Browsing by Author "Luger, Karolin, advisor"
Now showing 1 - 13 of 13
Results Per Page
Sort Options
Item Open Access Biochemical, biophysical and functional characterization of histone chaperones(Colorado State University. Libraries, 2014) Zhang, Ling, author; Luger, Karolin, advisor; Krapf, Diego, committee member; Nyborg, Jennifer, committee member; van Orden, Alan, committee member; Stargell, Laurie, committee memberNucleosomes, the basic repeating unit of chromatin, are highly dynamic. Nucleosome dynamics allow for various cellular activities such as replication, recombination, transcription and DNA repair, while maintaining a high degree of DNA compaction. Each nucleosome is composed of 147 bp DNA wrapping around a histone octamer. Histone chaperones interact with histones and regulate nucleosome assembly and disassembly in the absence of ATP. To understand how nucleosome dynamics are regulated, it is essential to characterize the functions of histone chaperones. The first project of my doctoral research focused on the comparison of different nucleosome assembly proteins employing various biochemical and molecular approaches. Nucleosome assembly proteins (Nap) are a large family of histone chaperones, including Nap1 and Vps75 in Saccharomyces cerevisiae, and Nap1 (also Nap1L1), Nap1L2-6 (Nap1-like 2-6, with Nap1L4 being Nap2) and Set in metazoans. The functional differences of nucleosome assembly proteins are thus interesting to explore. We show that Nap1, Nap2 and Set bind to histones with similar and high affinities, but Nap2 and Set do not disassemble non-nucleosomal DNA-histone complexes as efficiently as Nap1. Also, nucleosome assembly proteins do not display discrepancies for histone variants or different DNA sequences. In the second project, we identified Spn1 as a novel histone chaperone and look into new functions of Spn1 on the regulation of chromatin structural states. Spn1 was identified as a transcription regulator that regulates post-recruitment of RNA polymerase II in yeast. We demonstrated that Spn1 is a H3/H4 histone chaperone, a novel finding that was not observed previously. Spn1 also interacts with Nap1, and forms ternary complexes with Nap1 and histones. We also show that Spn1 has chromatin assembly activity and N- and C- terminal domains of Spn1 are required for its histone chaperone properties. At the same time, we had an interesting observation that Spn1 potentially has topoisomerase/nuclease activity, which is dependent on magnesium ions. This activity of Spn1 can also help answer questions raised by in vivo assays related to Spn1, including its correlation with telomere length, the heat sensitivity in the reduction of function yeast strains, and the elongated lifespan in the Spn1ΔNΔC strain. Our studies on the functional comparison of nucleosome assembly proteins revealed their distinct roles in the regulation of nucleosome dynamics. Our findings on the histone chaperone functions and nuclease/topoisomerase activities disclosed new roles of Spn1 in chromatin regulation, by regulating histone-DNA interaction and also maintenance of DNA integrity.Item Open Access Biochemical, biophysical and structural study of the nucleosome-MeCP2 complex(Colorado State University. Libraries, 2009) Yang, Chenghua, author; Luger, Karolin, advisorMethyl-CpG Binding Protein (MeCP2) is an abundant chromatin associated protein that is important in maintaining human health; mutations in this protein cause Rett Syndrome, a neurodevelopmental disease that is a common cause of mental retardation and autism in females. MeCP2 was initially identified as a protein that recognizes the genetic DNA methyl-CpG mark and it was thought to repress gene transcription by recruiting histone deacetylases. Recent studies show that MeCP2 can both repress and activate gene transcription. It also binds chromatin in the absence of the methylation mark, suggesting that its mode of action is more complex than previously assumed. The observation that MeCP2 compacts nucleosomal arrays in vitro and mediates silent chromatin loop formation in vivo suggests a novel mechanism by which MeCP2 regulates gene expression. To further characterize the interplay between MeCP2 and chromatin, it is important to understand the interactions between MeCP2 and nucleosomes, the fundamental component of chromatin. We used biochemical and biophysical approaches to study the interplay between MeCP2 and nucleosomes. Gel mobility assays showed that although MeCP2 can interact with a nucleosome with or without extra nucleosomal DNA, it has a higher affinity for nucleosomes with extra nucleosomal DNA. The N-terminal portion of human MeCP2 (amino acids 78-305) is sufficient to establish this interaction. Size-exclusion chromatography combined with multi-angle light scattering and fluoresecence resonance energy transfer (FRET) assays demonstrated that this interaction occurs at a 1:1 molar ratio and that MeCP2 brings