Nucleosome unwrapping measurement.

(A) Experimental scheme. The red and green stars represent labelled Cy5 (acceptor) and Cy3 (donor) fluorophores, respectively. Biotin, B, and digoxigenin, D, are used to tether the nucleosome-lambda DNA construct to the surface and the bead, respectively. (B, C, D, E): Representative stretching traces of the outer turn (ED1) for nucleosomes reconstituted from the 601 sequence (B) and from the 601 sequence with containing a mismatch at different positions: on the outer turn (C), at the junction of the outer turn and inner turn (D) and at the inner turn (E). The red and green dots on the DNA bends represent labelled Cy5 and Cy3 fluorophores. The elongated circles enclosing red and green dots represent the ED labeling position. The black diamonds on the DNA bends represent the mismatch position with R18 and R39 on histone=facing minor grooves and R56 on a histone-facing major groove.

Unwrapping force of mismatch-containing nucleosomes is higher for subsequent stretching cycles.

(A) Representative single-molecule stretching traces at two stretching cycles from the sample molecule, probe by the ED1 FRET pair in the 601-R18 nucleosome. (B) Averaging FRET vs. Force for many molecules at the first three stretching cycles (purple) and the subsequent stretching cycles (orange). Histone proteins were expressed in xenopus. The error bars represent S.D. of n = 25 and 11 traces for the first 3 stretching cycles (purple) and for the cycle 5th and the subsequent stretching cycles (orange), respectively.

Enhancement of nucleosome mechanical stability by DNA mismatch.

Average of FRET vs. Force for ED1 probe (A) and ED2 probe (B) for the 601 nucleosome (black) and for the first stretching cycle of the mismatch containing nucleosome 601-R39 (purple). Histone proteins were expressed in xenopus. The error bars represent S.D. of n = 25 and 7 for the ED1 probe of the 601 and 601-R39 nucleosomes (A) and n = 20 and 39 for the ED2 probe of the 601 and 601-R39 nucleosomes (B), respectively.

Mismatch position-dependence of nucleosome unwrapping.

Average of FRET vs. Force for ED1 probe for the 601 nucleosome (black) and the mismatch-containing nucleosome 601-R39 (purple), 601-R18 (blue) and 601-R56 (red). Histone proteins were expressed in xenopus. The error bars represent S.D. of n = 25, 11, 7 and 10 for the 601, 601-R18, 601-R39 and 601-R56 nucleosomes, respectively.

C-C mismatch enhances DNA flexibility.

(A): Single molecule cyclization assay: The DNA construct with 10-nucleotide complementary sticky ends is immobilized on a PEG passivated imaging chamber. DNA looping is induced using the imaging buffer containing 1M NaCl followed by time course TIRF imaging. To calculate the looping time, the fraction of looped molecules (high FRET) as a function of time is fitted to an exponential function, e-t/(looping time) (right panel for one run of experiments). (B, C) Fitted looping time for the right half of the 601 construct without and with mismatches (B) and with the biotin position being moved by 16 nt (C). Error bars represented the S.E.M with n = 3 technical replicates.

DNA flexibility enhancement is dependent on mismatch type.

Looping times for DNA containing a single mismatch (one of eight types each) and an intact DNA without a mismatch. Also shown are ensemble FRET efficiencies (EFRET) from Ref. 6 as a measure of DNA buckling for the same type of mismatch.

Unwrapping of yeast vs. xenopus reconstituted nucleosomes:

Average of FRET vs. Force for nucleosomes reconstituted from xenopus (red) vs yeast (black and gray) histone proteins with DNA labeled by outer turn probes ED1 (A), ED2 (B) and inner turn probe INT (C). The error bars represent S.D. of n = 17 (Xenopus) and 5 (Yeast) nucleosomes with the ED1 probe (A), n = 20 (Xenopus), 6 (Yeast – strong) and 4 (Yeast-weak) nucleosomes with the ED2 probe (B), and n = 22 (Xenopus) and 6 (Yeast) nucleosomes with the INT probe (C), respectively.