Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.

Nobile, Carlo; Nickol, J; Martin, R G

doi:10.1128/mcb.6.8.2916

Cited by 33 publications

(12 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our interpretation is that a histone octamer can associate with the curved DNA in a stable way despite there being >146 bp of DNA in the resulting nucleosome. Precedents exist for the asymmetric association of short linear DNA with a histone core (25,26). Moreover, DNA fragments as small as 80 bp can remain stably incorporated into a nucleosome (27).…”

Section: Resultsmentioning

confidence: 99%

Initiation of transcription on nucleosomal templates.

Wolffe

Drew

1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We describe a simple method that uses curved DNAs to move nucleosomes relative to a sequence of interest.With this method, small changes in the association of a T7 RNA polymerase promoter with a nucleosome are shown to lead to significant changes in transcription efficiency.The assembly of nucleosomes onto promoter sequences in vitro is inhibitory to transcription initiation (1-4). Although the exact reason for this repression is unknown, DNA wound around the histone core is believed to be inaccessible to DNA-binding proteins such as RNA polymerases (5). In contrast, once transcription is initiated, RNA polymerase can progress through a nucleosomal template (2, 6, 7).Evidence from in vivo experiments suggests that precisely positioned nucleosomes may be important in repressing gene transcription (8-11). It is not known precisely which DNA sequences need to be incorporated into a nucleosome to prevent the interaction of other, sequence-specific DNAbinding proteins. Linker DNA, which separates nucleosomes, is accessible to RNA polymerase (12, 13). However, the length oflinker DNA that is required for RNA polymerase to bind and initiate transcription has not been defined.In this study, we have used the preference of nucleosomes to bind to curved DNAs (14,15) to alter the position of a nucleosome relative to the promoter for bacteriophage T7 RNA polymerase. Small changes in the position of promoter sequences with respect to the boundaries of a nucleosome are shown to have large effects on transcription efficiency. MATERIALS AND METHODSSynthetic DNA Curves. Sequence-directed bending of DNA has been detected by electrophoretic and electron microscopic analysis (16)(17)(18). These experimental observations led to the development of structural models for bent DNA, allowing the design and synthesis of extended DNA curves (19,20). Intrinsic bending of DNA molecules results from local structural polymorphism in regions of homopolymeric dA-dT, especially when these sequences phase with the helical periodicity of DNA [i.e., 10.5 base pairs (bp) in solution].The five different DNA double helices listed below were cloned into the BamHI site of a plasmid containing the T7 promoter and a chloramphenicol acetyltransferase (CAT) gene (Fig. 1). This plasmid (T7-CAT) is a derivative of pUC18 (5081-bp total length; (C. Collis, P. L. Molloy, G. W. Both and H.R.D., unpublished work). Clones were selected that had a single orientation with the BamHI site recreated to the right of the curved DNA insert. In each of the DNA molecules an additional two base pairs were inserted that adjusted the periodicity of a repeat (GGCCTAAAT) to 10.5 bp per helical turn of DNA. These base pairs are marked with asterisks. _3Individual fragments have the repeats staggered by two bases relative to each other. This alters the inclination of the curved DNA to the promoter. The distance from the beginning of the curve to the T7 promoter also changes, a fact we make use of later.In order to radiolabel these DNA fragments, 10 jig of plasmid DNA was res...

show abstract

Section: Resultsmentioning

confidence: 99%

Initiation of transcription on nucleosomal templates.

Wolffe

Drew

1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…It is likely that the formation of a cruciform would occur only if the (AT)24 tract was positioned in linker DNA (12,52,53) (Fig. 3 and 8).…”

Section: Discussionmentioning

confidence: 99%

Evidence for torsional stress in transcriptionally activated chromatin.

Leonard

Patient

1991

Mol. Cell. Biol.

View full text Add to dashboard Cite

The existence of torsional stress in eukaryotic chromatin has been controversial. To determine whether it could be detected, we probed the structure of an alternating AT tract. These sequences adopt cruciform geometry when the DNA helix is torsionally strained by negative supercoiling. The single-strand-specific nuclease P1 was used to determine the structure of an alternating AT sequence upstream of the Xenopus P-globin gene when assembled into chromatin in microinjected Xenopus oocytes. The pattern of cleavage by P1 nuclease strongly suggests that the DNA in this chromatin template is under torsional stress. The cruciform was detected specifically in the most fully reconstituted templates at later stages of chromatin assembly, suggesting that negative supercoiling is associated with chromatin maturation. Furthermore, the number of torsionally strained templates increased dramatically at the time when transcription of assembled chromatin templates began. Transcription itself has been shown to induce supercoiling, but the requisite negative supercoiling for cruciform extrusion by (AT). in oocytes was not generated in this way since the characteristic P1 cutting pattern was retained even when RNA polymerase elongation was blocked with a-amanitin. Thus, torsional stress is associated with transcriptional activation of chromatin templates in the absence of ongoing transcription.A role for DNA supercoiling in gene activation is suggested by the observations that initiation of transcription is accompanied by local unwinding of DNA (71) and that such strand separation is facilitated by negative DNA supercoiling (reviewed in reference 39). Furthermore, both the formation of stable initiation complexes and the number of initiated transcripts have been shown to be greater on negatively supercoiled than on relaxed templates in vitro (64). However, although superhelicity has been shown to influence transcription of a number of prokaryotic genes in vivo (62), the situation in eukaryotes is much less clear.Both prokaryotic (88) and eukaryotic (59) chromosomes appear to be organized as a series of independently supercoiled loops or domains. In prokaryotes, the supercoiling is only partially constrained by association with protein (11), so that partitioning between the writhing and twisting of the DNA helix can still take place. Two sources of supercoiling in bacteria are DNA gyrase, which generates negative supercoils (23), and the process of transcription, which introduces both negative and positive supercoils (90). The overall level of unconstrained supercoiling appears to be the result of the combined effects of transcription, DNA gyrase, and the relaxing enzyme DNA topoisomerase I (63,65,78 into chromatin in a site-specific manner. However, there are a number of other potential sources of local supercoiling in eukaryotic chromatin, including the breakdown of higherorder structure (57), the loss of nucleosomes from nucleasehypersensitive sites (32, 69), unfolding of the nucleosome itself (13, 61), histone acetylation (54),...

show abstract

“…Nucleosome structure is resistant to microccocal nuclease (MNase) digestion, leaving a footprint of about 150bp that reflects the position of a nucleosome (7). Therefore, determining the boundaries of these footprints can indicate the positions of nucleosomes in the genome.…”

Section: Introductionmentioning

confidence: 99%