REPAIR AND EFFECTS OF THE 8-OXOG LESION IN DNA

Eukaryotic DNA is packaged in a condensed state with histone proteins. The minimal structural unit within packaged eukaryotic DNA is the nucleosome core particle (NCP). The NCP consists of a 146 bp DNA fragment wrapped around an octamer of histone core proteins. Nucleosome core particle formation induces DNA structural changes and reduced DNA accessibility providing a very different setting than that commonly modeled by in vitro studies. In vitro reconstituted NCP provide a controlled environment that more closely models eukaryotic DNA than studies using naked DNA. Reconstituted NCP studies of DNA damage have exhibited a spectrum of effects compared to naked DNA ranging from protective, enhanced, and no effect. The nature of the effect appears to be related to the type of oxidant, its sterics and interactions with the histone surface and altered DNA structure. While there is differences in efficiencies of oxidation of nucleobases throughout the nucleosome, nucleobase oxidation is still widespread within the genome. DNA repair processes that combat global DNA oxidation are crucial to cell survival. One major cellular repair mechanism that is employed to remove DNA damage is base excision repair (BER). The BER pathway involves the concerted activity of a small number of proteins which catalyze individual reactions in a chemical pathway that repairs single nucleotide lesions. In vivo, the majority of DNA is wrapped around histones and the repair machinery of BER has to work within or around the structure of the nucleosome and deal with a distorted DNA structure and reduced accessibility due to the presence of bulky histone proteins. To address the questions of DNA damage and repair in the nucleosome an in-vitro nucleosomal system was established by reconstituting purified histones and a 154 bp wrapping fragment from the Xenopus borealis 5S rRNA gene to form individual nucleosome core particles (NCP). The effect of nucleosome formation on chromium- mediated DNA damage and the efficiency of BER glycosylase cleavage of the lesion 8-oxoG were investigated. Base excision of 8-oxoG by Fpg and hOGG1 indicated that: i) the position of the lesion 8-oxoG in naked DNA can influence BER activity; ii) nucleosomal formation decreases the activity of these BER enzymes by as much as 2.5 fold with a rotational dependence exhibiting increased cleavage towards the more accessible lesion; iii) the rotational dependence for both Fpg and hOGG1 was almost identical, however hOGG1 showed better cleavage in the nucleosome setting relative to free DNA at earlier time points. An additional study was done to examine the potential of 8-oxoG lesions to mimic cytosine methylation effects with regard to the activity of a methyl-sensitive endonuclease. Using enzyme cleavage assays the effects of placing an 8-oxoG or methylated cytosine into the recognition sequence of a restriction endonuclease, NotI, were investigated. Results indicate identical inhibitory effects between 8-oxoG and cytosine methylation, hinting at a potential role of 8-oxoG in epigenetics

Targeting poly(ADP-ribose) polymerase activity for cancer therapy

Poly(ADP-ribosyl)ation is a ubiquitous protein modification found in mammalian cells that modulates many cellular responses, including DNA repair. The poly(ADP-ribose) polymerase (PARP) family catalyze the formation and addition onto proteins of negatively charged ADP-ribose polymers synthesized from NAD+. The absence of PARP-1 and PARP-2, both of which are activated by DNA damage, results in hypersensitivity to ionizing radiation and alkylating agents. PARP inhibitors that compete with NAD+ at the enzyme’s activity site are effective chemo- and radiopotentiation agents and, in BRCA-deficient tumors, can be used as single-agent therapies acting through the principle of synthetic lethality. Through extensive drug-development programs, third-generation inhibitors have now entered clinical trials and are showing great promise. However, both PARP-1 and PARP-2 are not only involved in DNA repair but also in transcription regulation, chromatin modification, and cellular homeostasis. The impact on these processes of PARP inhibition on long-term therapeutic responses needs to be investigated

Localization and function of histone methylation at active genes in "Drosophila"

