Browsing by Author "Turchi, John"
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Item Consequences of telomerase inhibition and telomere dysfunction in BRCA1 mutant cancer cells(2014-03-12) Phipps, Elizabeth Ann; Herbert, Brittney-Shea; Grimes, Brenda R.; Sledge, George W.; Turchi, John; White, Kenneth E.Telomere maintenance is a critical component of genomic stability. An increasing body of evidence suggests BRCA1, a tumor suppressor gene with a variety of functions including DNA repair and cell cycle regulation, plays a role in telomere maintenance. Mutations in BRCA1 account for approximately half of all hereditary breast and ovarian cancers, and the gene is silenced via promoter methylation and loss of heterozygosity in a proportion of sporadic breast and ovarian cancers. The objective of this study was to determine whether GRN163L, a telomerase inhibitor, currently in clinical trials for the treatment of cancer, has enhanced anti-cancer activity in BRCA1 mutant breast/ovarian cancer cell lines compared to wild-type cancer cells. BRCA1 mutant cancer cells were observed to have shorter telomeres and increased sensitivity to telomerase inhibition, compared to cell lines with wild-type BRCA1. Importantly, GRN163L treatment was synergistic with DNA-damaging drugs, suggesting potential synthetic lethality of the BRCA1 cancer subtype and telomerase inhibition In a related study to examine the roles of BRCA1/2 in telomere maintenance, DNA and RNA extracted from peripheral blood were used to investigate the age-adjusted telomere lengths and telomere-related gene expression profiles of BRCA1 and BRCA2 individuals compared to individuals who developed sporadic cancer and healthy controls. BRCA1 mutation carriers and breast cancer patients showed the shortest average telomere lengths compared to the other groups. In addition, distinct genomic profiles of BRCA mutation carriers were obtained regarding overexpression of telomere-related genes compared to individuals who developed sporadic or familial breast cancer. In summary, telomerase inhibition may be a viable treatment option in BRCA1 mutant breast or ovarian cancers. These data also provides insights into further investigations on the role of BRCA1 in the biology underlying telomere dysfunction in cancer development.Item Defining the Role of Lysine Acetylation in Regulating the Fidelity of DNA Synthesis(2020-12) Ononye, Onyekachi Ebelechukwu; Balakrishnan, Lata; Watson, John; Baucum, AJ; Turchi, John; Bochman, MatthewAccurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.Item Determining molecular mechanisms of DNA Non-Homologous End Joining proteins(2010-12) Pawelczak, Katherine S.; Wek, Ronald C.; Turchi, John; Lee, Suk-Hee; Takagi, YuichiroDNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR) are complex lesions and if not repaired, these breaks can lead to genomic instability, chromosomal abnormalities and cell death. IR-induced DSB often have DNA termini modifications including thymine glycols, ring fragmentation, 3' phosphoglycolates, 5' hydroxyl groups and abasic sites. Non-homologous end joining (NHEJ) is a major pathway responsible for the repair of these complex breaks. Proteins involved in NHEJ include the Ku 70/80 heterodimer, DNA-PKcs, processing proteins including Artemis and DNA polymerases µ and λ, XRCC4, DNA ligase IV and XLF. The precise molecular mechanism of DNA-PK activation and Artemis processing at the site of a DNA DSB has yet to be elucidated. We have investigated the effect of DNA sequence and structure on DNA-PK activation and results suggest a model where the 3' strand of a DNA terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ. Artemis, a nuclease implicated in processing of DNA termini at a DSB during NHEJ, has been demonstrated to have both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity. Evidence suggests that either the enzyme contains two different active sites for each of these distinct processing activities, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. These results reveal novel mechanisms of two critical NHEJ proteins, and further enhance our understanding of DNA-PK and Artemis activity and their role in NHEJ.Item Exploring a structural protein-drug interactome for new therapeutics in lung cancer(Royal Society of Chemistry, 2014-03-04) Peng, Xiaodong; Wang, Fang; Li, Liwei; Bum-Erdene, Khuchtumur; Xu, David; Wang, Bo; Sinn, Tony; Pollok, Karen; Sandusky, George; Li, Lang; Turchi, John; Jalal, Shadia I.; Meroueh, Samy; Department of Biochemistry & Molecular Biology, IU School of MedicineThe pharmacology of drugs is often defined by more than one protein target. This property can be exploited to use approved drugs to uncover new targets and signaling pathways in cancer. Towards enabling a rational approach to uncover new targets, we expand a structural protein-ligand interactome () by scoring the interaction among 1000 FDA-approved drugs docked to 2500 pockets on protein structures of the human genome. This afforded a drug-target network whose properties compared favorably with previous networks constructed using experimental data. Among drugs with the highest degree and betweenness two are cancer drugs and one is currently used for treatment of lung cancer. Comparison of predicted cancer and non-cancer targets reveals that the most cancer-specific compounds were also the most selective compounds. Analysis of compound flexibility, hydrophobicity, and size showed that the most selective compounds were low molecular weight fragment-like heterocycles. We use a previously-developed screening approach using the cancer drug erlotinib as a template to screen other approved drugs that mimic its properties. Among the top 12 ranking candidates, four are cancer drugs, two of them kinase inhibitors (like erlotinib). Cellular studies using non-small cell lung cancer (NSCLC) cells revealed that several drugs inhibited lung cancer cell proliferation. We mined patient records at the Regenstrief Medical Record System to explore the possible association of exposure to three of these drugs with occurrence of lung cancer. Preliminary in vivo studies using the non-small