the extra nucleosomal DNA ends in a closer proximity. Small angle X-ray scattering (SAXS) revealed the formation of a more compact complex when MeCP2 interacts with nucleosome with (versus without) extra nucleosomal DNA, indicating that the extra nucleosomal DNA is important in organizing the MeCP2-nucleosome complex. Our data suggest a model in which MeCP2 compacts chromatin by changing the extra nucleosomal DNA path. X-ray crystallography is also used to characterize the nucleosome-MeCP2 complex. Crystals of the nucleosomes with extra nucleosomal DNA in complex with MeCP2 were obtained and diffracted to 5.2 Å. Although MeCP2 dissociated from the crystals after soaking in cryo-protectant, the electron density map reveals the path of extra nucleosomal DNA which may be organized by MeCP2.Item Open Access Biophysical, structural, and functional studies of histone binding proteins(Colorado State University. Libraries, 2010) Sudhoff, Keely B., author; Luger, Karolin, advisor; Chen, Chaoping, committee member; Henry, Charles, committee member; Woody, Robert, committee member; Hansen, Jeffrey C., committee memberEukaryotic genomes are extensively compacted with an equal amount of histone proteins to form chromatin. A high level of control over chromatin structure is required to regulate critical cellular processes such as DNA replication, repair, and transcription. To achieve this feat, cells have developed a variety of means to locally or globally modulate chromatin structure. This can involve covalent modification of histones, the incorporation of histone variants, remodeling by ATP-dependent remodeling enzymes, histone chaperone-mediated assembly/disassembly, or any combination of the above activities. To understand how chromatin structure is affected by histones, it is essential to characterize the interactions between histones and their associated proteins. In Saccharomyces cerevisiae, the multi-subunit SWR1 complex mediates histone variant H2A.Z incorporation. Swc2 (Swr1 complex 2) is a key member of the SWR1 complex and is essential for binding and transfer of H2A.Z. Chz1 (Chaperone for H2A.Z/H2B) can deliver H2A.Z/H2B heterodimers to the SWR1 complex in vitro. Swc2 1-179 (a domain of Swc2 that retains histone binding and the apparent preference for variant dimers) and Chz1 are intrinsically disordered, but become more ordered upon interaction with histones. Quantitative measurements done under physiological in vitro conditions demonstrate that Chz1 and Swc2 1-179 are not histone variant-specific. They bind to histones with an affinity lower than that of previously described histone chaperones, and lack the ability to act on nucleosomes or other histone-DNA complexes. Small-angle X-ray scattering demonstrates that the intrinsic disorder of the proteins allows them to adopt a multitude of structural states, perhaps facilitating many different interactions and functions. We show that Swc2 1-179, despite its overall acidic charge, can bind double stranded DNA, in particular, 3-way and 4-way junction DNA. These junctions are thought to mimic the central intermediates found in DNA damage repair. This characteristic is unique to Swc2 1-179. Consistent with this unexpected activity, yeast phenotypic assays have revealed a role for SWC2 in DNA damage repair, as indicated by sensitivity to DNA damaging agent methane methylsulfonate. Importantly, our data has exposed a novel role for Swc2 in DNA damage repair. In an independent study, we investigated the histone chaperone Vps75, a Nap1 homolog. Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone H3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. We show that deletion of VPS75 results in dramatic and diverse mutant phenotypes, in contrast to the lack of effects observed for the deletion of NAP1. The flexible C-terminal domain of Vps75 is important for the in vivo functions of Vps75 and modulates Rtt109 activity in vitro. Our data highlight the functional specificity of Vps75 in Rtt109 activation.Item Open Access Functional characterization of nucleosome assembly proteins(Colorado State University. Libraries, 2021) Krzizike, Daniel, author; Luger, Karolin, advisor; Kennan, Alan, committee member; Nyborg, Jennifer, committee member; Stargell, Laurie, committee member; Woody, Robert, committee memberThe amount of DNA found within the human body will span from the earth to the sun ~50 times. With the DNA providing the genetic blueprint of all living things, it needs to be packaged in a way that allows accessibility. The first step in this packaging involves nucleosomes, large macromolecular complexes made up of histone proteins and DNA. Nucleosomes must remain dynamic as they are constantly assembled and disassembled for processes such as DNA replication, repair, and transcription. Both assembly and disassembly occur in a specific stepwise manner orchestrated by multiple proteins employed by the cell. Specifically, histone