viral replicases, however, their specific roles during (+)RNA virus replication are far less understood. I used TBSV as a model (+) RNA virus, and established a cell-free TBSV replication system using artificial membranes prepared from different phospholipids. I showed that phosphatidylethanolamine (PE) is required for full cycle replication of the viral RNA.Moreover, PE is enriched at the sites of TBSV replication in plant and yeast cells, and was up-regulated during TBSV replication. Furthermore, up-regulation of total cellular PE content in yeast due to deletion of CHO2 leads to dramatically stimulated TBSV replication. Overall, I identified PE as the key lipid component of membranes required for TBSV replication, and my data highlighted that PE, an abundant phospholipid in all eukaryotic cells, not only serves as a structural component of membrane bilayers, its interaction with the viral replication proteins also stimulates (+)RNA virus replication. Further experiments indicated both early secretory pathway and endocytic pathway are involved in PE re-distribution to site of replication. In addition to lipids and subcellular membranes, certain host proteins are also involved in (+) RNA virus replication and VRC assembly. I identified Hop-like stress- inducible protein 1 (Sti1p), which interacts with heat shock protein 70, is required for the inhibition of CIRV replication. My findings indicate that Hop/Sti1 co-chaperone could act as a virus restriction factor in case of mitochondrial CIRV, but not against peroxisomal tombusvirus.

Checkpoint kinase 2 (CHK2) is an effector kinase of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'α subunit of protein phosphatase 2A (PP2A), a major protein serine/threonine phosphatase of the cell, as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'α binds to the SQ/TQ cluster domain of CHK2, which is a target of ATM phosphorylation. CHK2 is able to bind to many B' subunits as well as the PP2A C subunit, indicating that it can bind to the active PP2A enzyme. The induction of DNA double-strand breaks by ionizing radiation (IR) as well as treatment with doxorubicin causes dissociation of the B'α and CHK2 proteins, however, it does not have an effect on the binding of B'α to CHK1. IR-induced dissociation is an early event and occurs in a dose-dependent manner. CHK2 and B'α can re-associate hours after DNA damage and this is not dependent upon the repair of the DNA. Dissociation is dependent on ATM activity and correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after IR. CHK2 is able to phosphorylate B'α in vitro; however, in vivo, irradiation has no effect on PP2A activity or localization. Alternatively, PP2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity and protein stability. These data reveal a novel mechanism for PP2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP2A/CHK2 complex in later time points after damage, which may help to attenuate the signal. This mechanism of CHK2 negative regulation by PP2A joins a growing list of negative regulations of DNA damage response proteins by protein serine/threonine phosphatases.

DNA double-strand breaks (DSBs) are lethal in eukaryotic cells if left unrepaired. In Saccharomyces cerevisiae the Mre11/Rad50/Xrs2 (MRX) complex is required for repair of DSBs through homologous recombination and nonhomologous end joining. Although Mre11 complexes exhibit 3'[rightwards arrow]5' exonuclease activity and endonuclease activity on DNA hairpin and single-stranded DNA overhang substrates in vitro, the role of the MRX complex in homologous recombination in vivo is not well understood. It has been shown to be specifically required for the processing of protein-conjugated DNA ends at DSBs during meiosis and hairpin-capped DSBs in mitotic cells and has been suggested that the Mre11 nuclease functions to remove damaged DNA ends. Recently, the Sae2 protein has been demonstrated to be involved in hairpin-capped DSBs and DNA end processing along with MRX in vivo. However, the Sae2 protein has no known homologs outside of fungi and no obvious motifs to suggest the function(s) of the Sae2 protein. We have purified recombinant Sae2 and MRX and report that the Sae2 protein itself is a single-stranded DNA endonuclease. The Sae2 protein stimulates the 3[rightwards arrow]5' exonuclease activity of the MRX complex. Also, the MRX complex can stimulate Sae2 nuclease activity to cleave ssDNA adjacent to DNA hairpin structures. The Sae2 protein also binds independently to double-stranded DNA and forms higher order protein-DNA complexes with MRX. These results provide biochemical evidence for functional cooperatively between MRX and Sae2 on DSBs and hairpin-capped DNA ends. / text

