Sequence-related structural DNA anomalies affect restriction enzyme activity and DNA polymerase I binding

DNA serves as a target molecule for several types of enzymes and may assume a wide variety of structural motifs depending upon the local sequence. The BssHII restriction site (GC)3 resides in a 9bp region of alternating pyrimidine and purine residues within the &phis;X174 genome. Such sequences are known to demonstrate non-canonical helical behavior under the appropriate conditions. The kinetics of BssHII cleavage was investigated in supercoiled and linear plasmid DNA, and in a 323bp DNA fragment obtained via amplification of &phis;X174. The rate of enzyme cleavage was enhanced in the supercoiled form and in the presence of 50μM cobalt hexamine. Similarly, cobalt hexamine was also found to enhance TaqI activity directly adjacent to the (GC)3 region. ^ Initial DNA polymerase I binding studies (including a gel mobility shift assay and a protection assay) indicated a notable interaction between DNA polymerase I and the BssHII site. An in-depth study revealed that equilibrium binding of DNA polymerase I to the T7 RNA polymerase promoter was comparable to that of the (GC)3 site, however the strongest interaction was observed with a cruciform containing region. Increasing the ionic strength of the solution environment, including the addition of DNA polymerase I reaction buffer significantly decreased the equilibrium dissociation constant values. ^ It is suggested that the region within or around the BssHII site experiences a conformational change generating a novel structure under the influence of supercoiled tension or 50μM cobalt hexamine. It is proposed that this transition may enhance enzyme activity and binding by providing an initial enzyme-docking site—the rate-limiting step in restriction enzyme kinetics. The high binding potential of DNA polymerase I for each of the motifs described, is hypothesized to be due to recognition of the structural DNA anomalies by the 3′–5′ exonuclease domain. ^

Buried voices: Genres and types of female authorship during the Spanish American colonial period (sixteenth to eighteenth centuries)

This dissertation analyzes various types of non-canonical texts authorized by women from a wide spectrum of classes and races in the Spanish colonies. The female voice, generally absent from official colonial documents of the sixteenth, seventeenth and eighteen centuries, left a gap in the complex subject of women's history and social participation. Through the study of personal letters, autobiographies, journals, court documents, inquisitorial transcripts, wills and testaments, edicts, orders, proclamations and posters, that voice is recovered. Thus, the Indigenous, Spaniards and African women and their descendants who lived during this period left their written legacy and proof of participation. Beginning with a thorough history of the native woman's interest in writing, this study focuses on how women of all social levels utilized the few means of writing available at their disposal to display a testimonial, critical and sometimes fictional narrative of their surroundings. ^ This investigation concludes that it is necessary to change the traditional image of the passive women of the colonies, subjected to a patriarchal authority and unable to speak or grow on their own. The documents under study, introduced women who were able to self represent themselves as followers of the tradition while at the same time their writings were denying that very same statement. They passed from the private arena to the public one with discourses that confessed their innermost feelings and concerns, challenged the authority of the Inquisitor or the Governor, exposed their sexual freedom and transvestite narratives, successfully developed stratagems that challenged the official ideology of the oppressive religious environment and established their own authority reaching at last the freedom of their souls. ^