Biochemistry, Biophysics, and Structural Biology Commons^™

Polθ Activity Modulates Sensitivity To Standard Therapies In Dnmt3a-Deficient Leukemia, Bac Viet Le, Umeshkumar Vekariya, Monika M. Toma, Margaret Nieborowska-Skorska, Marie-Christine Caron, Malgorzata Gozdecka, Zayd Haydar, Martin Walsh, Jayashri Ghosh, Elaine Vaughan-Williams, Paulina Podszywalow-Bartnicka, Anna-Mariya Kukuyan, Sylwia Ziolkowska, Jessica Atkins, Emir Hadzijusufovic, Gurushankar Chandramouly, Reza Nejati, Katarzyna Piwocka, Richard T. Pomerantz, George S. Vassiliou, Brian J.P. Huntly, Peter Valent, Mariusz Wasik, Alfonso Bellacosa, Jean-Yves Masson, Gaorav P. Gupta, Grant A. Challen, Tomasz Skorski

Department of Biochemistry and Molecular Biology Faculty Papers

Myeloid malignancies carrying somatic DNMT3A mutations (DNMT3Amut) are refractory to standard therapy. DNMT3Amut leukemia cells accumulate toxic DNA double-strand breaks (DSBs) and stalled replication forks, rendering them dependent on DNA damage response (DDR). We report here that DNA polymerase theta (Polθ), a key element in DSB repair by end-joining (Polθ-mediated end-joining [TMEJ]) and in fork restarting, promotes survival and proliferation of DNMT3Amut leukemia cells. Polθ is overexpressed in DNMT3Amut leukemia cells due to abrogation of PARP1 PARylation-dependent UBE2O E3 ligase-mediated ubiquitination and proteasomal degradation of Polθ. In addition, PARP1-mediated recruitment of the SMARCAD1-MSH2/MSH3 repressive complex to DSBs is diminished in …

Induced Mutagenesis In West Nile Virus Non-Structural Protein 3 With A Discussion Of The Mutation's Effects On Viral Properties, Caden Connealy

Honors Program: Senior Projects (Public)

West Nile Virus (WNV), a member of the Flaviviridae family and introduced to the Western hemisphere in 1999, presents a unique and emerging threat to human health. Infection in humans is often mild but can occasionally result in severe neurological impairment and even death. West Nile Virus non-structural protein 3 (NS3) plays an important role in the process of viral genome amplification. More specifically, the C-terminal domain of the protein folds into a helicase enzyme, which is responsible for the “unwinding” of double-stranded RNA intermediates and RNA secondary structure on the WNV genome. This is a vitally important step in …

Post-Transcriptional Methylation Of Mitochondrial-Trna Differentially Contributes To Mitochondrial Pathology, Sunita Maharjan, Howard Gamper, Yuka Yamaki, Thomas W. Christian, Robert Y. Henley, Nan-Sheng Li, Takeo Suzuki, Tsutomu Suzuki, Joseph A. Piccirilli, Meni Wanunu, Erin L. Seifert, Douglas C. Wallace, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA would also stabilize its pathogenic variants is unknown. Here we show that the N¹-methylation of guanosine at position 9 (m¹G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has a destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated, as removal of the m¹G9 …

The C-Terminal 4cxxc-Type Zinc Finger Domain Of Cdca7 Recognizes Hemimethylated Dna And Modulates Activities Of Chromatin Remodeling Enzyme Hells, Akeo Shinkai, Hideharu Hashimoto, Chikako Shimura, Hiroaki Fujimoto, Kei Fukuda, Naoki Horikoshi, Masaki Okano, Hitoshi Niwa, Erik W Debler, Hitoshi Kurumizaka, Yoichi Shinkai

