Indiana University-Purdue University Indianapolis (IUPUI) / DNA is subject to various types of damage that can impair cellular function or cause cell death. DNA damage blocks normal cellular processes such as replication and transcription and can have catastrophic consequences for the cell and for the organism. It has long been thought that the G1/S cell cycle checkpoint allows time for DNA repair by delaying S-phase entry. The p53 tumor suppressor pathway regulates the G1/S checkpoint by regulating the cyclin-dependent kinase inhibitor p21Waf1/Cip1, but p53 also regulates the nucleotide excision DNA repair protein XPC. Here, using p53-null cell lines we show that additional mechanisms stabilize XPC protein and promote NER in concert with the G1/S checkpoint. At least one mechanism to stabilize and destabilize XPC involves ubiquitin-mediated degradation of XPC, as the ubiquitin ligase inhibitor MG-132 blocked XPC degradation. The retinoblastoma protein, RB, in its unphosphorylated form actually stabilized XPC and promoted NER as measured by host-cell reactivation experiments. The data suggest that XPC protein and XPC-mediated NER is tightly linked to the G1/S checkpoint even in cells lacking functional p53.

Radiation-induced damage to the genomic DNA of cells may lead to errors in transcription and replication and, if not repaired correctly, these may result in mutations, genomic instability and cell death. Laser microbeams have generally been used by many research groups to investigate the real-time dynamics of protein recruitment in response to DNA insults in mammalian cells; however, such irradiations induce a plethora of DNA damage (including UV base damage, base damage, SSBs and DSBs and complex damage). A novel experimental setup has been designed capable of following the dynamics of protein recruitment in response to DNA insults in mammalian cells shortly following submicrosecond- pulsed electron irradiation of living mammalian cells, not possible using conventional irradiation techniques. This arrangement was developed based on a 6 MeV electron pulse linear accelerator, to deliver sparsely ionising radiation, coupled to an automated, time-lapse inverted epifluorescence microscope imaging system. An integrated robotic system contained within a physiological environment of 37°C and 5% CO₂ was used to transfer remotely and repetitively custom-designed cell dishes containing the mammalian cells between irradiation and imaging locations. Following the development of the linear accelerator and associated imaging devices, preliminary ‘proof-of-principle’ investigations were carried out using living HT1080 mammalian cells containing YFP-tagged 53BP1, an established biomarker of DSB, to follow the recruitment and loss of 53BP1 to sites of radiation-induced DNA damage in real-time. This novel experimental setup has allowed for the first time observations of the appearance and disappearance of radiation-induced foci in the same cell population at very early times. These single-foci studies have provided evidence for the formation of not only promptly formed DSBs but also late appearing DNA damage signalled by 53BP1. These data highlight different classes of DSBs formed in response radiation damage. Additionally, the role of cell cycle on the repair kinetics was undertaken using HT1080- 53BP1-YFP cells which also express Geminin-mCherry under appropriate selection. Geminin is increasingly expressed from early S-phase onwards, but is degraded following mitosis. Geminin-associated fluorescence can be used as a marker of progression through the cell cycle. 53BP1 repair kinetics were characterised in response to radiation damage in combination with ATM and PARP inhibitors. These studies provided supporting evidence for the existence of different classes of DSBs, potentially assigned to radiation-induced replication breaks and DSBs formed by enzymatic conversion of clustered damage. These preliminary ‘proof-of-principle’ findings using DNA damage repair as an example, emphasize the use of this novel technology to explore the dynamics of numerous other biochemical processes in living cells in real-time with the knowledge of being able to quantify the range of damage induced by IR coupled with accurate dosimetry. The knowledge obtained may be used to identify potential biological targets coupled with drug discovery for translation into adjuncts for radiotherapy.

No description available.