Eukaryotic cells repair DNA double-strand breaks (DSBs) through both non-homologous and homologous recombination pathways. The initiation of homologous recombination requires the generation of 3' overhangs, which are essential for the formation of Rad51 protein-DNA filaments that catalyze subsequent steps of strand invasion. Experiments in budding yeast show that resection of the 5' strand at a DSB is delayed in strains lacking any components of the Mre11/Rad50/Xrs2 (MRX) complex¹ . In meiosis, a specific class of hypomorphic mutants of mre11 and rad50 (Rad50S) are completely deficient in 5' resection and leave Spo11 covalently attached to the 5' strands of DNA breaks². Similar to mre11S and rad50S mutants, sae2 deletion strains fail to resect 5' strands at meiotic DSBs and accumulate covalent Spo11 adducts³;⁴. In addition, Sae2 and MRX were also found to function cooperatively to process hairpin-capped DNA ends in vivo in yeast. sae2 and mrx null strains show a severe defect in processing these structures and accumulate hairpin-capped DNA ends⁵;⁶. The Longhese laboratory has also shown that Sae2 deletion strains show a delay in 5' strand resection, similar to rad50S strains⁷. Recently, Bettina Lengsfeld in our laboratory demonstrated that Sae2 itself possesses nuclease activity and that MRX and Sae2 act cooperatively to cleave single-stranded DNA adjacent to DNA hairpin structures⁸. In vitro characterization of Sae2 showed that the central and N-terminal domains are required for MRX-independent nuclease activity and that the C-terminus is required for cooperative activities with MRX. Sae2 also acts independently of MRX as a 5' flap endonuclease on branched structures in vitro. Our studies investigate whether MRX, Sae2, and Exo1 function cooperatively in DNA resection using recombinant, purified proteins in vitro. We developed assays utilizing strand-specific Southern blot analysis to visualize DNA end processing of model DNA substrates using recombinant proteins in vitro. Our results demonstrate that MRX and Sae2 cooperatively resect the 5' end of a DNA duplex together with the Exo1 enzyme, supporting a role for these factors in the early stages of homologous recombination and repair. / text