Department of Biochemistry and Molecular Biology Faculty Papers

The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG …

Mutant Androgen Receptor Induces Neurite Loss And Senescence Independently Of Are Binding In A Neuronal Model Of Sbma, Jordyn Karliner, Y Liu, Diane Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Spinal and bulbar muscular atrophy (SBMA) is a slowly progressing neuromuscular disease caused by a polyglutamine (polyQ)-encoding CAG trinucleotide repeat expansion in the androgen receptor (AR) gene, leading to AR aggregation, lower motor neuron death, and muscle atrophy. AR is a ligand-activated transcription factor that regulates neuronal architecture and promotes axon regeneration; however, whether AR transcriptional functions contribute to disease pathogenesis is not fully understood. Using a differentiated PC12 cell model of SBMA, we identified dysfunction of polyQ-expanded AR in its regulation of neurite growth and maintenance. Specifically, we found that in the presence of androgens, polyQ-expanded AR inhibited neurite …

Genetic Diversity Of 1,845 Rhesus Macaques Improves Genetic Variation Interpretation And Identifies Disease Models, Jun Wang, Meng Wang, Ala Moshiri, R Alan Harris, Muthuswamy Raveendran, Tracy Nguyen, Soohyun Kim, Laura Young, Keqing Wang, Roger Wiseman, David H O'Connor, Zach Johnson, Melween Martinez, Michael J Montague, Ken Sayers, Martha Lyke, Eric Vallender, Tim Stout, Yumei Li, Sara M Thomasy, Jeffrey Rogers, Rui Chen

Faculty, Staff and Students Publications

Understanding and treating human diseases require valid animal models. Leveraging the genetic diversity in rhesus macaque populations across eight primate centers in the United States, we conduct targeted-sequencing on 1845 individuals for 374 genes linked to inherited human retinal and neurodevelopmental diseases. We identify over 47,000 single nucleotide variants, a substantial proportion of which are shared with human populations. By combining rhesus and human allele frequencies with established variant prediction methods, we develop a machine learning-based score that outperforms established methods in predicting missense variant pathogenicity. Remarkably, we find a marked number of loss-of-function variants and putative deleterious variants, which …

Rewiring The Sex-Determination Pathway During The Evolution Of Self-Fertility., Yongquan Shen, Shin-Yi Lin, Jonathan Harbin, Richa Amin, Allison Vassalotti, Joseph Romanowski, Emily Schmidt, Alexis Tierney, Ronald E Ellis

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Although evolution is driven by changes in how regulatory pathways control development, we know little about the molecular details underlying these transitions. The TRA-2 domain that mediates contact with TRA-1 is conserved in Caenorhabditis. By comparing the interaction of these proteins in two species, we identified a striking change in how sexual development is controlled. Identical mutations in this domain promote oogenesis in Caenorhabditis elegans but promote spermatogenesis in Caenorhabditis briggsae. Furthermore, the effects of these mutations involve the male-promoting gene fem-3 in C. elegans but are independent of fem-3 in C. briggsae. Finally, reciprocal mutations in these genes show …

Fused In Sarcoma Regulates Glutamate Signaling And Oxidative Stress Response, Chiong-Hee Wong, Abu Rahat, Howard C Chang

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Mutations in fused in sarcoma (fust-1) are linked to ALS. However, how these ALS causative mutations alter physiological processes and lead to the onset of ALS remains largely unknown. By obtaining humanized fust-1 ALS mutations via CRISPR-CAS9, we generated a C. elegans ALS model. Homozygous fust-1 ALS mutant and fust-1 deletion animals are viable in C. elegans. This allows us to better characterize the molecular mechanisms of fust-1-dependent responses. We found FUST-1 plays a role in regulating superoxide dismutase, glutamate signaling, and oxidative stress. FUST-1 suppresses SOD-1 and VGLUT/EAT-4 in the nervous system. FUST-1 also regulates synaptic AMPA-type glutamate receptor …

