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We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.

Observations that ␤-sheet proteins form amyloid fibrils under at least partially denaturing conditions has raised questions as to whether these fibrils assemble by docking of preformed ␤-structure or by association of unfolded polypeptide segments. By using ␣-helical protein apomyoglobin, we show that the ease of fibril assembly correlates with the extent of denaturation. By contrast, monomeric ␤-sheet intermediates could not be observed under the conditions of fibril formation. These data suggest that amyloid fibril formation from apomyoglobin depends on disordered polypeptide segments and conditions that are selectively unfavorable to folding. However, it is inevitable that such conditions often stabilize protein folding intermediates.

Abbreviations used in this paper: CENP, centromere protein; FCS, fl uorescence correlation spectroscopy; mRFP, monomeric red fl uorescent protein; PCNA, proliferating cell nuclear antigen.The online version of this paper contains supplemental material.

Can a transcriptional activator known to bend DNA be functionally replaced by a sequence‐directed bend in Escherichia coli? To investigate this question, a partially truncated promoter was used, deleted of its ‐35 region and of its CRP binding site, leaving only two Pribnow boxes as functional elements. Synthetic and naturally occurring curved DNA sequences introduced upstream from these elements could restore transcription at either one of the two natural starts. Some of these hybrid promoters turned out to be more efficient than the CRP activated wild‐type gal promoter in vivo. Control experiments performed with very similar sequences devoid of any curvature produced weak promoters only. Minimal changes in the location of the centre of curvature or perturbation in the amount of curvature strongly affected the level of expression. No significant stimulation of transcription could be detected in vitro. Furthermore, both gal P1 and P2 starts could be activated in vivo but also in vitro via a properly positioned CRP binding site. This partial analogy suggests that bending induced by the cAMP‐CRP complex upon binding to its site may be biologically relevant to the mechanism of transcriptional activation.

SUMMARY In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms in mammalian cells. Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50 -80%) are highly mobile (recovery halftime: t 1/2 Ϸ 0.9 s; diffusion coefficient: D Ϸ 0.6 -0.7 m 2 s ؊1 ). Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t 1/2 Ϸ 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5-7% of very slow HP1 molecules. The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells. During mitosis 8 -14% of GFP-HP1␣, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1␣ in heterochromatin of mitotic chromosomes. These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle. INTRODUCTIONThe genomic DNA within the eukaryotic nucleus is organized into structurally distinct domains that regulate gene expression and chromosome behavior (Lamond and Earnshaw, 1998). Chromosomes are composed of two types of domains: heterochromatin and euchromatin (Cohen and Lee, 2002;Grewal and Moazed, 2003). Constitutive heterochromatic domains at centromeres and telomeres consist of repetitive DNA and are largely transcriptionally silent. Euchromatin defines the gene-rich and transcriptionally active region of the cell nucleus (Grewal and Elgin, 2002). Heterochromatin mediates many diverse functions in the cell nucleus, including centromere function, gene silencing, regulation of gene expression, and nuclear organization. At centromeres, heterochromatin is required for proper sister chromatid cohesion and mitotic segregation (Bernard et al., 2001;Peters et al., 2001; Nonaka et al., 2002;Hall et al., 2003). Smaller heterochromatin domains are involved in epigenetic regulation of gene expression during development and cellular differentiation (Cavalli, 2002;Grewal and Moazed, 2003). Heterochromatic inactivation of one of the two X chromosomes, giving rise to the Barr body, is essential in dosage compensation in somatic cells of female mammals (Avner and Heard, 2001). The link between heterochromatin and transcriptional silencing has been firmly established by detailed analysis of the phenomenon PEV (position effect variegation), in which a gene is silenced by positioning it abnormally close to heterochromatin (Wallrath and Elgin, 1995).The establishment of heterochromatin requires the physical coupling of histone-modifying activities and structural proteins at specific genomic sites (Richards and Elgin, 2002). The "histone code" hypothesis predicts tha...