Histone‐ and DNA sequence‐dependent stability of nucleosomes studied by single‐pair FRET

Abstract

Opening of the nucleosome structure is essential for accessing genomic DNA. To study the mechanism of this process, we monitor the distance between various fluorescently labeled positions on mononucleosomes by single-molecule F€ orster resonance energy transfer (FRET). Here, we compare nucleosomes reconstituted from recombinant mouse, Xenopus, and yeast histones. As DNA sequences we compared, the effect of 5S rDNA, MMTV-B sequence, and Widom 601 DNA. The stability, as measured by the salt concentration at the opening transition midpoint, is lowest for yeast, followed by Xenopus and mouse. The 601 DNA sequence builds much more stable nucleosomes and the distribution of FRET efficiencies is narrower than for those reconstituted on 5S rDNA or MMTV-B sequences. The opening pathway through an intermediate state, as found for Xenopus histones, could be verified for the mouse and yeast systems and for the different DNA sequences, suggesting a general mechanism for accessing nucleosomal DNA.

Asymmetric Unwrapping of Nucleosomes under Tension Directed by DNA Local Flexibility

“…In order to obtain clearly interpretable data on local nucleosome dynamics we chose the nucleosome positioning sequence 601 (Lowary and Widom, 1998), which has been used for previous high resolution single molecule studies (Bintu et al, 2011,2012; Böhm et al, 2011; Brower-Toland et al, 2002; Deindl et al, 2013; Gansen et al, 2009; Hall et al, 2009; Hodges et al, 2009; Kruithof and van Noort, 2009; Mack et al, 2012; Mihardja et al, 2006; North et al, 2012; Sheinin et al, 2013; Shundrovsky et al, 2006; Sudhanshu et al, 2011; Tóth et al, 2013). A nucleosome was anchored to a PEG-coated glass surface on one end of the DNA and pulled via a λ-DNA tethered to the other end by an optical trap (Figure 1A).…”

“…These mechanical properties of DNA are affected by sequence composition and a variety of modifications (Hagerman, 1988; Mirsaidov et al, 2009; Rief et al, 1999; Severin et al, 2011; Vafabakhsh and Ha, 2012; Widom, 2001). The DNA sequence has a profound effect on nucleosome positioning, structure and stability (Chua et al, 2012; North et al, 2012; Tóth et al, 2013; Widom, 2001), but how it affects nucleosome dynamics is poorly understood.…”

A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT

“…To test this hypothesis, we investigated whether a higher affinity nucleosome positioning sequence would rescue the HBRΔ mutant assembly defect. We chose the Widom 601 DNA, an artificial DNA sequence that binds to histones with significantly higher affinity than 5S rDNA (42,43). Mononucleosome assembly reactions showed that FACT can assemble both wild-type and HBRΔ mutant histones onto 601 DNA with comparable efficiency (Supplementary Figure S12A), indicating that the strong histone affinity of 601 DNA can compensate for the assembly defect caused by the HBR deletion.…”

The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres

“…Our evidence that CentO confers translational and rotational phasing on rice CENH3 nucleosomes suggests that regular positioning of cenH3 nucleosomes is advantageous for centromere formation. The 10-bp periodicity of WW dinucleotides is believed to stabilize nucleosomes through reducing the deformation energy required to wrap DNA around them (37), and the strongly positioning Widom 601 sequence derived from α-satellite stabilizes conventional nucleosomes relative to other DNA sequences, such as 5S rDNA (47). These observations suggest that satellites may evolve to stabilize cenH3 nucleosomes against the pulling forces they undergo during chromosome segregation.…”