Kinome Reprogramming Is A Targetable Vulnerability In Esr1 Fusion-Driven Breast Cancer, Xuxu Gou, Beom-Jun Kim, Meenakshi Anurag, Jonathan T Lei, Meggie N Young, Matthew V Holt, Diana Fandino, Craig T Vollert, Purba Singh, Mohammad A Alzubi, Anna Malovannaya, Lacey E Dobrolecki, Michael T Lewis, Shunqiang Li, Charles E Foulds, Matthew J Ellis

Faculty, Staff and Students Publications

Transcriptionally active ESR1 fusions (ESR1-TAF) are a potent cause of breast cancer endocrine therapy (ET) resistance. ESR1-TAFs are not directly druggable because the C-terminal estrogen/anti-estrogen-binding domain is replaced with translocated in-frame partner gene sequences that confer constitutive transactivation. To discover alternative treatments, a mass spectrometry (MS)-based kinase inhibitor pulldown assay (KIPA) was deployed to identify druggable kinases that are upregulated by diverse ESR1-TAFs. Subsequent explorations of drug sensitivity validated RET kinase as a common therapeutic vulnerability despite remarkable ESR1-TAF C-terminal sequence and structural diversity. Organoids and xenografts from a pan-ET-resistant patient-derived xenograft model that harbors the ESR1-e6>YAP1 TAF were …

Structural Underpinnings Of Mutation Rate Variations In The Human Genome, Zian Liu, Md Abul Hassan Samee

Faculty, Staff and Students Publications

Single nucleotide mutation rates have critical implications for human evolution and genetic diseases. Importantly, the rates vary substantially across the genome and the principles underlying such variations remain poorly understood. A recent model explained much of this variation by considering higher-order nucleotide interactions in the 7-mer sequence context around mutated nucleotides. This model's success implicates a connection between DNA shape and mutation rates. DNA shape, i.e. structural properties like helical twist and tilt, is known to capture interactions between nucleotides within a local context. Thus, we hypothesized that changes in DNA shape features at and around mutated positions can explain …

Identification And Characterization Of Genetic Elements That Regulate A C-Di-Gmp Mediated Multicellular Trait In Pseudomonas Fluorescens, Collin Kessler

Electronic Theses and Dissertations

Microbial communities contain densely packed cells where competition for space and resources are fierce. These communities are generally referred to as biofilms and provide advantages to individual cells against immunological and antimicrobial intervention, dehydration, and predation. High intracellular pools of cyclic diguanylate monophosphate (c-di-GMP) cause cells to aggregate during biofilm formation through the production of diverse extracellular polymers. Genes that encode c-di-GMP catalytic enzymes are commonly mutated during chronic infections where opportunists display enhanced resistance to phagocytosis and antibiotics. Our lab uses an emergent multicellular trait in the model organism Pseudomonas fluorescens Pf0-1 to study the emergence of c-di-GMP mutations …

Evolutionary Action Of Mutations Reveals Antimicrobial Resistance Genes In Escherichia Coli, David C Marciano, Chen Wang, Teng-Kuei Hsu, Thomas Bourquard, Benu Atri, Ralf B Nehring, Nicholas S Abel, Elizabeth A Bowling, Taylor J Chen, Pamela D Lurie, Panagiotis Katsonis, Susan M Rosenberg, Christophe Herman, Olivier Lichtarge

Faculty, Staff and Students Publications

Since antibiotic development lags, we search for potential drug targets through directed evolution experiments. A challenge is that many resistance genes hide in a noisy mutational background as mutator clones emerge in the adaptive population. Here, to overcome this noise, we quantify the impact of mutations through evolutionary action (EA). After sequencing ciprofloxacin or colistin resistance strains grown under different mutational regimes, we find that an elevated sum of the evolutionary action of mutations in a gene identifies known resistance drivers. This EA integration approach also suggests new antibiotic resistance genes which are then shown to provide a fitness advantage …