In the eukaryotic nucleus, DNA is bound by an octamer of four core histones forming the fundamental repeating unit of chromatin, called the nucleosome. Presenting a barrier to virtually all DNA-templated events, nucleosomal packaging is subject to dynamic alterations. Nucleosomal histone modifications have emerged as a major determinant of chromatin structure and gene expression. Genome-wide and local profiling of chromatin structure in Drosophila cells reveals a complex landscape of histone methylation marks along the body of active genes. Methylation of lysine 4 and lysine 79 of histone H3 coincide at promoters and gradually decrease towards the 3’ end. Conversely, H3 lysine 36 methylation states show very different distribution patterns. Dimethylation of H3K36 peaks downstream of promoter-proximal K4 methylation, whereas trimethylation accumulates towards the 3’ end of genes. These topographic differences do not reflect deposition-coupled targeting by histone variant H3.3 but instead argue for discrete regulation and function of active methylation marks during transcription elongation. Indeed, H3K36 di- and trimethylation states rely on two distinct HMTs and display opposite effects on H4K16 acetylation at autosomal genes. This crosstalk is reminiscent of K36me3-dependent deacetylase recruitment in budding yeast, yet it is more intricate as dimethylation appears to signal for increased H4K16 acetylation. Apart from its autosomal function, H3K36me3 has a separate role to enhance H4K16 acetylation at the dosage-compensated X chromosome in male Drosophila cells. This additional function most likely involves MSL complex recruitment to dosage compensated genes. Together, our results reveal a complex pattern of histone methylation marks at active genes, which may enable dynamic chromatin changes during transcription elongation in higher eukaryotes. Furthermore, the context-dependent readout of H3K36me3 implies that methylation marks act as general signaling platforms, which impart their specificity by recruiting effector proteins to characteristic landmarks along the transcription unit

Genetics Association and Epigenetic Changes in COPD

Genome-wide association studies (GWASs) have successfully identified susceptibility loci associated with COPD. The genes mapped on these loci, eg The FAM13A gene (family with sequence similarity 13, member A), provide a new approach to understand the COPD pathology. Furthermore, heavy smoking is reported to correlate with altered methylation and epigenetic changes of multiple genes in small airway cells. These changes have been shown to be associated with the severity of COPD. It is likely that smoking-induced changes in epigenetic control of gene expression result in genetically vulnerable individual’s results in reduced tissue repair, tissue damage and persistent inflammation associated with COPD pathophysiology

The Elongator Complex Interacts with PCNA and Modulates Transcriptional Silencing and Sensitivity to DNA Damage Agents

Histone chaperones CAF-1 and Asf1 function to deposit newly synthesized histones onto replicating DNA to promote nucleosome formation in a proliferating cell nuclear antigen (PCNA) dependent process. The DNA replication- or DNA repair-coupled nucleosome assembly pathways are important for maintenance of transcriptional gene silencing and genome stability. However, how these pathways are regulated is not well understood. Here we report an interaction between the Elongator histone acetyltransferase and the proliferating cell nuclear antigen. Cells lacking Elp3 (K-acetyltransferase Kat9), the catalytic subunit of the six-subunit Elongator complex, partially lose silencing of reporter genes at the chromosome VIIL telomere and at the HMR locus, and are sensitive to the DNA replication inhibitor hydroxyurea (HU) and the damaging agent methyl methanesulfonate (MMS). Like deletion of the ELP3, mutation of each of the four other subunits of the Elongator complex as well as mutations in Elp3 that compromise the formation of the Elongator complex also result in loss of silencing and increased HU sensitivity. Moreover, Elp3 is required for S-phase progression in the presence of HU. Epistasis analysis indicates that the elp3Δ mutant, which itself is sensitive to MMS, exacerbates the MMS sensitivity of cells lacking histone chaperones Asf1, CAF-1 and the H3 lysine 56 acetyltransferase Rtt109. The elp3Δ mutant has allele specific genetic interactions with mutations in POL30 that encodes PCNA and PCNA binds to the Elongator complex both in vivo and in vitro. Together, these results uncover a novel role for the intact Elongator complex in transcriptional silencing and maintenance of genome stability, and it does so in a pathway linked to the DNA replication and DNA repair protein PCNA

Mapping DNA structure & protein-DNA interactions using hydroxyl radical footprinting & high-throughput sequencing