The entire epidermis of patients with vitiligo exhibits accumulation of up to 10-3M concentrations of hydrogen peroxide (H₂O₂) (Schallreuter, Moore et al. 1999). Over the last decade our group and others have focused on the effect of H₂O₂-mediated oxidative stress on the function of many proteins and peptides due to oxidation of target amino acid residues in their structure including L-methionine, L-tryptophan, L-cysteine and seleno cysteine (Rokos, Beazley et al. 2002; Gillbro, Marles et al. 2004; Hasse, Kothari et al. 2005; Schallreuter, Chavan et al. 2005; Spencer, Chavan et al. 2005; Chavan, Gillbro et al. 2006; Elwary, Chavan et al. 2006; Gibbons, Wood et al. 2006; Schallreuter, Bahadoran et al. 2008; Shalbaf, Gibbons et al. 2008; Wood, Decker et al. 2009). Moreover, it was shown that patients with vitiligo possess up regulated wild type functioning p53 protein in their skin (Schallreuter, Behrens- Williams et al. 2003). The reason behind this up regulation has remained unclear (Schallreuter, Behrens-Williams et al. 2003). Therefore the aim of this thesis was to get a better understanding of these puzzling data. Along this project different techniques have been used including Western blot, dot blot, immuno precipitation, immuno fluorescence, EMSA and computer modelling. In this thesis we confirmed the previous result on up regulation of p53 in vitiligo and we showed that p90MDM2, the master regulator for p53 protein is not different in patients and healthy controls. Therefore we decided to test for expression of p76MDM2 which mediates the inhibition of p90MDM2-p53 binding. Our results show for the first time the presence and over expression of p76MDM2 protein in vitiligo compared to 3 healthy individuals. This result could provide an explanation, why up regulated p53 is not degraded in this disease. Since epidermal H₂O₂ accumulation has been extensively documented in vitiligo, we wanted to know whether other ROS could also contribute to the overall oxidative stress in this scenario. Therefore we turned our interest to nitric oxide (NO) and its possible effects on p53 protein. In order to elucidate this role in more detail, the expression levels of epidermal nitric oxide synthesase (iNOS) and the oxidation product of NO and O2 - i.e peroxynitrite (ONOO-) were investigated. Our data revealed over expression of iNOS and nitrated tyrosine residues, the foot print for ONOO-. Moreover, we show for the first time the presence of abundant nitration of p53 protein in vitiligo. In addition using purified p53 from E. coli strain (BL21/DE3) and mutant p53 protein from HT-29 cells (colon cancer cells), we show that nitration takes place in a dose and time dependent manner. On this basis we investigated the effect of both H₂O₂ and ONOO- on p53-DNA binding capacity employing EMSA, since this is the most acceptable technique to follow the binding between proteins and DNA. Our results revealed that ONOO- abrogated p53-DNA binding capacity at concentrations >300 μM, meanwhile oxidation of p53 protein with H₂O₂ at the same concentrations does not affect binding capacity. Importantly, a much higher p53- DNA binding capacity was observed after exposure to both ONOO- and H₂O₂. Taken together, p53 is regulated by both ROS (H₂O₂) and RNS (ONOO-). Next we identified the presence of phosphorylated and acetylated p53 in vitiligo. Phosphorylation of ser 9 and ser 15 residues of the protein are associated with over expressed ATM protein kinase, while acetylation of lys 373, 382 residues correlates with increased PCAF expression. We show that up regulated p53 is associated with over expressed p21 (cyclin dependent kinase inhibitor 1) and induced PCNA 4 expression. Hence, we can conclude that p53 in patients with vitiligo is up regulated, activated and functional. Finally we show up regulated BCL-2 supporting the long voiced absence of increased apoptosis in vitiligo. Given that patients with vitiligo have no increased risk for solar induced skin cancer and increased photo damage (Calanchini-Postizzi and Frenk 1987; Westerhof and Schallreuter 1997; Schallreuter, Tobin et al. 2002), despite the presence of increased DNA damage as evidenced by increased 8-oxoG levels in the skin and in the plasma, our findings suggest that both p53 and PCNA provide a powerful machinery to mediate DNA repair via hOgg1, APE1 and DNA polymerase ß (Shalbaf 2009). On this basis it is tempting to conclude that DNArepair is the overriding mechanism to combat oxidative stress in this disease.

B cells have unique machinery to make up a large pool of antibody repertoire. After V(D)J recombination in early B cell development, the rearranged immunoglobulin genes are further diversified by somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Acitvation induced cytidine deaminase (AID) is a key initiating factor for SHM, GC and CSR. A majority of research data supports the model that AID modifies Ig genes at the DNA level by deaminating cytosines to uracils. The mutagenic activity of AID is largely restricted to Ig genes to avoid genomic instability in general. The specificity cannot be attributed to the primary sequence of the Ig genes since unrelated DNA is mutated by AID in the context of Ig genes. A clue to this problem is that AID function is dependent on transcription. Since not all transcribed genes are mutated by AID, there must be something special about the transcription of Ig genes, and the reasoning has prompted extensive analysis of Ig promoters and enhancers. We addressed this question in chicken B cell line DT40. We identified a 2.4-kilobase regulatory region which is important for AID function both within and outside of Ig locus. This regulatory region contains binding sites for multiple transcription factors. Mutation of these binding sites impairs AID mediated gene conversion. In addition, ablation of NF-κB family member, c-Rel and p50, reduces the AID targeting function of this regulatory region. Since the implicated transcription factors have been reported to associate with histone acetylases, the regulatory region may function by facilitating the access of AID to target DNA. To test this hypothesis, we used the I-SceI endonuclease and dam methylase as probes for chromatin structure. We found that the regulatory region does not increase chromatin accessibility to these probes. In fact, the regulatory region appears to interfere with the cleavage of target DNA by I-SceI. Another possible role of the regulatory region could be direct recruitment of AID to Ig genes. To test this hypothesis, we utilized Dam identification method. Surprisingly, we found that the regulatory region facilitates AID targeting to the Igλ locus. / text

