Fig 13.

Mus81 is solubilized from the chromatin pellet after sonication. (A) After cell lysis of cross-linked cells (WDHY2456; same results for WDHY3267 are not shown), whole-cell extract (WCE), soluble (SOL), and pellet (PEL) fractions were collected. The pellet fraction was washed three times, first in lysis buffer (W1) and then with an additional two washes in lysis buffer (W2S and W3S) or DNase buffer (W2D and W3D) for sonication and DNase experiments, respectively. The pellet fraction was then resuspended in either DNase or lysis buffer for the respective treatments with 5 units DNase, a single round of sonication, or four rounds of sonication (10 s at 15% impulse) or no treatment. Posttreatment, the samples were centrifuged into a soluble (Psol) and pellet (Ppel) fractions. All samples were precipitated with trichloroacetic acid, and protein concentrations were determined by Bradford analysis. Twenty-five micrograms of protein or the entire volume from each fraction was loaded on a 4 to 20% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose and blotted using anti-histone H3 and anti-Mus81 antibodies. (B) DNA was extracted from the samples for panel A at the step of posttreatment for the total pellet sample and then the soluble and pellet fractions to examine DNA distribution. (C) The total amount of DNA was calculated for each sample to analyze the DNA after the respective treatments.