Mutations In Hcfc1 And Ronin Result In An Inborn Error Of Cobalamin Metabolism And Ribosomopathy, Tiffany Chern, Annita Achilleos, Xuefei Tong, Matthew C Hill, Alexander B Saltzman, Lucas C Reineke, Arindam Chaudhury, Swapan K Dasgupta, Yushi Redhead, David Watkins, Joel R Neilson, Perumal Thiagarajan, Jeremy B A Green, Anna Malovannaya, James F Martin, David S Rosenblatt, Ross A Poché

Faculty, Staff and Students Publications

Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously …

Recurrent High-Impact Mutations At Cognate Structural Positions In Class A G Protein-Coupled Receptors Expressed In Tumors, Eunna Huh, Jonathan Gallion, Melina A Agosto, Sara J Wright, Theodore G Wensel, Olivier Lichtarge

Faculty, Staff and Students Publications

G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated …

Expression And Purification Of Cytochrome 2u1 And Canine 1a2 And Metabolism Of Caffeine By Cytochrome 1a2, Brenda M. Wekesa

Graduate Theses/Dissertations

Cytochrome P450 enzymes are a large family of membrane heme proteins involved in eliminating drugs and the synthesis or elimination of steroids, eicosanoids, and vitamins. CYP2U1 is one of the poorly characterized P450 enzymes classified as orphan cytochrome P450 with unknown exact biological function. It is expressed in the brain and thymus as a catalyst in the hydroxylation of arachidonic acid. Unlike other cytochrome P450s, CYP2U1 is a bit unusual with forty extra amino acids. If a mutation occurs, it can lead to an early onset inherited disorder of the central nervous system called Hereditary Spastic Paraplegias (HSP). Most of …

Replication Protein A (Rpa) Targeting Of Uracil Dna Glycosylase (Ung2), Derek Chen, Brian P Weiser

Rowan-Virtua Research Day

Replication Protein A (RPA) is a single stranded DNA binding protein which stabilizes ssDNA for replication and repair. One function of RPA is to bind the DNA repair enzyme uracil DNA glycosylase (UNG2) and direct its activity towards ssDNA dsDNA junctions.

UNG2 removes uracil bases from DNA which can appear through dUMP misincorporation or through cytosine deamination. If uracil is present instead of a cytosine, then the original GC pair becomes a GU pair. The uracil will then base pair to adenine in the replicated daughter strand. This results in a GC → AT mutation that could contribute to cancer …

Pathological Mechanisms Of Vacuolar Aggregate Myopathy Arising From A Casq1 Mutation, Amy D Hanna, Chang Seok Lee, Lyle Babcock, Hui Wang, Joseph Recio, Susan L Hamilton

Faculty, Staff and Students Publications

Mice with a mutation (D244G, DG) in calsequestrin 1 (CASQ1), analogous to a human mutation in CASQ1 associated with a delayed onset human myopathy (vacuolar aggregate myopathy), display a progressive myopathy characterized by decreased activity, decreased ability of fast twitch muscles to generate force and low body weight after one year of age. The DG mutation causes CASQ1 to partially dissociate from the junctional sarcoplasmic reticulum (SR) and accumulate in the endoplasmic reticulum (ER). Decreased junctional CASQ1 reduces SR Ca

