Overview AFM image of unbound B1-DC381 (a) and 2cer (b) DNA molecules, with indication of the contour length (L) and end-to-end distance (R), and a histogram of the contour length distribution of these molecules with an overlay of a normal distribution curve.

(a) Schematic presentation of a wrapped PepA·carAB complex showing the bending angles (100°–120°) obtained with complexes formed with the two carAB fragments as determined by the Bending Analysis Program using end-to-end distance measurements, and the corresponding calculated bending angles taking wrapping in the opposite orientation into account (240°–260°). (b) Schematic presentation of a wrapped PepA·carAB complex showing the measured angle of 86° between the in- and outgoing DNA arms (tangent method) of complexes formed with the B1-DC381 fragment, the calculated net apparent bending angle (94°) and the corresponding calculated bending angle assuming that the DNA is being wrapped in the opposite direction (266°). (c) AFM image of a globular PepA·DNA complex with indication of the angle between the in- and outgoing DNA arms as measured with the tangent method. (d) Histogram of the bending angle distribution obtained by the tangent method (180°—experimentally measured angle between in- and outgoing DNA arms), with an overlay of a normal distribution curve.

(a) AFM images of two linear 2cer DNA fragments with one and two complexed regions, respectively. (b) Gallery of AFM images of PepA·2cer complexes showing a DNA loop and a single complexed region with a two-lobe shape, from which protrude the DNA arms. (c) AFM images of three similar PepA·2cer complexes presented as surface plots at a 15° viewing angle to emphasize the topography. (d) Histogram of the contour length of the DNA loop formed by bringing together the two cer sites in a wrapped nucleoprotein complex with a two-lobe shape. (e) Histogram of lengths of protruding arms from the globular complexed region showing two peaks, at 33 nm and 59 nm. (f) Histogram of the volume analysis of unbound PepA with an overlay of a normal distribution curve. (g) Histogram of volume measurements of looped 2cer DNA·PepA complexes, showing a bi-modal distribution. (h) AFM image of two 2cer DNA fragments held together by PepA.

Cartoon presentation of PepA·DNA complexes. (a) Front and side view of a nucleoprotein complex with the carAB control region wrapped around a single PepA hexamer. In this model the operator DNA contacts two C-terminal groove segments (colored in yellow) and the adjacent N-terminal DNA binding paths (in red, at the edges of the triangle). The third groove segment is not contacted and runs in a direction opposite to that of the overlying DNA, comprising the PurR binding site (colored in green). (b) Modified Sträter model for the interwrapped synaptic PepA·cer site recombination complex as presented previously by Reijns et al. (20). In this model, two cer sites and the flanking region (colored in green and magenta) are brought together by two PepA hexamers. The DNA is wrapped and contacts the PepA hexamers in such a manner that each cer site contacts one C-terminal-groove and the adjacent N-terminal DNA-binding paths on each PepA hexamer. In this cartoon a single hexameric ArgR molecule (purple triangle) is sandwiched between the two PepA hexamers. (c) Model with a different stoichiometry proposed by Reijns et al. (20) for the interwrapped cer synaptic complex in which two cer sites are brought together in a nucleoprotein complex containing a single PepA hexamer. Two crossings of the DNA are trapped on two of the three faces of the hexameric PepA triangle. A single hexameric ArgR molecule might contribute to the stabilization of the bend.

(a) Schematic view of the carAB control region showing the carP1 and carP2 promoters, the binding sites for the various regulatory and architectural proteins, and the size and the borders of the fragments used in the AFM study. PEPA1 and PEPA2 represent two continuous DNA stretches that are protected against DNase I digestion upon PepA binding; a line indicates the global zone showing alternating protection and regularly spaced sites hyperreactive for DNase I upon PepA binding (4). The length of the flanking regions, calculated on the basis of the footprints, is indicated with dotted lines. Similarly, we indicated the experimentally determined length of the arms and wrapped region (indicated in bold). The approximate length of the wrapped region is based on the average of different measurements (read-through and total visible contour length). (b) Organization of the two cer sites in direct repeat in plasmid pGIC009, and indication of the 2cer and 1cer fragments used in AFM studies. The binding sites for ArgR, XerC and XerD are boxed; a line represents the global zone exhibiting protection and hyperreactivity for DNase I upon PepA binding to a linear DNA fragment containing two cer sites in direct repeat; dotted lines indicate the predicted non-bound regions. Two alternatives for the arm lengths as determined by AFM are indicated (in bold) (3). (c) View along the 3-fold molecular axis of hexameric PepA, in which the two trimers are indicated in green and blue, respectively. The N-terminal domains point towards the corners of the triangle, the C-terminal domains cluster together at the center. Adapted from ref. (18).

(a) Overview AFM image of PepA binding to the 642-bp carAB fragment B1-DC381. (b) Four examples of typical wrapped PepA·DNA complexes formed with the same carAB fragment, with indication of the read-through contour length and the visible contour length. (c, d) Histograms with an overlay of a normal distribution curve of the read-through and visible contour length, respectively, of 156 PepA·DNA complexes formed with the B1-DC381 fragment and of 150 complexes formed with the fragment B1–B2, shown in (e) and (f). (g) Unbound PepA molecules. (h) Example of a rare interwrapped χ-like complex comprising two DNA and two PepA molecules.

(a and b)Histograms of the volume analyses of unbound and DNA-bound (B1-DC381 fragment) PepA molecules, with an overlay of a normal distribution curve.

(a) Three AFM images of 1cer·PepA complexes with a linear shape. (b) Three AFM images of 1cer·PepA complexes showing a DNA loop of variable size.