chaperones have been implicated in almost every aspect of nucleosome dynamics such as shuttling histones into the nucleus, histone storage, and both nucleosome assembly and disassembly in an ATP-independent manner. While the structures of many histone chaperones have been determined, the mechanism of how they regulate nucleosome dynamics is still largely unknown. I investigated the mechanism of the nucleosome assembly protein family (Nap family) through several biochemical approaches. The Nap family of proteins are implicated in histone homeostasis through interactions with core histones, histone variants, and linker histones. They are conserved among all eukaryotes from yeast to humans. Members of the Nap family contain a conserved core region flanked by highly disordered N- and C-terminal tails varying in length and charge between species. Using yNap1, we investigated how these tails impact the overall function in regards to histone binding, histone selectivity among core histones and histone variants, and in mediating histone-DNA interactions. We found that the tails are critical for overall function, with the charge of the tails being crucial in regulation. We also investigated Vps75, another member of the Nap family. Similar to Nap1, Vps75 binds core histones, but also stimulates the acetylation activity of Rtt109, a histone acetyltransferase. In light of a recent debate regarding the stoichiometry with which these Nap members bind their histone cargo, we characterized the Vps75-histone interaction using core histones H2A-H2B and H3-H4. Comparing Vps75 with yeast Nap1, we found that the mechanism of histone binding is not conserved among these Nap family members. Further expanding on Vps75, we investigated the interaction with Rtt109 in both the presence and absence of H3-H4. We discovered dimeric Vps75 is capable of binding either one histone tetramer or two units of Rtt109 with the ternary complex consisting of only one unit of Rtt109 and one H3-H4 tetramer. While characterizing Nap family members I became very familiar with Analytical Ultracentrifugation (AUC). AUC is a powerful in-solution technique that provides first-principle hydrodynamic information to determine size, shape, and molecular interactions, making it ideal for the characterization of proteins, DNA, and the interactions among them. As our lab traditionally used AUC to obtain van Holde-Weischet plots, an excellent graphical representation of homogeneity or heterogeneity, we incorporated new analysis techniques for improved accuracy in molecular mass and gross shape determination. Using the added-on fluorescence detection system, we obtained a level of sensitivity and selectivity that was otherwise not possible. Using the powerful method of analytical ultracentrifugation combined with fluorescent studies, we provide insight into the regulation mechanism of Nap family members along with establishing a framework to study other macromolecular complexes.Item Open Access Parsing PARP: the enzymatic and biophysical characterization of poly (ADP-Ribose) polymerases I and II(Colorado State University. Libraries, 2015) Hepler, Maggie R. D., author; Luger, Karolin, advisor; Bailey, Susan, committee member; Yao, TingTing, committee memberThe ADP-ribosyl transferase (ART) family is a prominent group of at least seventeen enzymes comprised of mono (ADP-ribose) transferases (MARTs) and poly (ADP-ribose) polymerases (PARPs). Each family member contains a conserved PARP signature motif in the catalytic domain. Enzymatically active proteins, in the presence of co-factor NAD+, catalyze individual or multiple ADP-ribose groups onto themselves or other proteins in automodification and heteromodification, respectively. The act of ADP-ribosylation implicates the ART family in a multitude of cellular processes including, but not limited to, transcription, apoptosis, DNA damage, metabolism, and inflammation. The founding member of the ART family is PARP-1, a first responder to DNA damage and regulator of active gene expression. In its inactive state and as a chromatin architectural protein, PARP-1 tightly binds chromatin, thereby regulating cellular activities, signifying the importance of PARP-1 and chromatin interaction. Importantly, PARP-1 must be activated and automodified in order to bind histones and gain nucleosome assembly function. Structurally similar and in many ways thought to be functionally redundant, PARP-2 is also thought to primarily function in the DNA damage response. PARP-2 has a non-canonical DNA binding domain, and therefore it is able to recognize different types of DNA structures in comparison to PARP-1, which could suggest a unique role for PARP-2 in repair. PARP-2 has not been extensively studied in a chromatin or gene regulation context due to this assumed redundancy. Given the pronounced functional changes in PARP-1 upon automodification, it is important to better understand what exactly triggers