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The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.

Bacterial chromosome (nucleoid) conformation dictates faithful regulation of gene transcription. The conformation is condition-dependent and is guided by several nucleoid-associated proteins (NAPs) and at least one nucleoid-associated noncoding RNA, naRNA4. Here we investigated the molecular mechanism of how naRNA4 and the major NAP, HU, acting together organize the chromosome structure by establishing multiple DNA-DNA contacts (DNA condensation). We demonstrate that naRNA4 uniquely acts by forming complexes that may not involve long stretches of DNA-RNA hybrid. Also, uncommonly, HU, a chromosome-associated protein that is essential in the DNA-RNA interactions, is not present in the final complex. Thus, HU plays a catalytic (chaperone) role in the naRNA4-mediated DNA condensation process., Competing Interests: The authors declare no conflict of interest.

Information readout in the DNA minor groove is accompanied by substantial DNA deformations, such as sugar switching between the two conformational domains, B-like C2'-endo and A-like C3'-endo. The effect of sugar puckering on the sequence-dependent protein-DNA interactions has not been studied systematically, however. Here, we analyzed the structural role of A-like nucleotides in 156 protein-DNA complexes solved by X-ray crystallography and NMR. To this end, a new algorithm was developed to distinguish interactions in the minor groove from those in the major groove, and to calculate the solvent-accessible surface areas in each groove separately. Based on this approach, we found a striking difference between the sets of amino acids interacting with B-like and A-like nucleotides in the minor groove. Polar amino acids mostly interact with B-nucleotides, while hydrophobic amino acids interact extensively with A-nucleotides (a hydrophobicity-structure correlation). This tendency is consistent with the larger exposure of hydrophobic surfaces in the case of A-like sugars. Overall, the A-like nucleotides aid in achieving protein-induced fit in two major ways. First, hydrophobic clusters formed by several consecutive A-like sugars interact cooperatively with the non-polar surfaces in proteins. Second, the sugar switching occurs in large kinks promoted by direct protein contact, predominantly at the pyrimidine-purine dimeric steps. The sequence preference for the B-to-A sugar repuckering, observed for pyrimidines, suggests that the described DNA deformations contribute to specificity of the protein-DNA recognition in the minor groove.

The HMG-box domain of the human male sex-determining factor SRY, hSRY(HMG) (comprising residues 57-140 of the full-length sequence), binds DNA sequence-specifically in the minor groove, resulting in substantial DNA bending. The majority of point mutations resulting in 46X,Y sex reversal are located within this domain. One clinical de novo mutation, M64I in the full-length hSRY sequence, which corresponds to M9I in the present hSRY(HMG) construct, acts principally by reducing the extent of DNA bending. To elucidate the structural consequences of the M9I mutation, we have solved the 3D solution structures of wild-type and M9I hSRY(HMG) complexed to a DNA 14mer by NMR, including the use of residual dipolar couplings to derive long-range orientational information. We show that the average bend angle (derived from an ensemble of 400 simulated annealing structures for each complex) is reduced by approximately 13 degrees from 54(+/-2) degrees in the wild-type complex to 41(+/-2) degrees in the M9I complex. The difference in DNA bending can be localized directly to changes in roll and tilt angles in the ApA base-pair step involved in interactions with residue 9 and partial intercalation of Ile13. The larger bend angle in the wild-type complex arises as a direct consequence of steric repulsion of the sugar of the second adenine by the bulky S(delta) atom of Met9, whose position is fixed by a hydrogen bond with the guanidino group of Arg17. In the M9I mutant, this hydrogen bond can no longer occur, and the less bulky C(gamma)m methyl group of Ile9 braces the sugar moieties of the two adenine residues, thereby decreasing the roll and tilt angles at the ApA step by approximately 8 degrees and approximately 5 degrees, respectively, and resulting in an overall difference in bend angle of approximately 13 degrees between the two complexes. To our knowledge, this is one of the first examples where the effects of a clinical mutation involving a protein-DNA complex have been visualized at the atomic level.

High resolution chemical footprinting and cross-linking experiments have provided a basis for elucidating the overall architecture of the complex between the core DNA binding domain of p53 (p53DBD, amino acids 98-309) and the p21/waf1/cip1 DNA response element implicated in the G1/S phase cell cycle checkpoint. These studies complement both a crystal structure and earlier biophysical studies and provide the first direct experimental evidence that four subunits of p53DBD bind to the response element in a regular staggered array having pseudodyad symmetry. The invariant guanosines in the highly conserved C(A/T)|(T/A)G parts of the consensus half-sites are critical to the p53DBD-DNA binding. Molecular modeling of the complex using the observed peptide-DNA contacts shows that when four subunits of p53DBD bind the response element, the DNA has to bend approximately 50 degrees to relieve steric clashes among different subunits, consistent with recent DNA cyclization studies. The overall lateral arrangement of the four p53 subunits with respect to the DNA loop comprises a novel nucleoprotein assembly that has not been reported previously in other complexes. We suggest that this kind of nucleoprotein superstructure may be important for p53 binding to response elements packed in chromatin and for subsequent transactivation of p53-mediated genes.