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

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

Candidate Variants in DNA Replication and Repair Genes in Early-Onset Renal Cell Carcinoma Patients Referred for Germline Testing

Background: Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined. Methods: Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes. Results: Analysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of [Formula: see text]H2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased [Formula: see text]H2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities. Conclusions: Together, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC

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

The Human Protein PRR14 Tethers Heterochromatin to the Nuclear Lamina during Interphase and Mitotic Exit

The nuclear lamina is a protein meshwork that lies under the inner nuclear membrane of metazoan cells. One function of the nuclear lamina is to organize heterochromatin at the inner nuclear periphery. However, very little is known about how heterochromatin attaches to the nuclear lamina and how such attachments are restored at mitotic exit. Here, we show that a previously unstudied human protein, PRR14, functions to tether heterochromatin to the nuclear periphery during interphase, through associations with heterochromatin protein 1 (HP1) and the nuclear lamina. During early mitosis, PRR14 is released from the nuclear lamina and chromatin and remains soluble. Strikingly, at the onset of anaphase, PRR14 is incorporated rapidly into chromatin through HP1 binding. Finally, in telophase, PRR14 relocalizes to the reforming nuclear lamina. This stepwise reassembly of PRR14 suggests a function in the selection of HP1-bound heterochromatin for reattachment to the nuclear lamina as cells exit mitosis

Human UBN1 Is an Ortholog of Yeast Hpc2p and Has an Essential Role in the HIRA/ASF1a Chromatin-Remodeling Pathway in Senescent Cells▿

Cellular senescence is an irreversible proliferation arrest, tumor suppression process and likely contributor to tissue aging. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Given its likely contribution to tumor suppression and tissue aging, it is essential to identify all components of the SAHF assembly pathway. Formation of SAHF in human cells is driven by a complex of histone chaperones, namely, HIRA and ASF1a. In yeast, the complex orthologous to HIRA/ASF1a contains two additional proteins, Hpc2p and Hir3p. Using a sophisticated approach to search for remote orthologs conserved in multiple species through evolution, we identified the HIRA-associated proteins, UBN1 and UBN2, as candidate human orthologs of Hpc2p. We show that the Hpc2-related domain of UBN1, UBN2, and Hpc2p is an evolutionarily conserved HIRA/Hir-binding domain, which directly interacts with the N-terminal WD repeats of HIRA/Hir. UBN1 binds to proliferation-promoting genes that are repressed by SAHF and associates with histone methyltransferase activity that methylates lysine 9 of histone H3, a site that is methylated in SAHF. UBN1 is indispensable for formation of SAHF. We conclude that UBN1 is an ortholog of yeast Hpc2p and a novel regulator of senescence

The conformational feasibility for the formation of reaching dimer in ASV and HIV integrase: a molecular dynamics study

<p>Retroviral integrases are reported to form alternate dimer assemblies like the core–core dimer and reaching dimer. The core–core dimer is stabilized predominantly by an extensive interface between two catalytic core domains. The reaching dimer is stabilized by N-terminal domains that reach to form intermolecular interfaces with the other subunit’s core and C-terminal domains (CTD), as well as CTD–CTD interactions. In this study, molecular dynamics (MD), Brownian dynamics (BD) simulations, and free energy analyses, were performed to elucidate determinants for the stability of the reaching dimer forms of full-length Avian Sarcoma Virus (ASV) and Human Immunodeficiency Virus (HIV) IN, and to examine the role of the C-tails (the last ~16–18 residues at the C-termini) in their structural dynamics. The dynamics of an HIV reaching dimer derived from small angle X-ray scattering and protein crosslinking data, was compared with the dynamics of a core–core dimer model derived from combining the crystal structures of two-domain fragments. The results showed that the core domains in the ASV reaching dimer express free dynamics, whereas those in the HIV reaching dimer are highly stable. BD simulations suggest a higher rate of association for the HIV core–core dimer than the reaching dimer. The predicted stability of these dimers was therefore ranked in the following order: ASV reaching dimer < HIV reaching dimer < composite core–core dimer. Analyses of MD trajectories have suggested residues that are critical for intermolecular contacts in each reaching dimer. Tests of these predictions and insights gained from these analyses could reveal a potential pathway for the association and dissociation of full-length IN multimers.</p

Mapping the Epitope of an Inhibitory Monoclonal Antibody to the

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