Proteogenomic And Metabolomic Characterization Of Human Glioblastoma, Liang-Bo Wang, Alla Karpova, Marina A Gritsenko, Jennifer E Kyle, Song Cao, Yize Li, Dmitry Rykunov, Antonio Colaprico, Joseph H Rothstein, Runyu Hong, Vasileios Stathias, Macintosh Cornwell, Francesca Petralia, Yige Wu, Boris Reva, Karsten Krug, Pietro Pugliese, Emily Kawaler, Lindsey K Olsen, Wen-Wei Liang, Xiaoyu Song, Yongchao Dou, Michael C Wendl, Wagma Caravan, Wenke Liu, Daniel Cui Zhou, Jiayi Ji, Chia-Feng Tsai, Vladislav A Petyuk, Jamie Moon, Weiping Ma, Rosalie K Chu, Karl K Weitz, Ronald J Moore, Matthew E Monroe, Rui Zhao, Xiaolu Yang, Seungyeul Yoo, Azra Krek, Alexis Demopoulos, Houxiang Zhu, Matthew A Wyczalkowski, Joshua F Mcmichael, Brittany L Henderson, Caleb M Lindgren, Hannah Boekweg, Shuangjia Lu, Jessika Baral, Lijun Yao, Kelly G Stratton, Lisa M Bramer, Erika Zink, Sneha P Couvillion, Kent J Bloodsworth, Shankha Satpathy, Weiva Sieh, Simina M Boca, Stephan Schürer, Feng Chen, Maciej Wiznerowicz, Karen A Ketchum, Emily S Boja, Christopher R Kinsinger, Ana I Robles, Tara Hiltke, Mathangi Thiagarajan, Alexey I Nesvizhskii, Bing Zhang, D R Mani, Michele Ceccarelli, Xi S Chen, Sandra L Cottingham, Qing Kay Li, Albert H Kim, David Fenyö, Kelly V Ruggles, Henry Rodriguez, Mehdi Mesri, Samuel H Payne, Adam C Resnick, Pei Wang, Richard D Smith, Antonio Iavarone, Milan G Chheda, Jill S Barnholtz-Sloan, Karin D Rodland, Tao Liu, Li Ding, Clinical Proteomic Tumor Analysis Consortium

Faculty, Staff and Students Publications

Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM …

Tbx5-Encoded T-Box Transcription Factor 5 Variant T223m Is Associated With Long Qt Syndrome And Pediatric Sudden Cardiac Death, Alexandra M Markunas, Perathu K R Manivannan, Jordan E Ezekian, Agnim Agarwal, William Eisner, Katherina Alsina, Hugh D Allen, Gregory A Wray, Jeffrey J Kim, Xander H T Wehrens, Andrew P Landstrom

Faculty, Staff and Students Publications

Long QT syndrome (LQTS) is a genetic disease resulting in a prolonged QT interval on a resting electrocardiogram, predisposing affected individuals to polymorphic ventricular tachycardia and sudden death. Although a number of genes have been implicated in this disease, nearly one in four individuals exhibiting the LQTS phenotype are genotype-negative. Whole-exome sequencing identified a missense T223M variant in TBX5 that cosegregates with prolonged QT interval in a family with otherwise genotype-negative LQTS and sudden death. The TBX5-T223M variant was absent among large ostensibly healthy populations (gnomAD) and predicted to be pathogenic by in silico modeling based on Panther, PolyPhen-2, Provean, …

Adaptive Thermogenesis Enhances The Life-Threatening Response To Heat In Mice With An Ryr1 Mutation, Hui J Wang, Chang Seok Lee, Rachel Sue Zhen Yee, Linda Groom, Inbar Friedman, Lyle Babcock, Dimitra K Georgiou, Jin Hong, Amy D Hanna, Joseph Recio, Jong Min Choi, Ting Chang, Nadia H Agha, Jonathan Romero, Poonam Sarkar, Nicol Voermans, M Waleed Gaber, Sung Yun Jung, Matthew L Baker, Robia G Pautler, Robert T Dirksen, Sheila Riazi, Susan L Hamilton

Faculty, Staff and Students Publications

Mutations in the skeletal muscle Ca2+ release channel, the type 1 ryanodine receptor (RYR1), cause malignant hyperthermia susceptibility (MHS) and a life-threatening sensitivity to heat, which is most severe in children. Mice with an MHS-associated mutation in Ryr1 (Y524S, YS) display lethal muscle contractures in response to heat. Here we show that the heat response in the YS mice is exacerbated by brown fat adaptive thermogenesis. In addition, the YS mice have more brown adipose tissue thermogenic capacity than their littermate controls. Blood lactate levels are elevated in both heat-sensitive MHS patients with RYR1 mutations and YS mice due to …