NM23-H1 represents the first identified metastasis suppressor, exhibiting reduced expression in breast carcinoma and melanoma, and an ability to inhibit metastatic growth without significant impact on the transformed phenotype. Although its molecular mechanism of action is not fully understood, NM23-H1 possesses at least three enzymatic activities that may mediate metastasis suppressor function. It catalyzes nucleoside diphosphate kinase (NDPK) activity, as well as protein histidine kinase and 3’-5’ exonuclease activities. As 3’-5’ exonucleases are generally required for maintenance of genomic integrity, this activity represents a plausible mediator to underlie the metastasis suppressor function of NM23-H1 protein. To investigate the relevant activity of NM23-H1 in metastasis suppression, we constructed a panel of NM23-H1 mutant variants with selective enzymatic lesions. Previous studies have identified some key amino acid residues important for the enzymatic characteristics of NM23-H1. However, none of them are selective for disrupting the 3’-5’ exonuclease activity. In this study, we show that a substitution of Glu5 to alanine results in a dramatic, selective loss in 3’-5’ exonuclease property without significant affecting other enzymatic activities. To measure the extent to which the exonuclease function opposes mutation and metastasis, NM23-deficient and metastatic cell lines with forced expression of NM23-H1 variants are analyzed in nude mice. In spontaneous metastasis models, NM23-H1 mutants deficient in 3‘-5’ exonuclease activity significantly disrupt the capacity of metastasis suppression of wild-type protein, indicating that the 3’-5’ exonuclease activity of NM23-H1 is necessary for the spontaneous metastasis-suppressing effects. As 3'-5' exonucleases are generally associated with DNA repair process, we have also studied the contributions of yeast NM23 homologue YNK1 to genomic integrity in Saccharomyces cerevisiae. Consistent with an antimutator function, ablation of YNK1 significantly results in increased mutation rates following exposure to UV irradiation and the alkylating agent methyl methanesulfonate (MMS). The impaired DNA-damage response of ynk1Δ cells suggests a role of human homologue NM23 in DNA repair. More evidence is being collected in our laboratory to demonstrate a role for NM23-H1 in maintaining genomic integrity. Collectively, our findings of DNA repair activity of NM23-H1 will contribute to the understandings of the mechanisms in metastasis suppression and new drug discoveries.

The DNA repair capabilities of rainbow trout (Salmo gairdneri) were studied vising the method of autoradiography. Trout were fed a semi-purified control diet containing 0 ppm, 50 ppm, or 300 ppm cyclopropenoid fatty acids (CPFA) for 6-9 weeks. Liver slices were prepared and exposed in vitro to a control treatment, ultraviolet irradiation (UV), ethidium bromide (EB), UV/EB in succession, or aflatoxin B₁. The degree of DNA repair was analyzed in terms of net grains per cell. Except following the EB treatment, fish on the control diet revealed an absence of ongoing DNA repair. Trout fed 50 ppm CPFA exhibited a consistently low level of repair over time following the in vitro control treatment. Fish fed 300 ppm CPFA revealed a relatively higher degree of ³H-Me-thymidine incorporation indicative of induced DNA repair following the in vitro control treatment, and the degree of repair increased with time on the diet. UV-irradiation caused a marked increase in the degree of induced DNA repair in 300 ppm CPFA fish at 6 and 7.5 weeks, and in 50 ppm CPFA fish at 7.5 weeks. Follcwing UV-irradiation, liver slices were exposed to EB, a DNA intercalating agent used to inhibit normal DNA replication. However, in contrast to the desired effect, EB caused a marked decrease in the degree of repair synthesis observed in 300 ppm CPFA fish at 6 and 7.5 weeks. Indicative of intercalation, the in vitro EB treatment caused a moderate degree of ³H-Me-thymidine incorporation in fish fed the control diet. Repair was also induced in 300 ppm CPFA fish following exposure to EB at 6 and 7.5 weeks. Aflatoxin B₁ induced DNA repair to various degrees in fish on all diets at 7.5 and 9 weeks. In comparison to the in vitro control treatment, it was observed that the degree of induced DNA repair was decreased significantly - "completely" following the UV, UV/EB, and EB treatments - in fish fed the 300 ppm CPFA diet for 9 weeks. In view of the low level of DNA repair observed in rainbow trout using autoradiography, the repair capabilities were studied using a more sensitive assay, bromodeoxyuridine (BrdU) photolysis. Isolated hepatocytes were prepared from fish fed the various diets and exposed in vitro to a control treatment, UV-irradiation, or 4-nitroquinoline-N-oxide. The obtained results were nonconclusive indicating technical improvements on the assay need to be made. / Graduation date: 1988