<p>In diploid yeast cells, mitotic recombination is very important for repairing double-strand breaks (DSB). When repair of a DSB results in crossovers, it may cause loss of heterozygosity (LOH) of markers centromere-distal to the DSB in both daughter cells. Gene conversion events unassociated with crossovers cause LOH for an interstitial section of a chromosome. Alternatively, DSBs can initiate break-induced replication (BIR), causing LOH in only one of the daughter cells. Mapping mitotic LOH contributes to understanding of mechanisms for repairing DSBs and distribution of these recombinogenic lesions. Methods for selecting mitotic crossovers and mapping the positions of crossovers have recently been developed in our lab. Our current approach uses a diploid yeast strain that is heterozygous for about 55,000 SNPs, and employs SNP-Microarrays to map LOH events throughout the genome. These methods allow us to examine selected crossovers on chromosome V and unselected mitotic recombination events (crossovers, gene conversion events unassociated with crossovers, and BIR events) at about 1 kb resolution across the genome.</p><p>Mitotic recombination can be greatly induced by UV radiation. However, prior to my research, the nature of the recombinogenic lesions and the distribution of UV-induced recombination events were relatively uncharacterized. Using SNP microarrays, we constructed maps of UV-induced LOH events in G1-synchronized cells. Mitotic crossovers were stimulated 1500-fold and 8500-fold by UV doses of 1 J/m2 and 15 J/m2, respectively, compared to spontaneous events. Additionally, cells treated with 15 J/m2 have about eight unselected LOH events per pair of sectors, including gene conversions associated and unassociated with crossovers as well as BIR events. These unselected LOH events are distributed randomly throughout the genome with no particular hotspots; however, the rDNA cluster was under-represented for the initiation of crossover and BIR events. Interestingly, we found that a high fraction of recombination events in cells treated with 15 J/m2 reflected repair of two sister chromatids broken at roughly the same position. In cells treated with 1 J/m2, most events reflect repair of a single broken sister chromatid (Chapter 2). </p><p>The primary pathway to remove pyrimidine dimers introduced by UV is the nucleotide excision repair (NER) pathway. In NER, the dimer is excised to generate a 30-nucleotide gap that can be replicated to form DSBs if not filled in before DNA replication. The NER gap can also be expanded by Exo1p to form single stranded gaps greater than one kilobase. Alternatively, in the absence of NER, unexcised dimers could result in blocks of DNA replication forks. Resolving the stalled replication fork could lead to recombinogenic breaks. In Chapter 3 and Chapter 4, we analyzed recombination events in strains defective in various steps of processing of UV-induced DNA damage, including exo1 and rad14 mutants. </p><p>In Chapter 3, I show that Exo1p-expanded NER gaps contribute to UV-induced recombination events. Interestingly, I also found that Exo1p is also required for the hotspot activity of a spontaneous crossover hotspot involving a pair of inverted Ty repeats. In addition to its role of expanding a nick to a long single-stranded gap, Exo1p is also a major player in DSB end resection. Therefore, I examined the gene conversion tract lengths in strains deleted for EXO1. I found that, although crossover-associated gene conversion tracts become shorter in the exo1 mutant as expected, noncrossover tract lengths remained unaffected. As a result, noncrossover tracts are longer than crossover tracts in the exo1 mutant while the opposite result was observed in the wild-type strains. I proposed models to rationalize this observation.</p><p>In Chapter 4, to investigate whether the substantial recombinogenic effect in UV in G1-synchronized cells requires NER, we mapped UV-induced LOH events in NER-deficient rad14 diploids treated with 1 J/m2. Mitotic recombination between homologs was greatly stimulated, which suggests that dimers themselves can also cause recombination without processing by NER. We further show that UV-induced inter-homolog recombination events (noncrossover, crossover and BIR) depend on the resolvase Mus81p, and are suppressed by Mms2p-mediated error-free post-replication repair pathway. </p><p>The research described in Chapters, 2, 3, and 4 are in the publications Yin and Petes (2013), Yin and Petes (2014), and Yin and Petes (2015), respectively.</p> / Dissertation

Viruses are the only organisms known to store their genetic information solely in the form of RNA, and have thus evolved unique machinery to replicate an RNA genome and initiate viral gene expression in the infected cell. The large polymerase protein (L) of negative-strand (NS) RNA viruses is a particularly intriguing model for viral replication, where all of the enzymatic activities required for mRNA transcription, RNA modification, and genomic RNA replication are contained within a single polypeptide. Whereas the host cell requires a suite of enzymes to accomplish these tasks, L alone is the catalytic engine driving NS RNA viral replication. Here we demonstrate purification of functional L protein from Machupo virus (MACV) and reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified recombinant components. Through single-molecule electron microscopy analysis of MACV L, we provide the first structural information of viral L proteins. Comparative analysis with nonsegmented NS RNA viral L proteins reveals how the various enzymatic domains are arranged into a conserved architecture shared by both polymerases. Our in vitro RNA synthesis data defines the basis of arenavirus sequence-specific polymerase recruitment and how inter-termini interactions regulate template recognition. Moreover, we discover a new role for the arenaviral matrix protein in regulating viral RNA synthesis by locking a polymerase-template complex. The inhibitory matrix-L-RNA assembly functionally links transcription regulation and polymerase packaging, and reveals a mechanism for NS RNA viruses to ensure polymerase incorporation during virion maturation. Reconstitution of RNA synthesis in vitro establishes a new framework to understand the arenaviral polymerase complex, and our structural and biochemical experiments provide a basis for mechanistic analysis of the NS RNA viral replication machinery.

