In order to better characterize linker histone interactions with DNA, avian erythrocyte-specific linker H5 and the trypsin-resistant globular domain of H5 (GH5) were used in DNA binding studies. To begin, H5 displayed a considerably higher binding
affinity for DNA than the isolated globular domain (GH5), supporting the importance of the...
This dissertation examines the interaction of the linker histone with DNA and with nucleosomes. The first goal of the project was to characterize the interaction of the linker histone with DNA. Three factors previously reported to influence the linker histone's interaction with DNA were examined: ratio of linker histone to...
The DNA-binding domains of E. coli uracil-DNA glycosylase (Ung) and human replication protein A (hRPA) were studied using a general protocol developed in our laboratory for probing protein-DNA interactions. The procedure involves purification and mass spectrometric analysis of the nucleopeptide-products of a tryptically digested UV-crosslinked protein-nucleic acid complex. In the...
PCR-based codon-specific random mutagenesis and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg276 in the conserved leucine-loop of the core catalytic domain of human uracil-DNA glycosylase (UNG). Each Arg276 mutant was then overproduced in E. coli cells and purified to apparent homogeneity by...
Although the Escherichia coli host has almost all of the enzymes necessary to
synthesize nucleotides needed for bacteriophage T4 DNA replication, phage genes
expressed early in infection encode enzymes for de novo DNA precursor biosynthesis
and salvage from degraded host DNA. Eight early enzymes and two host enzymes
comprise the...
With respect to a multienzyme complex of deoxyribonucleoside triphosphate (dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory has demonstrated the existence of a multienzyme complex in T4-infected E. coli, named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP synthesis to DNA replication and...
The solution structure determination of DNA molecules has been an important part of structural biology. NMR solution structures are a complement to structures solved via X-ray crystallography; the two methods are the only ways of obtaining three dimensional coordinates of macromolecules. Because of the nature of the molecule, the solution...
Intrinsically disordered proteins (IDP) are a class of proteins that lack a three-dimensional structure and their prevalence and diverse functions in the cell have only been discovered relatively recently. The intermediate chain (IC) subunit of the microtubule motor protein complex dynein contains an N-terminal disordered region, N-IC, which is central...
The symbiotic sea anemone Exaiptasia diaphana (Aiptasia) is a model organism used to better understand cnidarian-algal symbiosis and coral bleaching (dysbiosis). Many biological processes are hypothesized to be involved in coral dysbiosis, and the innate immunity is a key area of interest. Rab proteins are small proteins with GTPase domains,...
Protein-protein interactions underlie all biological processes and are a field of study that has wide implications throughout many other fields including medicine, genetics, biology, and ecology. Proteins are the building blocks and primary actors of life. They work together to accomplish virtually every task within a cell, including, metabolism, signal...
Cells represent microcosms of spatial and temporal structural organization, with the achievement of internal spatial organization relying upon a collection of macromolecular motor complexes to transport and localize components throughout the cell. Cytoplasmic dynein is one such motor complex, and is the principal ATP-dependent motor for retrograde transport along microtubules...
The enzymes of dNTP synthesis in T4 infection associate to form a multienzyme complex, the T4 dNTP synthetase complex, facilitating the flow of metabolites en route to dNTPs, and their subsequent flow into DNA. Study of protein-protein interactions helps one to understand how the enzymes in the complex are organized...
COUP-TFI, an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily, plays important roles in homeostasis and the CNS development, including differentiation, patterning, axonal projection, cell migration, cortical arealization and the temporal specification of neural stem cells. A number of COUP-interacting proteins have been described previously, and the majority of...
Hub proteins bind a large number of partners to facilitate structural changes and downstream protein interactions. LC8, a highly conserved protein homodimer, is a unique hub that regulates the activity of proteins in a wide range of cellular processes by binding to intrinsically disordered regions. With many of these systems,...
Epigenetic mechanisms are important for control of plant development, and may play a particularly important role in trees given their long life cycles and distinctive and stable tissue types. To help understand the role of epigenetics in tree development, we produced transgenic poplars with reduced activity of the DDM1 genes,...
The Pentatricopeptide Repeat (PPR) gene family produces RNA-binding proteins that target organellar transcripts. The PPR family is expanded in land plants, with nearly 450 genes identified in Arabidopsis thaliana. In plants with a Cytoplasmic Male Sterility (CMS) phenotype, members of the PPR family can act as a RESTORER OF FERTILITY...