No description available.

No description available.

In sperm chromatin, DNA exists in a highly condensed state reaching a final volume roughly twenty times that of a somatic nucleus. For the vast majority (>90%) of sperm DNA in mammals, somatic-like histones are first replaced by transition proteins which in turn are replaced by arginine-rich protamines. This near crystalline organization of the DNA in mature sperm is thought crucial for both the transport and protection of genetic information since all DNA repair mechanisms are shut down. Recent studies show that increased DNA damage is linked to dysfunctions in replacing histones with protamines resulting in mispackaged DNA. This increased DNA damage correlates not only to infertility but also impacts normal embryonic development. This damage is currently poorly characterized, but is known to involve oxidative base damage by reactive oxygen species (ROS). Using a variety of biophysical methods, the effect of DNA condensation by polycations on the on free radical access and DNA damage in the packaged state was investigated. In Chapter 2, gel electrophoresis was used to quantify the ability of free radicals to damage both unpackaged and packaged DNA. DNA condensed by polycations shows significantly reduced levels of indirect damage from exposure to free radicals. Combining previous work on packaging density, it is also shown that differences in the packaged state, even by a few Angstroms, can result in significantly different degrees of damage to the DNA. In Chapter 3, we investigate the effects of protamine concentration on the ability to condense and protect DNA. Insufficient protamination is known to be a potential source of protamine dysfunction in mammalian sperm chromatin. Using gel retardation assays and UV-Vis studies, we examined the ability for DNA to condense with protamine at varying nitrogen to phosphate (N:P) charge ratios. Initial results on damage as a function of N:P are also discussed. Future work will more quantitatively determine the interrelationship between DNA packaging densities and the resulting accessibility of DNA to reactive oxygen species (ROS).

published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

No description available.

No description available.

The LexA repressor of Escherichia coli represses a set of genes that are expressed in response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires RecA protein. Under physiological conditions in vitro, RecA-dependent cleavage also occurs. At alkaline pH, however, the specific cleavage reaction occurs spontaneously without RecA, a reaction which is termed autodigestion. The LexA repressor is, therefore, thought to cleave itself with RecA acting to stimulate autodigestion. A set of lexA (Ind⁻) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function were isolated. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction. Many of the mutant proteins were purified in order to further characterize their properties in both autodigestion and RecA-mediated cleavage. All of these mutant proteins are found to be deficient in both cleavage reactions. A mutant protein, replacing Lys-156 to Arg, requires a higher pH condition than the wild-type protein does for both cleavage reactions. The results suggest that deprotonation of Arg-156, and by inference Lys-156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage; and that in the latter reaction RecA acts to reduce the pKa of Lys-156, allowing efficient cleavage of wild-type repressor under physiological conditions. Finally, several mutant proteins affecting amino acids around the cleavage site and the proposed nucleophile in the cleavage reaction (Ser-119) could not efficiently act as a competitive inhibitor in the RecA-mediated cleavage of wild-type repressor, presumably because they affect RecA binding.