An Autism-Linked Missense Mutation In Shank3 Reveals The Modularity Of Shank3 Function, Li Wang, Kaifang Pang, Kihoon Han, Carolyn J Adamski, Wei Wang, Lingjie He, Jason K Lai, Vitaliy V Bondar, Joseph G Duman, Ronald Richman, Kimberley F Tolias, Patrick Barth, Timothy Palzkill, Zhandong Liu, J Lloyd Holder, Huda Y Zoghbi

Faculty, Staff and Students Publications

Genome sequencing has revealed an increasing number of genetic variations that are associated with neuropsychiatric disorders. Frequently, studies limit their focus to likely gene-disrupting mutations because they are relatively easy to interpret. Missense variants, instead, have often been undervalued. However, some missense variants can be informative for developing a more profound understanding of disease pathogenesis and ultimately targeted therapies. Here we present an example of this by studying a missense variant in a well-known autism spectrum disorder (ASD) causing gene SHANK3. We analyzed Shank3's in vivo phosphorylation profile and identified S685 as one phosphorylation site where one ASD-linked variant has …

Expression Changes Confirm Genomic Variants Predicted To Result In Allele-Specific, Alternative Mrna Splicing, Peter Rogan

Biochemistry Publications

Splice isoform structure and abundance can be affected by either noncoding or masquerading coding variants that alter the structure or abundance of transcripts. When these variants are common in the population, these nonconstitutive transcripts are sufficiently frequent so as to resemble naturally occurring, alternative mRNA splicing. Prediction of the effects of such variants has been shown to be accurate using information theory-based methods. Single nucleotide polymorphisms (SNPs) predicted to significantly alter natural and/or cryptic splice site strength were shown to affect gene expression. Splicing changes for known SNP genotypes were confirmed in HapMap lymphoblastoid cell lines with gene expression microarrays …

A Dedicated Chaperone Mediates The Safe Transfer Of Mitoribosomal Proteins To Their Site Of Assembly, Gabrielle Ashley Hillman

Graduate School of Biomedical Sciences Theses and Dissertations

Mitochondrial ribosomes are functionally specialized for the synthesis of several essential inner membrane proteins of the respiratory chain. While remarkable progress has recently been made towards understanding the structure of mitoribosomes, the unique pathways and factors that facilitate their biogenesis remain largely unknown. This dissertation defines the physiological role of an evolutionarily conserved yeast protein called Mam33 in mitochondrial ribosome assembly. The biomedical relevance of this finding stems from the fact that mutations or changes in its expression of the human ortholog p32 result in mitochondrial dysfunction. In human patients, bi-allelic mutations cause severe multisystemic defects in mitochondrial energy metabolism, …

Transcription Factor Binding Site Clusters Identify Target Genes With Similar Tissue-Wide Expression And Buffer Against Mutations., Peter Rogan, Ruipeng Lu

Biochemistry Publications

Background: The distribution and composition of cis-regulatory modules composed of transcription factor (TF) binding site (TFBS) clusters in promoters substantially determine gene expression patterns and TF targets. TF knockdown experiments have revealed that TF binding profiles and gene expression levels are correlated. We use TFBS features within accessible promoter intervals to predict genes with similar tissue-wide expression patterns and TF targets using Machine Learning (ML). Methods: Bray-Curtis Similarity was used to identify genes with correlated expression patterns across 53 tissues. TF targets from knockdown experiments were also analyzed by this approach to set up the ML framework. TFBSs were …

Crispr Gene Editing In The Sea Squirt, Ciona Intestinalis, Evelyn Siler, Steven Irvine

Senior Honors Projects

Genetic manipulation has come a long way in the past ten years alone. Scientists have had access to gene editing techniques for decades, but until recently these methods have proven to be expensive and unpredictable. However, thanks to the development of a new, more efficient genome editing strategy called CRISPR/Cas9, more aggressive progress can now be made in genetics research.

