PARP-1 is the most abundantly expressed member of a family of proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins. Herein, we describe previously uncharacterized nucleosome binding properties of PARP-1 that promote the formation of compact, transcriptionally repressed chromatin structures. PARP-1 binds in a specific manner to nucleosomes and modulates chromatin structure through NAD+-dependent automodification, without modifying core histones or promoting the disassembly of nucleosomes. The automodification activity of PARP-1 is potently stimulated by nucleosomes, causing the release of PARP-1 from chromatin. The NAD+-dependent activities of PARP-1 are reversed by PARG, a poly(ADP-ribose) glycohydrolase, and are inhibited by ATP. In vivo, PARP-1 incorporation is associated with transcriptionally repressed chromatin domains that are spatially distinct from both histone H1-repressed domains and actively transcribed regions. Thus, PARP-1 functions both as a structural component of chromatin and a modulator of chromatin structure through its intrinsic enzymatic activity.

In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and aquatic ecosystems. We then relate the abundance of Shiga toxin in the environment to work that demonstrates that the genetic mechanisms underlying the production of Shiga toxin genes are modified and embellished beyond the classical microbial gene regulatory paradigms in a manner that apparently “fine tunes” the trigger to modulate the amount of toxin produced. Last, we highlight several recent studies examining microbe/protist interactions that postulate an answer to the outstanding question of why microbes might harbor and express Shiga toxin genes in the environment.

No abstract

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89 -91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH 2 group on the purine bases at the binding site center. The N2-NH 2 group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH 2 group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.It is well established that the sequence-specific binding of proteins to DNA involves specific contacts between DNA bases and protein side chains in a process known as "direct-readout." The precise alignment of DNA and protein can be specifically modulated by the sequence of DNA bases in the binding site that are not directly contacted by the protein. This phenomenon is known as indirect readout. In indirect readout, the affinity or specificity of a protein-DNA complex depends on sequence-dependent alterations in the conformation and/or conformational flexibility of the noncontacted bases in the DNA site.Since sequence-dependent DNA structural differences play a role in mediating protein-DNA complex formation, this implies that a protein binds only to a distorted DNA conformation. Noncontacted bases may be envisioned to affect the formation of a protein DNA complex in any of three ways: 1) altering the deformability of DNA; 2) altering the structure of the DNA in the bound complex, thereby changing the strength of particular protein-DNA contacts; or 3) altering the structure of the unbound DNA, thereby eliminating or requiring the imposition of energetically costly large-scale DNA conformational changes. Although indirect readout is part of the sequence recognition mechanism of many sequence-specific DNA binding proteins, little is known about the physical basis of how base sequence and/or the functional groups on the bases contribute to the sequence dependence of such DNA deformations. In addition, the mechanisms by which sequence-dependent differences in DNA conformation influence the strength or specificity of protein-DNA complexes is also unclear.434 repressor does not contact the functional groups on bases at the center of its binding site (1, 2; see also Fig. 1...

Shiga toxin (Stx) genes produce proteins that are pathogenic to humans, leading to severe gastrointestinal illness. This work focuses on examining the abundance and distribution of stx genes in relation to common microbial indicators in beach water and streams in the vicinity of Presque Isle State Park in Erie, PA. By use of quantitative PCR, the relative abundance levels of stx DNA in over 700 samples in the sampling area were determined. The results demonstrate that the abundance and distribution of stx genes are variable and do not correlate with the abundance of Escherichia coli bacteria, enterococci, or viral particles. These results suggest that microbial indicators of water quality are not adequate in predicting the occurrence of organisms that harbor stx genes and highlight the need for standardized pathogen-specific detection protocols for waters utilized for recreational swimming.