Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

Almouzni, Geneviève; Méchali, Marcel

doi:10.1002/j.1460-2075.1988.tb03334.x

Cited by 112 publications

(61 citation statements)

References 61 publications

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“…Those rearrangements may arise from the replication defects discussed above and/or may be linked to chromatin alterations. Indeed, in Xenopus extracts, Top1 is the dominant actor in chromatin assembly (36). Our micrococcal nuclease analyses showed comparable nucleosomal patterns in siTop1 cells and their normal counterpart (Fig.…”

Section: Discussionsupporting

confidence: 58%

Nonclassic Functions of Human Topoisomerase I: Genome-Wide and Pharmacologic Analyses

et al. 2007

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The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced f5-fold and Top2A compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone ;-H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed L-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function.

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Section: Discussionsupporting

confidence: 58%

Nonclassic Functions of Human Topoisomerase I: Genome-Wide and Pharmacologic Analyses

et al. 2007

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“…Either topo I or topo II can provide the relaxation activity required for replication-independent nucleosome assembly in budding yeast (Garinther and Schultz, 1997). Furthermore, nucleosome assembly in Xenopus egg extracts have suggested that in this system DNA relaxation is largely performed by topo I (Almouzni and Mechali, 1988). During chromatin assembly, histones are deposited on naked DNA and ( þ ) helical tension rises locally to balance the (À) writhe of DNA in nucleosomes.…”

Section: Discussionmentioning

confidence: 99%

Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA

Salceda¹,

Fernández²,

Roca

2006

EMBO J

101

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Eukaryotic topoisomerases I and II efficiently remove helical tension in naked DNA molecules. However, this activity has not been examined in nucleosomal DNA, their natural substrate. Here, we obtained yeast minichromosomes holding DNA under ( þ ) helical tension, and incubated them with topoisomerases. We show that DNA supercoiling density can rise above þ 0.04 without displacement of the histones and that the typical nucleosome topology is restored upon DNA relaxation. However, in contrast to what is observed in naked DNA, topoisomerase II relaxes nucleosomal DNA much faster than topoisomerase I. The same effect occurs in cell extracts containing physiological dosages of topoisomerase I and II. Apparently, the DNA strand-rotation mechanism of topoisomerase I does not efficiently relax chromatin, which imposes barriers for DNA twist diffusion. Conversely, the DNA cross-inversion mechanism of topoisomerase II is facilitated in chromatin, which favor the juxtaposition of DNA segments. We conclude that topoisomerase II is the main modulator of DNA topology in chromatin fibers. The nonessential topoisomerase I then assists DNA relaxation where chromatin structure impairs DNA juxtaposition but allows twist diffusion.

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“…Here, we focus on ATP-independent processes that dominate early stages of chromatin assembly and can be studied in extracts depleted of ATP (16,21). At low force, reduction of extension in time occurs as nucleosomes are formed, because the Ϸ150 bp wrapped around each nucleosome reduces the overall fiber extension by a length l Ϸ50 nm per nucleosome (10,14,16).…”

Section: Nucleosome Dynamics In Xenopus Egg Extractsmentioning

confidence: 99%

“…Nucleosomes can adsorb at any sterically permitted location on the DNA at rate r on . Adsorption is presumed to result in formation of a chromatosome covering 168 bp of DNA (histone octamer ϩ linker histone) (21). We consider this, less a small correction for thermal fluctuations, to be the experimentally measurable change in end-to-end distance.…”

Section: Theorymentioning

confidence: 99%

Nucleosome hopping and sliding kinetics determined from dynamics of single chromatin fibers in Xenopus egg extracts

Padinhateeri

Yan

Marko

2007

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

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Chromatin function in vivo is intimately connected with changes in its structure: a prime example is occlusion or exposure of regulatory sequences via repositioning of nucleosomes. Cell extracts used in concert with single-DNA micromanipulation can control and monitor these dynamics under in vivo-like conditions. We analyze a theory of the assembly-disassembly dynamics of chromatin fiber in such experiments, including effects of lateral nucleosome diffusion (''sliding'') and sequence positioning. Experimental data determine the force-dependent on-and off-rates as well as the nucleosome sliding diffusion rate. The resulting theory simply explains the very different nucleosome displacement kinetics observed in constant-force and constant-pulling velocity experiments. We also show that few-piconewton tensions comparable to those generated by polymerases and helicases drastically affect nucleosome positions in a sequence-dependent manner and that there is a long-lived structural ''memory'' of force-driven nucleosome rearrangement events.chromatin assembly ͉ chromatin disassembly I n the nucleus, chromatin undergoes continual structural rearrangement. Chromatin fibers have been observed to undergo large-scale diffusion-like motions (1, 2) and rapid local motions (3), possibly caused by the action of processive enzymes such as nucleic acid polymerases and helicases. At smaller scales, fluorescence recovery after photobleaching studies in vivo have shown histones to be mobile to some degree (4). Both large-scale conformational and nucleosomal rearrangements are biologically important, e.g., through their influence on gene regulation (5, 6).These in vivo results are complemented by biochemical experiments indicating that DNA can be transiently released from nucleosomes (7,8) and recent DNA-pulling experiments that show nucleosome disruption by forces ranging from a few to tens of piconewtons (9-12). Single-nucleosome or single-chromatinfiber experiments carried out in protein-free buffers provide useful quantifications of histone-DNA interaction strengths and force-driven opening rates. However, affinities and rates obtained from studies of isolated fibers may be very different relative from those occurring in vivo because of the very high levels of chromatin-acting enzymes found in the cell. This issue can be addressed by combining cell extracts with single-molecule methods for reading out folding and unfolding of protein-DNA complexes in real time, which permits observation of singlechromatin fiber dynamics under conditions close to those found in vivo (10,(13)(14)(15)(16).Here, we present a theory of chromatin fiber dynamics in Xenopus egg extracts, based on assembly, displacement, and lateral diffusion (''sliding'') of nucleosome units. Recent experimental data for sequence dependence of nucleosome affinities (17), combined with measurements of single-chromatin fiber assembly and disassembly in Xenopus egg extracts, constrain the theory sufficiently to determine force-dependent nucleosome on-and off-rates, the seque...

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Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

Cited by 112 publications

References 61 publications

Nonclassic Functions of Human Topoisomerase I: Genome-Wide and Pharmacologic Analyses

Nonclassic Functions of Human Topoisomerase I: Genome-Wide and Pharmacologic Analyses

Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA

Nucleosome hopping and sliding kinetics determined from dynamics of single chromatin fibers in Xenopus egg extracts

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