cell lung cancer (NCLSC) xenograft model showed that losartan- and astemizole-treated mice had tumors that weighed 50 (p < 0.01) and 15 (p < 0.01) percent less than the treated controls. These results set the stage for further exploration of these drugs and to uncover new drugs for lung cancer therapy.Item Genome-destabilizing and Mutagenic Effects of Break-induced Replication in Saccharomyces cerevisiae(2011-05) Deem, Angela Kay; Stauffacher, Cyntha V.; Bard, Martin; Malkova, Anna; Randall, Stephen Karl, 1953-; Turchi, JohnDNA suffers constant damage, leading to a variety of lesions that require repair. One of the most devastating lesions is a double-strand break (DSB), which results in physical dissociation of two pieces of a chromosome. Necessarily, cells have evolved a number of DSB repair mechanisms. One mechanism of DSB repair is break-induced replication (BIR), which involves invasion of one side of the broken chromosome into a homologous template, followed by copying of the donor molecule through telomeric sequences. BIR is an important cellular process implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities. Furthermore, BIR uniquely combines processive replication involving a replication fork with DSB repair. This work employs a system in Saccharomyces cerevisiae to investigate genetic control, physical outcomes, and frameshift mutagenesis associated with BIR initiated by a controlled HO-endonuclease break in a chromosome. Mutations in POL32, which encodes a third, non-essential subunit of polymerase delta (Pol delta), as well as RAD9 and RAD24, which participate in the DNA damage checkpoint response, resulted in a BIR defect characterized by decreased BIR repair and increased loss of the broken chromosome. Also, increased incidence of chromosomal fusions determined to be half-crossover (HCO) molecules was confirmed in pol32 and rad24, as well as a rad9rad50S double mutant. HCO formation was also stimulated by addition of a replication-inhibiting drug, methyl-methane sulfonate (MMS), to cells undergoing BIR repair. Based on these data, it is proposed that interruption of BIR after it has initiated is one mechanism of HCO formation. Addition of a frameshift mutation reporter to this system allowed mutagenesis associated with BIR DNA synthesis to be measured. It is demonstrated that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2800-fold higher than normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. Pol proofreading and mismatch repair (MMR) are confirmed to correct BIR errors. Based on these data, it is proposed that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR. Pif1p, a helicase that is non-essential for DNA replication, and elevated dNTP levels during BIR also contributed to BIR mutagenesis. Taken together, this work characterizes BIR as an essential repair process that also poses risks to a cell, including genome destabilization and hypermutagenesis.Item The Mechanisms by Which Small Molecules Modulate the HSP60/10 Chaperonin System to Elicit Antimicrobial Effects(2023-06) Stevens, Mckayla Marie; Johnson, Steven; Turchi, John; Hoang, Quyen; Wek, Ronald; Absalon, SabrinaHeat Shock Protein 60/10 (HSP60/10, or GroEL/ES in bacteria) chaperonin systems play a critical role in protein homeostasis through facilitating proper folding of misfolded or partially folded polypeptides that are otherwise prone to aggregation. HSP60 chaperonins are highly conserved and essential in nearly all organisms studied thus far, making them a promising target for antibiotic development. Early high-throughput screens in the Johnson lab have identified five main scaffolds that, though hit-to-lead development, have been optimized for chaperonin inhibition and antimicrobial effects. While these initial studies have shown promising evidence to support the viability of a chaperonin-targeting antibiotic strategy, it was unclear whether the conservation of human HSP60 (48% identity to bacterial GroEL) would hinder this therapeutic strategy from advancing due to potential toxicity associated with off-target inhibition of the human homolog. Additionally, while chaperonin inhibition often correlated with cytotoxicity to the various pathogens studied, there was a clear need to investigate inhibitor mechanisms to 1) verify on-target effects, and 2) guide future development of more potent and selective chaperonin-targeting antibiotic candidates. Herein, we conduct a medium-throughput screening of known bioactive molecules, approved drugs, and natural products against both bacterial GroEL and human HSP60, demonstrating that most molecules exhibited low-to-no toxicity to human cells in culture, despite being near equipotent inhibitors of human HSP60 and E. coli GroEL in our refolding assays. Thus, sequence conservation between human HSP60 and bacterial GroELs does not necessarily predict toxicity in vivo. We then investigate inhibitory mechanisms of our most well-established inhibitor series, the phenylbenzoxazole (PBZ) series, identifying three binding sites whereby PBZ molecules modulate GroEL folding and ATPase functions in a site-specific manner, predominately through its ability to interact with its co-chaperone GroES. Finally, we demonstrate that two standard of care drugs for T. brucei infections, suramin and nifurtimox, may elicit their trypanocidal effects through inhibiting HSP60. Due to structural similarities, we then screened our N-acylhydrazone (NAH) and α,β-unsaturated ketone (ABK) series of HSP60 inhibitors against T. brucei, finding that they are highly potent and selective trypanocidal agents. Together, these studies further support HSP60 as a viable drug target for antibiotic development.Item Unraveling the Complexities of DNA-Dependent Protein Kinase Autophosphorylation(American Society for Microbiology (ASM), 2014-06) Neal, Jessica A.; Sugiman-Marangos, Seiji; VanderVere-Carozza, Pamela; Wagner, Mike; Turchi, John; Lees-Miller, Susan P.; Junop, Murray S.; Meek, Katheryn; Department of Medicine, IU School of MedicineDNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a “hinge” region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ.