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 DNA sites of binding, monovalent ions in the local environment, and conformation of the DNA molecules. Evidence obtained through gel mobility shift assays demonstrates the strong preference by the linker histone for DNA with superhelical torsion, i.e., supercoiling, and the negative cooperative mode of binding that the linker histone exhibits in association with supercoiled DNA. The second part of the dissertation examines the location of linker histone binding on the nucleosome, and documents the pronounced tendency of the linker histone to bind to two DNA duplex strands. A preparation of homogeneous nucleosome core particles, consisting of a defined 238 base pair DNA fragment and the core histone octamer positioned precisely on this DNA, was used as a substrate for the UV-induced crosslinking of the linker histone to the DNA of this nucleosome. By site-specific labeling of a single site on the DNA of the nucleosome, the linker histone was observed crosslinked at that labeled site, confirming that the linker histone binds at the pseudo-dyad axis of the nucleosome. This evidence was used to support a model of linker histone binding to the nucleosome that invokes the association of the linker histone with no fewer than two duplex strands of DNA of the nucleosome. / Graduation date: 2004

Graduation date: 2003

Deoxyribonucleoside triphosphate (dNTP) concentrations measured in cells are not symmetric. dGTP almost always represents only 5-10% of the total dNTP pools in cells. In an in vitro replication system involving semiconservative replication from an SV 40 origin, the mutation frequency of an M13 phagemid replicated by human cell extracts in reaction mixtures containing "biologically biased" dNTP pools estimated from HeLa cell nuclei is not significantly different from that seen when replication is done with equimolar dNTP concentrations. Significant reduction of dGTP pool while keeping other dNTPs at "biologically biased" dNTP concentrations during replication reaction also did not increase mutation frequency. In contrast, in vitro replication with dNTP concentrations calculated from normal diploid fibroblast cells, which are three- to four-fold lower in concentrations, showed a marked reduction of the observed mutation frequency, showing the importance of overall dNTP levels during replication on mutation frequency in vitro. When whole-cell dNTP concentrations in HeLa cells were measured during S-phase, dNTP levels underwent a transient decrease in the middle of S-phase. Average HeLa cells' dNTP levels were also found to correlate with average DNA accumulation rates during S-phase, although no detailed relationship can yet be deducted from the available data. No significant changes in the ratio of the four dNTP concentrations were found during S-phase. Mutation rates of green fluorescent protein (GFP) inserted in either middle or late-replicating region of a chromosome in HeLa cells also correspond to average DNA accumulation rates and dNTP levels during middle and late S-phase. The late-replicating GFP-HeLa cells have a higher mutation rate than the middle-replicating GFP-HeLa cells, as the average DNA accumulation rates and dNTP pool levels were also lower in the middle compared to late S-phase. Taken together, these observations indicate that dNTP levels could play a role in determining the S-phase DNA replication rate and also the replication fidelity in mammalian cells. / Graduation date: 2002

Graduation date: 1999

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 terminal tail domains in binding. This conclusion is based upon binding curves conducted in low-salt solution, and on the considerably-higher salt concentration required to prevent protein-DNA contact. Linker histones also induce DNA-protein aggregation in a process that was found to result in protein insolubility in 2% SDS, and included protein-protein interactions that did not require the terminal tail domains. In addition, DNA supercoiling appeared to impede the aggregation process; this that may be attributable to binding of linker histones in isolated clusters, as gauged by a limit in the number of observed dithiobis (succinimidyl) propionate (DSP)-crosslinkable contacts. In a related study, the property of GH5 to bind, then organize onto DNA was investigated. GH5 crosslinked onto DNA with dithiobis (succinimidyl propionate), then cleaved with chymotrypsin, displayed highly uniform contacts that appeared to involve the C-terminal four amino acids, and suggests protein-protein interactions are important for binding. This finding may be relevant since GH5 (and H5) were observed to self-associate free in solution in an arguably specific manner. Finally, the exposure of Phe 93 to chymotrypsin was used to identify the surface of the globular domain that contacts DNA for the binding of intact H5. Results suggests that the side of the protein opposite to the recognition helix preferentially binds to DNA, supporting a novel winged-helix protein DNA-binding mechanism. Furthermore, parallel studies with octamers reconstituted onto a DNA fragment with twelve copies of the 208 b.p. rDNA 5s gene from Lytechinus variegatus, shows that H5 had a high binding affinity with all detectable protein binding to the reconstituted complex. H5 binding conferred protection to a site located near the dyad axis from endonuclease digestion, supporting the contention that H5 binds near or at the nucleosome dyad axis. H5 binding also was observed to condense fibers as observed from agarose gel electrophoresis, although velocity analytical sedimentation studies indicate that H5 in itself was not sufficient to fully compact chromatin fibers; rather H5 and 30 mM NaCl, in combination, were required. Results indicate that the chromatin-reconstituted "208-12 DNA" makes an excellent model for analyzing the effect of linker proteins on chromatin morphology. / Graduation date: 1998

