Figure 3. The N-terminus of Chd1 mediates interactions with the DNA-binding domain.

(A) Sites covalently linked by the crosslinker BS³ were identified by mass spectrometry and are represented graphically using a plot generated with Xvis (Grimm et al., 2015). Thick red and green lines indicates crosslinks between the N-terminal and C-terminal regions. (B) Size exclusion chromatography (SEC) elution profiles of selected Chd1 fragments. Normalised elution profile of DBD (1009–1305) in purple, chromo-helicase with intact N-terminal region (1–1010) in green, and Chd1 (1–1305) in blue. The elution profile for a 3:1 mixture of the DBD with the chromoATPase (1–1010 + 1009–1305) is shown in orange. The chromoATPase elutes at low volume consistent with the formation of a complex between the N- and C-terminal fragments. (C) Normalised elution profile of Chromo-helicase missing the N-terminal 133 amino acids is coloured green. Other profiles are similar to as described in Figure 3A. Loss of the N-terminal 133 amino acids prevents association with the C-terminal DBD. (D) Similar SEC experiment performed using a Chd1 N-terminal fragment that includes the internal deletion △ 57–88. This also prevents association between the two halves of the protein.

DOI: http://dx.doi.org/10.7554/eLife.22510.013

Figure 3—figure supplement 1. Protein composition of SEC peaks.

Fractions from size exclusion column chromatography as illustrated in Figure 3B. (A) Chd1 1009–1305 elutes as a peak centred on fraction 10 (volume 2.7 ml), (B) Chd1 1–1010 elutes in fraction 5 (2.2 ml), (C) When a mixture of Chd1 1–1010+Chd1 1009–1305 is resolved by SEC, Chd1 1009–1305 elutes one fraction earlier, fraction 4 (2.1 mL), at a similar location to intact Chd1 1–1305 (D). A proportion of Chd1 1009–1305 elutes in higher molecular weight fractions consistent with dynamic equilibrium with the N-terminal fragment. Molecular weight markers are loaded in the left hand lane of each gel.

DOI: http://dx.doi.org/10.7554/eLife.22510.014