its enzymatic activity. Similarly, due to the functional redundancy of PARP-2, insight into activators of its enzymatic activity could indicate specificity and selectivity for the protein. However, determining the details of nuclear components that activate PARP-1 and PARP-2 are limited by the availability of a reliable quantitative and kinetic assay, as well as by the availability of defined substrates. These limitations hinder the separation of potent, and thus biologically relevant, activators from weak or non-specific activators. Utilizing a fluorescence based enzyme assay adapted for this system, kinetic parameters of PARP-1 and PARP-2 allosteric activators are reported here. As proof of principle and to test the reliability of the enzymatic assay, PARP-1 and PARP-2 activity was first tested with nucleic acids and other previously reported activators, such as nucleosomes and histones. Next, potentially novel activators were tested. Notably, PARP-1 is activated in the presence of its enzymatic product, PAR, indicating a mechanism by which PARP-1 could spread at sites of DNA damage and active gene expression. PARP-2 exhibits unique activation and specificity different from that of PARP-1 through its enzymatic preference for RNA. Further, PARP-1 remains the prominent chromatin related PARP due to the weak interaction, both activity and affinity, of chromatin with PARP-2. However, while PARP-1 and PARP-2 can act individually, affinity and activity studies demonstrate a PARP-1 and PARP-2 complex suggesting that these proteins can act sequentially and simultaneously with one another during a PAR-mediated recruitment and signaling cascade. Overall, these data indicate novel functions and mechanisms for PARP-1 and PARP-2 within the nucleus as critical responders to DNA damage and gene regulation.Item Open Access Poly (ADP-ribose) polymerase 1 (PARP1) and its DNA-binding characteristics(Colorado State University. Libraries, 2011) Kramer, Michael A., author; Luger, Karolin, advisor; Woody, Robert, committee member; Bailey, Susan M., committee memberThe poly(ADP-ribose) polymerase (PARP) family is evolutionarily diverse, containing 18 different protein members. Roles played by PARP1 in the cell appear to be significant in establishing cellular complexity, as a correlation exists between higher eukaryotes and prevalence of PARP family members. Each member of the PARP family contains a conserved catalytic domain, which upon activation cleaves molecules of NAD+ to form polymers of ADP-ribose, with the release of nicotinamide. Poly(ADP-ribosyl)ation reactions carried out by PARP family members have been found to function in regulation of cellular systems including DNA-damage repair, transcription, mitotic spindle formation, telomere maintenance and cell-death signaling. The most well established member of the PARP family is poly(ADP-ribose) polymerase 1 or PARP1. PARP1 has been found to associate with an assortment of DNA structures within the cell. Despite being able to complex with any DNA present in the cell, PARP1 displays a propensity to interact with sites of DNA-damage. As such, PARP1 has been found to play a major role in initiation of DNA-damage repair. Through its catalytic activity PARP1 recruits additional DNA-damage repair machinery and promotes exposure of the site of damage through chromatin relaxation. Due to its ability to regulate chromatin structure, PARP1 has also been frequently connected with transcription regulation. Variable regulation of transcription by PARP1 has been observed. Catalytically inactive PARP1 can function in a similar fashion as the protein H1 to condense chromatin. Alternatively, active PARP1 functions to relax chromatin surrounding promoter regions and recruit transcription machinery. PARP1 activity appears to be primarily regulated through its association with DNA. Little is known regarding PARP1-DNA-binding affinity. Here I present a high-throughput in-solution FRET-based assay that I utilize to better characterize PARP1's interaction with sites of DNA-damage. In addition, the PARP1-nucleosome complex was analyzed utilizing the same FRET-based assay. Discrepancies found between PARP1 binding affinities to various DNA-damage and mononucleosome constructs provide insight into a potential variable mode of interaction exhibited by PARP1.Item Open Access Role of polyglutamylation in nucleosome assembly protein 1 (NAP1) function(Colorado State University. Libraries, 2008) Subramanian, Vidya, author; Luger, Karolin, advisorThe organization of DNA into chromatin requires the systematic deposition of the histones onto the DNA template. Chromatin function requires the dynamic exchange of the histone components during replication and transcription. Deposition and exchange is mediated in part by a family of proteins generally referred to as histone chaperones. It has been shown recently that recombinant yeast NAP1 (yNAP1) is capable of promoting