Development of biochemical techniques to examine chromatin structure and protein-DNA interactions on a global scale has allowed for extensive characterization of functional and regulatory elements essential to cellular biological processes. In particular, chromatin accessibility and susceptibility to damage, coupled with high-throughput sequencing, have served as means for characterizing these elements. To better understand protein occupancy in relation to chromatin architecture, a technique that can impartially probe DNA structure at high resolution is required. The hydroxyl radical, generated from a modified Fenton reaction or ɣ-irradiation of water molecules, is a chemical tool used for probing nucleic acid structure, and capable of mapping protein-DNA binding sites at single-nucleotide resolution. Adapting hydroxyl radical footprinting for analysis by high-throughput sequencing (OH-seq) aims to provide a detailed profile of the chromatin landscape in whole genomes. Initial development of OH-seq was carried out on a model system using synthetic oligonucleotides to mimic a hydroxyl radical damage site. The single-strand break was enzymatically converted to a double-strand break to allow for end-repair and ligation to a sequencing adapter. This dissertation describes the further development of OH-seq in vitro, and the optimization of this technique for application to whole genomes in vivo. To show that OH-seq can successfully map protein-DNA interactions, the technique was tested on the well characterized λ repressor-operator complex. Analyses for sequencing libraries, tagging single- and double-strand breaks created from hydroxyl radical cleavage of plasmid DNA in the absence and presence of λ repressor, show footprints similar to those from previous studies. Application of OH-seq to human and S. cerevisiae genomes captured double-strand breaks in genomic DNA following ɣ-irradiation of cells. Analyses examining the damage profile across aggregated transcription start sites and nucleosome positions in the human genome reveal high damage at promoters, and highly periodic nucleosomal footprints. OH-seq profiles for select transcription factors in yeast show distinct footprints comparable to those from other genome-wide studies. These preliminary results show the potential OH-seq has for characterizing chromatin structure and protein-DNA interactions. Further optimization will make the technique a useful addition to the current repertoire of tools for studying genome structure and function.2018-08-11T00:00:00

Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes

DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of DNA into chromatin provides roadblocks to repair. Damaged DNA bases may be buried within nucleosomes, where they are inaccessible to repair enzymes and other DNA binding proteins. Previous in vitro studies by our lab have demonstrated that BER enzymes can function within this challenging environment, albeit in a reduced capacity. Exposure to ionizing radiation often results in multiple, clustered oxidative lesions. Near-simultaneous BER of two lesions located on opposing strands within a single helical turn of DNA of one another creates multiple DNA single-strand break intermediates. This, in turn, may create a potentially lethal double-strand break (DSB) that can no longer be repaired by BER. To determine if chromatin offers protection from this phenomenon, we incubated DNA glycosylases with nucleosomes containing clustered damages in an attempt to generate DSBs. We discovered that nucleosomes offer substantial protection from inadvertent DSB formation. Steric hindrance by the histone core in the nucleosome was a major factor in restricting DSB formation. As well, lesions positioned very close to one another were refractory to processing, with one lesion blocking or disrupting access to the second site. The nucleosome itself appears to remain intact during DSB formation, and in some cases, no DNA is released from the histones. Taken together, these results suggest that in vivo, DSBs generated by BER occur primarily in regions of the genome associated with elevated rates of nucleosome turnover or remodeling, and in the short linker DNA segments that lie between adjacent nucleosomes. DNA ligase IIIα (LigIIIα) catalyzes the final step in BER. In order to facilitate repair, DNA ligase must completely encircle the DNA helix. Thus, DNA ligase must at least transiently disrupt histone-DNA contacts. To determine how LigIIIα functions in nucleosomes, given this restraint, we incubated the enzyme with nick-containing nucleosomes. We found that a nick located further within the nucleosome was ligated at a lower rate than one located closer to the edge. This indicated that LigIIIα must wait for DNA to spontaneously, transiently unwrap from the histone octamer to expose the nick for recognition. Remarkably, the disruption that must occur for ligation is both limited and transient: the nucleosome remains resistant to enzymatic digest before and during ligation, and reforms completely once LigIIIα dissociates