In this dissertation I explore the ecology of bacteriophage T4, a virus of Escherichia coli. In particular, I argue that the life history of bacteriophage T4 can be divided into the growth and survival of T4 phages in three distinct environments. I argue that these environments are distinguished by at least two T4 phage sensory systems. These include (i) the sensing of secondary adsorption by infecting phages and (ii) the determination of the concentration of monovalent cations and free tryptophan in solution about free T4 phage particles. The first environment consists of high concentrations of uninfected, logarithmic phase E. coli cells. These concentrations are approximately 10⁶ E. coli cells/ml and greater. This environment occurs in the prefecal colonic lumen of animals. Here T4 phages exhibit unimpeded logarithmic growth. The second environment contains high concentrations of infected E. coli cells, low concentrations of uninfected E. coli cells, and high concentrations of free T4 phage particles. This second environment also occurs in the prefecal colonic lumen of animals and represents the maturation of environments supporting logarithmic T4 phage population growth. Such phage phenotypes as secondary exclusion and lysis inhibition characterize T4 phage growth in this environment. The third environment consists of extra-colonic waters. Here T4 phages avoid infecting E. coli cells and exhibit strategies that maximize their stability. These strategies in extra-colonic waters increase the potential of T4 phages to disseminate successfully from colon to colon. I employ this enhanced understanding of T4 phage ecology, outlined above, in an exploration of the ecology of the repair of DNA damage by T4 phages.

<p>Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.</p> / Dissertation

DNA double-strand breaks (DSBs) are highly genotoxic lesions, which when incorrectly repaired can lead to gross chromosomal rearrangements and tumourigenesis through oncogene activation or loss of heterozygosity at tumour suppressor loci. Chromatin structure and therefore histone modification can play a key role in the repair of these lesions through affecting the access of repair machinery to the break. To identify novel histone-modifying enzymes involved in DSB repair, nineteen histone-modifying mutants from an S. pombe deletion library were screened for altered DSB repair. Eleven genes were identified as required for normal DSB repair: set1⁺, set2⁺, mst2⁺, sir2⁺, set3⁺, set11⁺, hat1⁺, set13⁺, clr4⁺, gcn5⁺ and elp3⁺. Two genes, hat1⁺ and set13⁺ are required for efficient homologous recombination (HR). In addition, four genes set1⁺, set2⁺, set3⁺ and mst2⁺ are required for non-homologous end-joining (NHEJ). Analysis of set1Δ, set2Δ, set3Δ and mst2Δ deletion mutants showed impaired NHEJ was associated with significantly increased levels of repair by HR, supporting the existence of a competitive relationship between these mechanisms of repair. Further work focused on Set2, a H3K36 methyltransferase. In addition to a role for Set2 in NHEJ, a role for Set2 in promoting efficient DNA replication was identified. This is likely through regulation of MBF-dependent genes. However, this altered progression of DNA replication was not responsible for the repair phenotype seen. The methyltransferase activity of Set2 was required for its role in NHEJ and DNA replication. Further analysis of a H3K36R mutant identified an additional role for H3K36 in DSB repair. Results indicate that H3K36 is also acetylated by Gcn5; quantitative analysis of DSB repair in a gcn5Δ mutant, identified a role for Gcn5 in promoting HR repair. Modification of H3K36 by methylation and acetylation is mutually exclusive, and preliminary analysis of the levels of these modifications suggests that they are cell cycle controlled. The findings presented here support a model whereby differential modification of H3K36 by Gcn5 and Set2 defines the DNA repair mechanism utilised. Methylation of H3K36 by Set2, which peaks in G1, promotes NHEJ whereas acetylation of H3K36 by Gcn5, which peaks in S phase, promotes HR.