Translational Repression of Bacteriophage T4 DNA Polymerase Biosynthesis

The research described in this dissertation elucidated the mechanism by which bacteriophage T4 DNA polymerase regulates its own biosynthesis. Utilizing both in vivo and in vitro studies, I have shown that autogenous repression occurs at the level of translation. While T4 mutants defective in the structural gene for DNA polymerase (gene 43) overproduce the protein product (gp43) in vivo, they do not overproduce the corresponding mRNA. In vitro, purified DNA polymerase specifically inhibited the translation of its own transcripts. Further, it was demonstrated that gp43 binds its own mRNA at a site overlapping the ribosome initiation domain. Thus, T4 DNA polymerase is a specific translational repressor that presumably inhibits initiation of translation. The mRNA binding site (translational operator) for DNA polymerase includes 38-40 nucleotides upstream of the initiator AUG. The 5\u27 half of this translational operator contains a putative five base-pair stem and 8-base loop, whose existence is inferred from RNase digestion experiments and computer-assisted analysis of RNA folding. To ascertain the important RNA sequence and structural determinants for DNA polymerase binding, I carried out a mutational analysis of the translational operator via the in vitro construction of several operator variants. Operator mutants were subsequently assayed for the effect of each mutation on: 1) gp43/mRNA binding, in vitro 2) the in vivo levels of gp43 biosynthesis from plasmid encoded constructs and 3) in vivo level of gp43 synthesis in phage infections (carried out after introducing mutant operators into the phage genome by virus-plasmid recombination). Mutations that either disrupted the stem or altered particular loop residues, led to diminished binding of purified T4 DNA polymerase in vitro and to derepression of protein synthesis in vivo. Compensatory mutations that restored the stern pairing, with a sequence other than wild-type, restored in vitro binding but still exhibited a mutant phenotype in vivo. Results from loop substitutions suggest that the spatial arrangement of specific loop residues is a major criterion for specific binding of DNA polymerase to its mRNA operator. These studies demonstrate the effectiveness of genetic approaches in dissecting the rules that govern RNA-protein interactions

What are Stated Gaps in Injury Prevention and Treatment Available on College Campuses for Dancers?

This essay is dedicated to looking at the availability of medical assistance to dancers in the collegiate setting. Dance is a physically demanding activity with prolonged training timelines, however, there has been little dedication to the treatment and prevention of injuries in dance. The apparent lack of dedicated medical assistance dancers receive is what inspired this research. The objective was to better understand this lack and how it can be improved. It is important that dancers have resources available to help treat and prevent potential injuries, which will allow for longer and more fulfilling careers that do less chronic harm to their bodies. Based on a literature review contextualized further with first-person interviews, there are some opportunities that should be made accessible to collegiate dancers to help close this gap between the medical field and the dance industry. Recommendations include creating dance medicine facilities, providing cross training opportunities, creating experiences for athletic training or physical therapy students to learn about dancers as athletes, and prioritizing screenings for dancers. By creating resources that dancers can utilize and a comfortable environment where dancers can get help to treat and prevent injuries, this gap can be closed and could result in healthier dancers.AMSUNY CayugaHonors CollegeBAOakes, Stevi

What are Stated Gaps in Injury Prevention and Treatment Available on College Campuses for Dancers?

VoRSUNY BrockportN/

What are Stated Gaps in Injury Prevention and Treatment Available on College Campuses for Dancers?

This essay is dedicated to looking at the availability of medical assistance to dancers in the collegiate setting. Dance is a physically demanding activity with prolonged training timelines, however, there has been little dedication to the treatment and prevention of injuries in dance. The apparent lack of dedicated medical assistance dancers receive is what inspired this research. The objective was to better understand this lack and how it can be improved.It is important that dancers have resources available to help treat and prevent potential injuries, which will allow for longer and more fulfilling careers that do less chronic harm to their bodies. Based on a literature review contextualized further with first-person interviews, there are some opportunities that should be made accessible to collegiate dancers to help close this gap between the medical field and the dance industry. Recommendations include creating dance medicine facilities, providing cross training opportunities,creating experiences for athletic training or physical therapy students to learn about dancers as athletes, and prioritizing screenings for dancers. By creating resources that dancers can utilize and a comfortable environment where dancers can get help to treat and prevent injuries, this gap can be closed and could result in healthier dancers.NAN/AHonors CollegeBFAOakes, Stevi

Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

BACKGROUND: To further our understanding of the structure and function of HIV-1 integrase (IN) we developed and characterized a library of monoclonal antibodies (mAbs) directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. RESULTS: We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A) and IN (I267A/I268A), exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. CONCLUSION: It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather our findings are most consistent with a model whereby mAb33 binding distorts or constrains the structure of the C-terminal domain and/or blocks substrate binding indirectly. The DNA binding properties and non-specific nuclease activity of the I267A derivatives suggest that the C-terminal domain of IN normally plays an important role in aligning the viral DNA end for proper processing

Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors

<p>Abstract</p> <p>Background</p> <p>Integration of retroviral DNA into the host cell genome is an obligatory step in the virus life cycle. In previous reports we identified a sequence (amino acids 201–236) in the linker region between the catalytic core and C-terminal domains of the avian sarcoma virus (ASV) integrase protein that functions as a transferable nuclear localization signal (NLS) in mammalian cells. The sequence is distinct from all known NLSs but, like many, contains basic residues that are essential for activity.</p> <p>Results</p> <p>Our present studies with digitonin-permeabilized HeLa cells show that nuclear import mediated by the NLS of ASV integrase is an active, saturable, and ATP-dependent process. As expected for transport through nuclear pore complexes, import is blocked by treatment of cells with wheat germ agglutinin. We also show that import of ASV integrase requires soluble cellular factors but does not depend on binding the classical adapter Importin-α. Results from competition studies indicate that ASV integrase relies on one or more of the soluble components that mediate transport of the linker histone H1.</p> <p>Conclusion</p> <p>These results are consistent with a role for ASV integrase and cytoplasmic cellular factors in the nuclear import of its viral DNA substrate, and lay the foundation for identification of host cell components that mediate this reaction.</p

Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins.Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate.Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution

Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity

POLE driver mutations in the exonuclease domain (ExoD driver) are prevalent in several cancers, including colorectal cancer and endometrial cancer, leading to dramatically ultra-high tumor mutation burden (TMB). To understand whether POLE mutations that are not classified as drivers (POLE Variant) contribute to mutagenesis, we assessed TMB in 447 POLE-mutated colorectal cancers, endometrial cancers, and ovarian cancers classified as TMB-high >= 10 mutations/Mb (mut/Mb) or TMB-low <10 mut/Mb. TMB was significantly highest in tumors with POLE ExoD driver plus POLE Variant (colorectal cancer and endometrial cancer, P < 0.001; ovarian cancer, P < 0.05). TMB increased with additional POLE variants (P < 0.001), but plateaued at 2, suggesting an association between the presence of these variants and TMB. Integrated analysis of AlphaFold2 POLE models and quantitative stability estimates predicted the impact of multiple POLE variants on POLE functionality. The prevalence of immunogenic neoepitopes was notably higher in the POLE ExoD driver plus POLE Variant tumors. Overall, this study reveals a novel correlation between POLE variants in POLE ExoD-driven tumors, and ultra-high TMB. Currently, only select pathogenic ExoD mutations with a reliable association with ultra-high TMB inform clinical practice. Thus, these findings are hypothesis-generating, require functional validation, and could potentially inform tumor classification, treatment responses, and clinical outcomes. Significance: Somatic POLE ExoD driver mutations cause proofreading deficiency that induces high TMB. This study suggests a novel modifier role for POLE variants in POLE ExoD-driven tumors, associated with ultra-high TMB. These data, in addition to future functional studies, may inform tumor classification, therapeutic response, and patient outcomes

Systematic evaluation of underlying defects in DNA repair as an approach to case-only assessment of familial prostate cancer

Risk assessment for prostate cancer is challenging due to its genetic heterogeneity. In this study, our goal was to develop an operational framework to select and evaluate gene variants that may contribute to familial prostate cancer risk. Drawing on orthogonal sources, we developed a candidate list of genes relevant to prostate cancer, then analyzed germline exomes from 12 case-only prostate cancer patients from high-risk families to identify patterns of protein-damaging gene variants. We described an average of 5 potentially disruptive variants in each individual and annotated them in the context of public databases representing human variation. Novel damaging variants were found in several genes of relevance to prostate cancer. Almost all patients had variants associated with defects in DNA damage response. Many also had variants linked to androgen signaling. Treatment of primary T-lymphocytes from these prostate cancer patients versus controls with DNA damaging agents showed elevated levels of the DNA double strand break (DSB) marker ?H2AX (p < 0.05), supporting the idea of an underlying defect in DNA repair. This work suggests the value of focusing on underlying defects in DNA damage in familial prostate cancer risk assessment and demonstrates an operational framework for exome sequencing in case-only prostate cancer genetic evaluation

Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solution

<p>Abstract</p> <p>Background</p> <p>HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.</p> <p>Results</p> <p>A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg⁺⁺- and Mn⁺⁺-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn⁺⁺than Mg⁺⁺. Although the pH optima for these two enzymes are similar with Mn⁺⁺, they differ significantly in the presence of Mg⁺⁺, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn⁺⁺.</p> <p>Conclusion</p> <p>A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10–30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins.</p