CRISPR is not a machine or a physical tool, but rather it is a system that involves introducing a protein into a cell, along with a DNA segment that will attract the protein to a desired location on the DNA. …

Role Of Protein Charge Density On Hepatitis B Virus Capsid Formation, Xinyu Sun, Dong Li, Zhaoshuai Wang, Panchao Yin, Rundong Hu, Rundong Hu, Hui Li, Qiao Liu, Yunyi Gao, Baiping Ren, Jie Zheng, Yinan Wei, Tianbo Liu

Chemistry Faculty Publications

The role of electrostatic interactions in the viral capsid assembly process was studied by comparing the assembly process of a truncated hepatitis B virus capsid protein Cp149 with its mutant protein D2N/D4N, which has the same conformational structure but four fewer charges per dimer. The capsid protein self-assembly was investigated under a wide range of protein surface charge densities by changing the protein concentration, buffer pH, and solution ionic strength. Lowering the protein charge density favored the capsid formation. However, lowering charge beyond a certain point resulted in capsid aggregation and precipitation. Interestingly, both the wild-type and D2N/D4N mutant displayed …

Mutations Of Conserved Non-Coding Elements Of Pitx2 In Patients With Ocular Dysgenesis And Developmental Glaucoma., Meredith E. Protas, Eric Weh, Tim Footz, Jay Kasberger, Scott C. Baraban, Alex V. Levin, L. Jay Katz, Robert Ritch, Michael A. Walter, Elena V. Semina, Douglas B. Gould

Natural Sciences and Mathematics | Faculty Scholarship

Mutations in FOXC1 and PITX2 constitute the most common causes of ocular anterior segment dysgenesis (ASD), and confer a high risk for secondary glaucoma. The genetic causes underlying ASD in approximately half of patients remain unknown, despite many of them being screened by whole exome sequencing. Here, we performed whole genome sequencing on DNA from two affected individuals from a family with dominantly inherited ASD and glaucoma to identify a 748-kb deletion in a gene desert that contains conserved putative PITX2 regulatory elements. We used CRISPR/Cas9 to delete the orthologous region in zebrafish in order to test the pathogenicity of …

Quaternary Interactions And Supercoiling Modulate The Cooperative Dna Binding Of Agt, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ω_ave. We found …

Conservative Tryptophan Mutations In Protein Tyrosine Phosphatase Ptp1b And Its Effect On Catalytic Rate And Chemical Reaction, Teisha Richan

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein-tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphorylated tyrosines by a 2-step mechanism involving nucleophilic attack by cysteine and general acid catalysis by aspartic acid. In most PTPs the aspartic acid resides on a flexible protein loop, consisting of about a dozen residues, called the WPD loop. PTP catalysis rates span several orders of magnitude, and differences in WPD loop dynamics have recently been show to correlate with the rate of enzymatic catalysis. The rate of WPD loop motion could possibly be related to a widely conserved tryptophan residue on the WPD loop. Therefore, point mutants were made in PTP1B …

Dna Polymerase Zeta-Dependent Mutagenesis: Molecular Specificity, Extent Of Error-Prone Synthesis, And The Role Of Dntp Pools, Olga V. Kochenova

Theses & Dissertations

Despite multiple DNA repair pathways, DNA lesions can escape repair and compromise normal chromosomal replication, leading to genome instability. Cells utilize specialized low-fidelity Translesion Synthesis (TLS) DNA polymerases to bypass lesions and rescue arrested replication forks. TLS is a highly conserved two-step process that involves insertion of a nucleotide opposite a lesion and extension of the resulting aberrant primer terminus. The first step can be performed by both replicative and TLS DNA polymerases and, because of non-instructive DNA lesions, often results in a nucleotide misincorporation. The second step is almost exclusively catalyzed by DNA polymerase ζ …