Mechanism of DNA flexibility enhancement by HMGB proteins

Abstract

The mechanism by which sequence non-specific DNA-binding proteins enhance DNA flexibility is studied by examining complexes of double-stranded DNA with the high mobility group type B proteins HMGB2 (Box A) and HMGB1 (Box A+B) using atomic force microscopy. DNA end-to-end distances and local DNA bend angle distributions are analyzed for protein complexes deposited on a mica surface. For HMGB2 (Box A) binding we find a mean induced DNA bend angle of 78 degrees, with a standard error of 1.3 degrees and a SD of 23 degrees, while HMGB1 (Box A+B) binding gives a mean bend angle of 67 degrees, with a standard error of 1.3 degrees and a SD of 21 degrees. These results are consistent with analysis of the observed global persistence length changes derived from end-to-end distance measurements, and with results of DNA-stretching experiments. The moderately broad distributions of bend angles induced by both proteins are inconsistent with either a static kink model, or a purely flexible hinge model for DNA distortion by protein binding. Therefore, the mechanism by which HMGB proteins enhance the flexibility of DNA must differ from that of the Escherichia coli HU protein, which in previous studies showed a flat angle distribution consistent with a flexible hinge model.

Figure 1.

(a) 1 μm × 1.2 μm image of bare pBR322 dsDNA (contour length 1.5 µm). (b) 3D view of a single dsDNA molecule from (a). (c) Diagram showing local DNA bend angle calculation based on the angle between two adjacent line segments drawn between three adjacent points (step size l0 nm) along the contour.

Figure 2.

(a) The distribution of bare dsDNA contour lengths and its Gaussian fit. (b) The local bend angle distribution for bare dsDNA (segment step size 10 nm) and Gaussian fit.

Figure 3.

Local DNA bend angle versus segment step size. A linear fit is shown for the intermediate range of step sizes (from 24 to 66 nm).

Figure 4.

(a) Images for three individual HMGB protein–DNA complexes. (b) Height information is provided for the two lines shown on the image: slice 1 crosses the only the bare dsDNA. The peak height is no more than 0.1 nm. Slice 2 crosses two bound HMGB proteins and the peak heights are more than 0.55 nm. (c) 3D view of the single DNA molecule selected from (a) showing bound proteins as spikes.

Figure 5.

Distribution of protein-site bend angles for (a) HMGB (Box A) and (b) HMGB (Box A+B). The distribution shows an average induced DNA bend angle of 78° with a SD of 23° for HMGB (Box A) protein. HMGB (Box A+B) induces an average DNA bend angle of 67° with a SD of 21°.

Figure 6.

Distribution of the number of binding proteins per dsDNA molecule for (a) HMGB2 (Box A) and (b) HMGB1 (Box A+B).