Im Rahmen dieser Arbeit wurde Organisation, Expression und Funktion des humanen Peroxisomal-Testis-Spezifisch-1(PXT1)-Gens untersucht. Die mRNA des humanen PXT1-Gens enthält nicht wie bisher bekannt zwei Exons, sondern fünf Exons. Die Expression von drei putativen Exons stromaufwärts konnte in dieser Arbeit bestätigt werden. Die Ergebnisse qualitativer und quantitativer Real Time-PCR zeigen, dass sich das Exon 1 aus drei unterschiedlich gespleißten Einheiten (Exons 1a, 1b und 1c) zusammensetzt. Das humane PXT1-Gen unterliegt dem alternativen Spleißen, wovon die Exons 1b, 1c, 2 und 4 betroffen sind, was Sequenzanalysen zeigen. Sechs Transkripte konnten insgesamt identifiziert werden. Die zusätzlichen Exons haben Auswirkungen auf die Proteinstruktur aufgrund der Verlängerung des ORF, kodierend für einst 51 Aminosäuren, auf 134. Im längeren Protein wird die BH3 interacting domain (BID) nachgewiesen, von der eine proapoptotische Funktion bekannt ist. Aufgrund des alternativ gespleißten Exon 4 und der daraus resultierenden Leserasterverschiebung existiert ein verkürztes Protein, in dessen mRNA sich ein vorzeitiges Stopkodon befindet. Die proapoptotische Domäne ist nicht mehr nachweisbar. In silico-Analysen zeigen, dass die Sequenzen der Exons 1a und 1b von PXT1 sich mit dem KCTD20-Gen überlappen, das für einen Kaliumkanal kodiert.  Im Unterschied zum murinen, testisspezifischen Pxt1-Gen, ist das humane Homolog trotz Prädominanz im Testis auch schwächer in anderen Geweben nachweisbar.  Zur weiteren Klärung der proapoptotischen Funktion von Pxt1 in Keimzellen wurde am Mausmodell (Pxt1-Knockout-Maus) die Anzahl an DNA-Strangbrüchen untersucht. Im Vergleich zu den Kontrolltieren (C57BL/6J) zeigt die Pxt1-Knockout-Maus eine signifikant erhöhte Anzahl an Spermien mit DNA-Strangbrüchen. Dieses Ergebnis bestätigt die Annahme, dass das PXT1/Pxt1-Gen eine Art Entsorgungsfunktion für beschädigte Spermien ausübt. Im zeitlichen Verlauf zeigte sich aber, dass die Spermien der Knockout-Tiere nicht sensibler als die Wildtyp-Tiere auf DNaseI reagieren.  Als mögliches Kandidatengen für Mutationsanalysen bei Männern mit Fertilitätsstörungen wurden 55 Patienten mit Fertilitätsstörungen (Azoo- oder Oligozoospermie) auf Punktmutationen im PXT1 untersucht. Eine Mutation konnte nicht identifiziert werden. Des Weiteren wurde die DNA der Patienten auf Copy Number Variations analysiert. Sowohl heterozygote als auch homozygote Duplikationen konnten im Exon1, bestätigt mithilfe der arraybasierten Comparativen Genomischen Hybridisierung (aCGH), vereinzelt auch in Exon2 und Exon3 nachgewiesen werden. Zusätzlich konnte bei einem Patienten eine Deletion nachgewiesen werden. Die bestätigte Duplikation im Exon 1 besitzt aber keinen Krankheitswert, da sie in einem Kontrollkollektiv eine Prävalenz von 41% in heterozygoter und 10% in homozygoter Form besitzt.

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. To protect genomic integrity and ensure cellular homeostasis, cells initiate a complex signaling-based response that activates cell cycle checkpoints, coordinates DNA repair, regulates gene expression and, if necessary, induces apoptosis. The spatio-temporal control of this signaling pathway relies on a large number of post-translational modifications, including phosphorylation and regulatory ubiquitylation. In this thesis, I describe the discovery and characterization of the E3 ubiquitin ligase RNF168, which cooperates with the upstream E3 ubiquitin ligase RNF8 to form a cascade of regulatory ubiquitylation at damaged chromatin. One of the main functions of RNF8/RNF168-dependent chromatin ubiquitylation is to generate a molecular landing platform for the ubiquitin-dependent accumulation of checkpoint and DNA repair proteins such as 53BP1, the breast-cancer associated protein BRCA1 and the RNF168-paralog RNF169. I present evidence that the hierarchical recruitment of these proteins to DSB sites is, in large part, organized through the use of tandem protein interaction modules. These modules are composed of a ubiquitin-binding domain and an adjacent targeting motif called LRM, which specifies the recognition of RNF8- and RNF168-ubiquitylation substrates at damaged chromatin. I conclude that the LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the chromatin-based response to DSBs. Collectively, my results indicate that RNF168-mediated chromatin ubiquitylation is critical for the physiological response to DSBs in human cells. The importance of the ubiquitin-based response to DSBs is underscored by the finding that RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder, is caused by mutations in the RNF168 gene.

Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that assembles its membrane-bound replication complexes at mitochondria in plant cells. This process is accompanied by extensive inward invagination of the mitochondrial outer membrane, leading to the formation of cytosol-filled spherules, wherein viral RNA synthesis occurs. The mechanism by which CIRV is able to induce spherule formation is unknown, however growing evidence suggests that the host-cell ESCRT (Endosomal Sorting Complex Required for Transport) machinery – a multi-protein complex normally involved in late endosome maturation – may be involved. ESCRT consists of ~30 soluble proteins that form sub-complexes assembled at the late endosomal surface, and function in multivesicular body (MVB) biogenesis. While ESCRT is relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Hence, as an initial step towards understanding the potential role of ESCRT in CIRV replication, we examined the protein-protein interaction network, subcellular localization, and gene expression profiles of the Arabidopsis thaliana ESCRT components. Overall, the results from these studies suggest that ESCRT organization and function is relatively well conserved in plants compared to other eukaryotes. We also observed that ESCRT is important for CIRV replication, as expression of dominant-negative versions of several key ESCRT components reduced CIRV replication efficiency in plant cells. Moreover, the Arabidopsis ESCRT-I component, Vps23A is recruited from late endosomes to mitochondria in plant cells expressing the CIRV replicase protein, p36, and recruitment of Vps23A was shown to be mediated by sequences located at the N terminus of p36. It was also shown that recruitment of Vp23A to mitochondria by p36 does not require the Ubiquitin E2 Variant domain of Vps23A, which is in contrast to recruitment of ESCRT by retroviruses during viral budding in mammalian cells. Taken together, these results support the hypothesis that CIRV recruits ESCRT by a novel mechanism in order to carry out its replication, a finding that may lend important insight to aspects of normal ESCRT function in plants.

DNA double-strand breaks (DSBs) are highly cytolethal DNA lesions. To protect genomic integrity and ensure cellular homeostasis, cells initiate a complex signaling-based response that activates cell cycle checkpoints, coordinates DNA repair, regulates gene expression and, if necessary, induces apoptosis. The spatio-temporal control of this signaling pathway relies on a large number of post-translational modifications, including phosphorylation and regulatory ubiquitylation. In this thesis, I describe the discovery and characterization of the E3 ubiquitin ligase RNF168, which cooperates with the upstream E3 ubiquitin ligase RNF8 to form a cascade of regulatory ubiquitylation at damaged chromatin. One of the main functions of RNF8/RNF168-dependent chromatin ubiquitylation is to generate a molecular landing platform for the ubiquitin-dependent accumulation of checkpoint and DNA repair proteins such as 53BP1, the breast-cancer associated protein BRCA1 and the RNF168-paralog RNF169. I present evidence that the hierarchical recruitment of these proteins to DSB sites is, in large part, organized through the use of tandem protein interaction modules. These modules are composed of a ubiquitin-binding domain and an adjacent targeting motif called LRM, which specifies the recognition of RNF8- and RNF168-ubiquitylation substrates at damaged chromatin. I conclude that the LRM-based selection of ligands is a parsimonious means to build a highly discrete ubiquitin-based signaling pathway such as the chromatin-based response to DSBs. Collectively, my results indicate that RNF168-mediated chromatin ubiquitylation is critical for the physiological response to DSBs in human cells. The importance of the ubiquitin-based response to DSBs is underscored by the finding that RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder, is caused by mutations in the RNF168 gene.