ATP-independent histone exchange and nucleosome sliding in vitro, and this ability is specifically attributed to the highly acidic C terminal tail of the protein. Drosophila NAP1 (dNAP1) has a shorter acidic C terminus than yNAP1. Preliminary data in the lab suggests that recombinant wild-type dNAP1 is incapable of this nucleosome dissociation. Native dNAP1 purified from Drosophila embryos, on the other hand, is capable of nucleosome dissociation. In this study we reveal the presence of a unique post-translational modification, polyglutamylation in native dNAP1, which restores the nucleosome dissociation function to recombinant dNAP1. We have also been able to identify two target sequences, as well as the number of glutamyl units associated with these modifications using mass spectrometric analysis (MALDI & MS/MS). The modification at the CTAD (C-terminal acidic tail domain) could compensate for the lesser amount of acidic amino acid in dNAP1 and may account for the gain in nucleosome dissociation function. The second polyglutamylation site is located at the NLS (Nuclear Localization Sequence) (based on the conserved core domain of yNAP1 and dNAP1).Item Open Access Structural and functional insight into kinetochore protein CENP-N and its interaction with CENP-A nucleosome(Colorado State University. Libraries, 2018) Zhou, Keda, author; Luger, Karolin, advisor; Yao, Tingting, committee member; Deluca, Jennifer, committee member; Bailey, Susan, committee memberProper chromosome segregation during mitosis is one of the most important processes to ensure genome integrity. During this process, the microtubules are captured by a multi-unit complex called kinetochore. The kinetochore is assembled specifically at centromere through recognizing nucleosomes containing the histone H3 variant CENP-A. CENP-N and CENP-C are the only two kinetochore proteins that specifically recognize CENP-A nucleosomes. There are about 1 in 25 nucleosomes that contain CENP-A at the centromere. Therefore, how these two proteins 'ignore' the abundant H3 nucleosomes to interact selectively with a handful of centromeric CENP-A nucleosomes has important implications for genome stability during cell division. To obtain deep insight into the mechanism behind this, I solved the structure of CENP-A nucleosome in complex with CENP-N by single particle cryo electron microscopy (cryo-EM) at 4 Å. Through charge and space complementarity, the unique "RG" loop on CENP-A is decoded by CENP-N. CENP-N also engages in extensive interactions with a long segment of the distorted nucleosomal DNA double helix. These interactions were validated in vitro and in vivo.The DNA ends of CENP-A nucleosome which are disordered in the crystal structure are mostly visible in the cryo-EM structure when it is in complex with CENP-N. By micrococcal nuclease digestion assay, the CENP-A nucleosome DNA ends are shown to be less flexible when CENP-N is presented in solution, which is consistent with structural study. Since CENP-N does not interact with DNA ends directly, the less dynamics on the DNA ends indicate a more stable nucleosome. By quantitative electrophoretic mobility shift assay (EMSA) and electron microscopy, the stabilizing effect of CENP-N on CENP-A nucleosome was confirmed in vitro. However, this effect was not significant in vivo, which indicates that the CENP-A nucleosome stability in vivo is determined by multiple factors. Besides the change on DNA ends of CENP-A nucleosome, the orientation of H4 N-terminal tail is altered due to its interaction with CENP-N, with important implications for the multiple biological processes involving the H4 N-terminal tail, especially with respect to the formation of chromatin higher order structure The structural and functional studies in this thesis shed light on how CENP-N ensures that the kinetochore assembles specifically at the centromere.Item Open Access Structural and functional studies on the chromatin and nucleosome binding proteins(Colorado State University. Libraries, 2007) Chodaparambil, Jayanth Velandy, author; Luger, Karolin, advisorThe approximately two meters of eukaryotic DNA are compacted within the confines of the nucleus by hierarchical packing with an equal amount of histone proteins to form chromatin. The nucleosome is the fundamental repeating structural unit of chromatin. The nucleosome is the fundamental repeating structural unit of chromatin. Highly compacted DNA is very accessible to the transcription machinery. To understand the mystery behind the two opposing functions of the chromatin, it is essential for us to study nucleosome and chromatin structure in detail.Item Open Access Structural insights into chromatin assembly factor 1 and nucleosome assembly mechanism(Colorado State University. Libraries, 2018) Gu, Yajie, author; Luger, Karolin, advisor; Bailey, Susan, committee member; Peersen, Olve, committee member; Yao, Tingting, committee memberThe eukaryotic