Requirements for chromatin reassembly during transcriptional downregulation of a heat shock gene in Saccharomyces cerevisiae.

Heat shock genes respond to moderate heat stress by a wave of transcription. The induction phase is accompanied by the massive eviction of histones, which later reassemble with DNA during the ensuing phase of transcription downregulation. In this article, we identify determinants of this reassembly throughout the heat shock protein 104 gene (HSP104) transcription unit. The results show that, although histone H3 lacking amino acids 4-30 of its N-terminal tail (H3Delta4-30) is normally deposited, reassembly of H3Delta4-40 is obliterated with an accompanying sustained transcription. On mutation of the histone chaperones Spt6p and Spt16p, but not Asf1p, reassociation of H3 with DNA is compromised. However, despite a lasting open chromatin structure, transcription ceases normally in the spt6 mutant. Thus, transcriptional downregulation can be uncoupled from histone redeposition and ongoing transcription is not required to prevent chromatin reassembly

Distamycin inhibition of topoisomerase I-DNA interaction: a mechanistic analysis.

Inhibition of eukaryotic DNA topoisomerase I by the minor groove binding ligand, distamycin A, was investigated. Low concentrations of the ligand selectively prevented catalytic action at a high affinity topoisomerase I binding sequence. A restriction enzyme protection assay indicated that the catalytic cycle was blocked at the binding step. Distamycin binding sites on DNA were localized by hydroxyl radical footprinting. A strongly preferred site mapped to a homopolymeric (dA).(dT)-tract partially included in the essential topoisomerase I binding region. Mutational elimination of the stable helix curvature associated with this ligand binding site demonstrated that (i) the intrinsic bend was unessential for efficient binding of topoisomerase I, and (ii) distamycin inhibition did not occur by deformation of a stable band. Alternative modes of inhibition are discussed