Current epigenetic aspects the clinical kidney researcher should embrace

Chronic kidney disease (CKD), affecting 10-12% of the world's adult population, is associated with a considerably elevated risk of serious comorbidities, in particular, premature vascular disease and death. Although a wide spectrum of causative factors has been identified and/or suggested, there is still a large gap of knowledge regarding the underlying mechanisms and the complexity of the CKD phenotype. Epigenetic factors, which calibrate the genetic code, are emerging as important players in the CKD-associated pathophysiology. In this article, we review some of the current knowledge on epigenetic modifications and aspects on their role in the perturbed uraemic milieu, as well as the prospect of applying epigenotype-based diagnostics and preventive and therapeutic tools of clinical relevance to CKD patients. The practical realization of such a paradigm will require that researchers apply a holistic approach, including the full spectrum of the epigenetic landscape as well as the variability between and within tissues in the uraemic milieu

Histone modifications and the HtrA-like serine protease Nma111p regulate apoptosis in budding yeast

Histone modifications and the HtrA-like serine protease Nma111p regulate apoptosis in budding yeast David Walter, 2010, PhD Thesis, University of Basel Apoptosis is a form of programmed cell death that plays a central role in development and cellular homeostasis in higher eukaryotes. Knowledge about apoptotic regulation is particularly important for medical research, since apoptotic misregulation is implicated in many human diseases, such as Alzheimer’s and Huntington’s disease, immunodeficiency and cancer. Recent studies have established yeast as model to study the mechanisms of apoptotic regulation. Changes in chromatin configuration are implicated in apoptotic regulation both in yeast and in higher eukaryotes. One mechanism that alters chromatin configuration is the covalent modification of histones, which associate with DNA to form the nucleosome, the fundamental unit of chromatin. In my thesis work, I have identified and characterized distinct interrelated histone modifications on histone H2B and histone H3 as regulators of apoptosis in yeast (Chapter 2 and 3). Histone H2B ubiquitination at lysine K123 by the E3 ligase BRE1 is required in promoting methylation of histone H3 at lysine K4 and K79. These methylations are brought about by the conserved methyltransferases Set1p and Dot1p, respectively. We found that disruption of the E3 ligase BRE1 or the methyltransferase SET1, which causes a lack of histone H2B K123 ubiquitination and histone H3 K4 methylation, respectively, causes metacaspase Yca1p-dependent apoptosis (Chapter 2 and 3). In contrast, we found that disruption of DOT1, which causes a lack of histone H3 K79 methylation confers apoptosis resistance (Chapter 3). Moreover, we found that Dot1p is required for Yca1p-dependent cell death of ∆set1 cells (Chapter 3). How does disruption of DOT1 confer apoptosis resistance? Yeast cells that fail to methylate histone H3 K79 due to DOT1 disruption exhibit defects in the DNA damage response. Particularly, Dot1p mediated histone H3 K79 methylation is required for Rad9p-dependent checkpoint activation after DNA damage. In higher eukaryotes, the evolutionarily conserved DNA-damage response is a signaling cascade that senses DNA damage and activates cellular responses including apoptosis. Strikingly, we found that Rad9p is required for cell death of ∆set1 similar to Dot1p (Chapter 5), suggesting that Dot1p mediates apoptosis through its function in the DNA-damage response. Thus, we suggest that apoptosis in budding yeast is linked to the DNA damage response similar to apoptosis in higher eukaryotes. Together, these studies highlight the requirement of Dot1p-mediated histone H3 K79 methylation for an Yca1p-dependent cell death scenario and points to a novel role of the conserved histone H2B/H3 crosstalk in apoptosis regulation. Moreover, our results imply a requirement of the DNA damage response for apoptosis induction in budding yeast. Another objective of this thesis was the characterization of the functional role of the HtrA1-like serine protease Nma111p in yeast apoptosis (Chapter 4). Nma111p functions as a nuclear serine protease that is necessary for apoptosis under cellular stress conditions. We have examined the role of nuclear protein import in the function of Nma111p in apoptosis. Nma111p contains two small clusters of basic residues toward its amino terminus, both of which are necessary for efficient translocation into the nucleus. Nma111p does not shuttle between the nucleus and cytoplasm during either normal growth conditions or under environmental stresses that induce apoptosis. The amino-terminal half of Nma111p is sufficient to provide the apoptosis-inducing activity of the protein, and both the NLS sequences and catalytic serine 235 are necessary for this function. Together, we provide compelling evidence that intranuclear Nma111p activity is necessary for apoptosis in yeast