The yeast two-hybrid system was used to examine interactions between the nine proteins involved in baculovirus DNA replication. From the six proteins required for DNA replication, four protein-protein interactions were identified, including an interaction between LEF-1 and LEF-2, LEF-3 and itself, LEF-3 and P143 (Helicase), and an interaction between IE-1 and itself. The replication factors LEF-1 and LEF-2 interacted in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. Using the yeast two-hybrid system, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Deletion analysis of LEF-1 failed to reveal an interaction domain, suggesting that either multiple interaction domains exists or the deletions disrupted secondary structures required for the interaction. All of the deletions which were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction plays a significant role in DNA replication. The baculovirus single-stranded DNA binding protein, LEF-3, interacts with itself in yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. Deletions of LEF-3, which were unable to interact with full length LEF-3, also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to apparent homogeneity and analyzed by analytical ultracentrifugation, native PAGE and MALDI mass spectrometry, identifying the oligomeric structure of LEF-3 as a homotrimer. In addition to interacting with itself, LEF-3 also interacts with P143 in yeast two-hybrid assays, immunoprecipitation experiments, and co-purification from a single-stranded DNA agarose column. The yeast two-hybrid system was used to map the LEF-3 interaction domain to the N-terminal 165 amino acids of LEF-3. Deletion analysis of P143 failed to reveal a delimited interaction domain. C-terminal deletions of LEF-3 containing amino acids 1 to 165 were unable to interact with full length LEF-3, indicating that the interaction of LEF-3 with itself (trimerization) is not required for the interaction between LEF-3 with P143. / Graduation date: 1998

1S801 is a transposable element isolated from Pseudomonas syringae pathovar (pv.) phaseolicola, the causal agent of halo blight of bean. Fragments of the element are present in multiple copies on an indigenous plasmid, pMMC7105, of strain LR781, and have been implicated as sites of homologous recombination leading to imprecise excision of the chromosomally integrated form of the plasmid. The element, which has been completely sequenced, is 1512 base pairs in length and is unusual among transposable elements in that it does not have direct or inverted repeats in its termini. The terminal regions of the element were uncoupled from the two major open reading frames, and trans-acting activity of the putative transposase was demonstrated in Escherichia coli (recA). An alignment of the sequences of thirteen insertions defined the precise borders of the element, and demonstrated that it does not duplicate its targets upon insertion. The target specificity of IS801 is similar to, but more degenerate than, the target specificity of two transposable elements to which it is closely related, IS91 and IS1294. The consensus derived from the aligned target sequences is G/C-A/G-A-C/G, and the target tetramer is found immediately adjacent to the right terminus of the element upon transposition. IS91 was demonstrated to mobilize 1S801, but not with the specificity characteristic of 1S801. The structure of 1S801 and the characteristics of IS91-activated transposition of 1S801 are discussed in light of a proposed model for IS91 transposition, and it is suggested that 1S801 could have been derived from IS91 by a modification of its left end. Remnants of IS801 are present near avirulence genes of various P. syringae pathovars, suggesting that the element has been involved in genetic rearrangements in the vicinity of these loci. / Graduation date: 1996

The studies presented here use the B- to Z-DNA transition and Z-DNA crystallization as model systems to determine the contribution of solvent interactions to macromolecular structures. The substituent groups of naturally occurring and modified nucleotide bases affect the hydration and thus the stability of right-handed B-DNA and left-handed Z-DNA. The free energy for alternating pyrimidine-purine sequences in the B- and Z-conformations are quantitated as solvent free energies (SFE) from their solvent-accessible surfaces. The effect of methylation of the C5 carbon of pyrimidine bases on the stability of Z-DNA was analyzed in comparisons of d(TA) versus d(UA) dinucleotides. In the minor groove, the N2 amino group of purine bases was examined by comparing the stability of d(CG) versus d(CI), and d(TA) versus d(TA') dinucleotides as Z-DNA. These comparisons correctly accounted for the observed effects of the major and minor groove substituent groups on the relative stability of Z-DNA. These predictions were confirmed by comparing the amount of salt required to crystallize various hexanucleotides as Z-DNA. The relative contribution of solvent and steric interactions in DNA assembly were studied crystallographically using an asymmetric hexanucleotide which assumes two discrete and discernible orientations in the crystal lattice. How this sequence orients in the lattice is a direct measure of the DNA-DNA interactions at the surface of the crystal. These interaction free energies were directly correlated with differences in solvation for 5 sequences where there was effectively no differences in steric interactions for the two orientations. In the singular case where a large steric clash was expected, the orientation was in fact determined by this interaction. The comparisons indicate that both solvent and van der Waals interactions are significant in DNA assembly with van der Waa ls interactions dominating in situations with large steric interactions. The analyses of the B- to Z-DNA transition and Z-DNA crystallization based on SFEs support the significance of solvent interactions in determining the structure and assembly of macromolecules. / Graduation date: 1995

Graduation date: 1994

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 97-105). Also available in electronic version.