genome is highly packed with histones to form chromatin. The basic building unit of chromatin is the nucleosome, which consists of a histone octamer core, wrapped by 147 base pairs of DNA. The nature of the nucleosome structure presents a formidable barrier for DNA-related processes, especially for DNA replication. Therefore, the chromatin will undergo dramatic dynamics during replication, involving disassembly of old nucleosomes and distribution of both new and old histones to form nucleosomes onto both daughter DNA strands. These nucleosome dynamics suggest a challenge for the maintenance of histone density and epigenetic inheritance in the wake of DNA replication. Chromatin Assembly Factor-1 (CAF-1) is a conserved histone chaperone that directly interacts with the replication machinery via the polymerase processivity factor PCNA, and is involved in assembling nucleosomes behind the DNA replication fork. CAF-1 is essential for multicellular eukaryotes, while deletion of CAF-1 in yeast is not lethal, but results in increased sensitivity to DNA damage and aberrant telomeric silencing. Despite the significance of this histone chaperone, the structural organization of this complex remains largely unknown, and thus the mechanism underlying CAF-1-mediated nucleosome assembly is elusive. In this study, we identified the key peptides involved in CAF-1 subunit assembly by performing HDX-MS analysis followed by site-directed mutagenesis studies, which were confirmed by yeast genetic studies. This structural information allows us to further characterize functional domains within CAF-1, and provides unprecedented details for future structural studies using crystallization and/or cryo-EM. This work also shows how histones H3-H4 are bound by CAF-1, and how this histone binding regulates the nucleosome assembly activity by CAF-1. We also show that DNA is acting as a bridge to bring two histone-bound CAF-1 together, thereby promoting (H3-H4)2 tetramer formation as well as the tetramer hand-off between CAF-1 and DNA, resulting in the formation of tetrasome ((H3-H4)2 tetramer wrapped with DNA), the initial step for nucleosome assembly. Overall, this study provides a mechanistic explanation for efficient nucleosome assembly by CAF-1 following DNA replication, and highlights a direct nucleosome assembly mechanism by a histone chaperone for the first time. Moreover, the concerted mechanism of CAF-1-mediated nucleosome assembly suggests that two H3-H4 dimers are brought together right before the (H3-H4)2 tetramer deposition onto DNA, shedding light on the future directions of epigenetic maintenance regulation during replication.Item Open Access The biophysical, biochemical and structural characterization of Poly(ADP-ribose) Polymerase-1 (PARP-1) and its complexes with DNA-damage models and chromatin substrates(Colorado State University. Libraries, 2013) Clark, Nicholas James, author; Luger, Karolin, advisor; Bailey, Susan, committee member; DeLuca, Jennifer, committee member; Hansen, Jeffrey C., committee member; Woody, Robert, committee memberEukaryotic DNA is highly dynamic and must be compacted and organized with the help of cellular machines, proteins, into 'heterochromatin' state. At its basic level, chromatin is comprised of spool-like structures of protein complexes termed histones, which bind and organize DNA into larger fibrous structures. Cellular processes like transcription and DNA-damage repair require that chromatin be at least partially stripped of its protein components, which in turn allows for complete accessibility by DNA-repair or transcription machinery. A number of protein factors contribute to chromatin structure regulation. Poly(ADP-ribose) Polymerase-1 (PARP-1) is one of these proteins that exists in all eukaryotic organisms except for yeast. In its inactive form, it compacts chromatin, but performs its chromatin-opening function by covalently modifying itself and other nuclear proteins with long polymers of ADP-ribose in response to DNA damage. Thus, it also serves as a first responder to many types of DNA damage. The highly anionic polymers serve to disrupt protein-DNA interactions and thus allow for the creation of a temporary euchromatin structure. Contained herein are investigations aimed at addressing key questions regarding key differences between the interactions of PARP-1 and chromatin and its DNA-damage substrates. Our experiments show that human PARP-1 interacts with and is enzymatically activated to a similar level by a variety of different DNA substrates. In terms of chromatin, it appears that PARP-1 fails to interact with nucleosomes that do not have linker DNA. PARP-1 most effectively interacts with chromatin by simultaneously binding two DNA strands through contacts made by its two N-terminal Zn-finger domains. Small-Angle X-ray (SAXS) and Neutron Scattering (SANS) and molecular dynamics (MD) experiments were combined with biophysical and biochemical studies to better describe the structural effects of DNA binding on PARP-1. The average solution structure of PARP-1 indicates that the enzyme is a monomeric, non-spherical, elongated molecule with a radius of gyration (Rg) of ~55Å. The DNA-bound form of PARP-1 is also monomeric and binding DNA causes the molecule to become more elongated with an average Rg of ~80Å.Item Open Access The effects of the histone chaperone and histone modifications on nucleosome structure(Colorado State University. Libraries, 2016) Wang, Tao, author; Luger, Karolin, advisor; Stargell, Laurie, committee member; Yao, Tingting, committee member; Williams, Robert M., committee memberThe nucleosome, composed of 147-bp DNA and a histone octamer, is the basic unit of chromatin in eukaryotes, which is considered as a barrier for all DNA dependent processes. Understanding how nucleosome structure is regulated provides new insights into pivotal cellular processes. Histone modifications and histone chaperones have potential roles in the regulation of nucleosome structure. Here, I investigated the role of FACT in regulating nucleosome structure. FACT (FAcilitate Chromatin Transcription) is a conserved histone chaperone that is essential for gene transcription elongation. Our biochemical data show that FACT is not only a H2A-H2B chaperone, but also a H3-H4 chaperone. By binding H3-H4, FACT facilitates tetrasome assembly. In the presence of H2A-H2B, FACT facilitates H2A-H2B deposition onto tetrasomes and hexasomes, and thus promotes nucleosome assembly. FACT is also able to tether partial nucleosome components, composed of a histone hexamer and DNA, and results in forming an unstable complex. Interaction with H2A-H2B is essential for FACT binding to tetrasomal H3-H4. In order to hold a histone hexamer, FACT also stabilizes dimer-tetramer interaction. Previous study shows that H2BK120ub facilitates FACT function in gene transcription with the help of other transcription factors. Here, we show that H2AK119ub and H2BK120ub have no effects on FACT-(H2A-H2B) interaction and FACT assembly activity. The role of select histone modifications in nucleosome structure was also determined in this dissertation. Histone modifications selected in this work are located at the entry-exit region of nucleosomal DNA. By using biochemical approaches, we find that H3Y41E (mimic phosphorylation) and H3R45E (mimic phosphorylation) affect the shape of nucleosome by facilitating nucleosomal 'DNA breathing'.Item Open Access The influence of histone orthologues, histone variants and post-translational modifications on the structure and function of chromatin(Colorado State University. Libraries, 2008) Resch, Michael George, author; Hansen, Jeffrey C., advisor; Luger, Karolin, advisorTwo meters of DNA is packaged into the nucleus of each eukaryotic cell in the form of chromatin. DNA wraps around a protein histone octamer to form a nucleosome, the fundamental repeating unit of chromatin. The highly basic histone octamer contains two copies each of H2A, H2B, H3 and H4 to form the protein core of the nucleosome. There is a dynamic interplay of accessibility which compacts DNA yet allows access for fundamental cellular processes like transcription and DNA replication. This thesis investigates how histone variants and post-translational modifications contribute to the level of chromatin compaction. I demonstrated that defined nucleosomal arrays made with histones from multiple species oligomerize at different concentrations of MgCl2. A comparison of endogenous and recombinant Drosophila melanogaster histone octamers showed that this is unlikely due to posttranslational histone modifications, but likely a result of subtle changes in the sequences constituting the histone tails and structured surface of the histone octamer. I investigated the effect of incorporation of the centromere specific H3 histone variant centromere protein - A (CENP-A) into nucleosomes and nucleosomal arrays. Despite the fact that CENP-A shares only 60% sequence homology within the structured domain of major-type H3 (15% in the N-terminal domain), CENP-A (together with the other three core histones) forms nucleosomes and condensed nucleosomal arrays comparable to major-type H3. Post-translational modifications (PTM) contribute to the regulation of chromatin structure. I have analyzed the effect of H3 lysine 56 acetylation on nucleosome structure and chromatin condensation. This modification was previously thought to disrupt nucleosome structure. I developed methods to enzymatically acetylate large amounts of H3 specifically at Lys 56, and demonstrated that histone octamers containing H3-K56Ac form canonical nucleosomes. However, nucleosomal array condensation is compromised by this particular PTM. Together, these studies suggest that even subtle variations in histone sequence or post-translational modifications result in differences in chromatin higher order structure.