Chromatin-remodeling complexes regulate access to nucleosomal DNA by mobilizing nucleosomes in an ATP-dependent manner. In this study, we find that chromatin remodeling by SWI/SNF and ISW2 involves DNA translocation inside nucleosomes two helical turns from the dyad axis at superhelical location-2. DNA translocation at this internal position does not require the propagation of a DNA twist from the site of translocation to the entry/exit sites for nucleosome movement. Nucleosomes are moved in 9- to 11- or approximately 50-base-pair increments by ISW2 or SWI/SNF, respectively, presumably through the formation of DNA loops on the nucleosome surface. Remodeling by ISW2 but not SWI/SNF requires DNA torsional strain near the site of translocation, which may work in conjunction with conformational changes of ISW2 to promote nucleosome movement on DNA. The difference in step size of nucleosome movement by SWI/SNF and ISW2 demonstrates how SWI/SNF may be more disruptive to nucleosome structure than ISW2.

Linker DNA was found to be critical for the specific docking of ISW2 with nucleosomes as shown by mapping the physical contacts of ISW2 with nucleosomes at base-pair resolution. Hydroxyl radical footprinting revealed that ISW2 not only extensively interacts with the linker DNA, but also approaches the nucleosome from the side perpendicular to the axis of the DNA superhelix and contacts two disparate sites on the nucleosomal DNA from opposite sides of the superhelix. The topography of the ISW2-nucleosome was further delineated by finding which of the ISW2 subunits are proximal to specific sites within the linker and nucleosomal DNA regions by site-directed DNA photoaffinity labeling. Although ISW2 was shown to contact B63 bp of linker DNA, a minimum of 20 bp of linker DNA was required for stable binding of ISW2 to nucleosomes. The remaining B43 bp of flanking linker DNA promoted more efficient binding under competitive binding conditions and was functionally important for enhanced sliding of nucleosomes when ISW2 was significantly limiting.

SUMMARY ISWI-family enzymes remodel chromatin by sliding nucleosomes along DNA, but the nucleosome translocation mechanism remains unclear. Here we use single-molecule FRET to probe nucleosome translocation by ISWI-family remodelers. Distinct ISWI-family members translocate nucleosomes with a similar stepping pattern maintained by the catalytic subunit of the enzyme. Nucleosome remodeling begins with a 7-bp step of DNA translocation followed by 3-bp subsequent steps towards the exit side of nucleosomes. These multi-bp, compound steps are comprised of 1-bp substeps. DNA movement on the entry side of the nucleosome occurs only after 7 bp of exit-side translocation and each entry-side step draws in a 3-bp equivalent of DNA that allows three additional base pairs to be moved to the exit side. Our results suggest a remodeling mechanism with precise coordination at different nucleosomal sites featuring DNA translocation towards the exit side in 1-bp steps preceding multi-bp steps of DNA movement on the entry side.

A photocrosslinking nucleotide, 5‐[N‐(p‐azidobenzoyl)‐3‐aminoallyl]‐deoxyuridine monophosphate (N3Rd‐UMP), has been used to identify four polypeptides that are associated with the large Saccharomyces cerevisiae RNA polymerase III transcription factor TFIIIC, and to map the locations of these subunits along DNA when TFIIIC binds to the S.cerevisiae SUP4 tRNA(Tyr) gene. The 145 kd subunit of TFIIIC is primarily accessible to photocrosslinking from the vicinity of the box B + internal promoter element; 95 and 55 kd subunits are located on opposite sides of the DNA helix in the vicinity of the box A internal promoter element; a 135 kd subunit is less strongly crosslinked to the box A region and to a DNA segment between boxes B and A. DNA probes containing more than one N3RdUMP residue can form crosslinks between polypeptide chains. The specific circumstances of formation and the apparent mol. wts of two of these products lead to the tentative suggestion that a protomer of TFIIIC may contain two 95 kd subunits.

The SWI/SNF complex disrupts and mobilizes chromatin in an ATP-dependent manner. SWI/SNF interactions with nucleosomes were mapped by DNA footprinting and site-directed DNA and protein cross-linking when SWI/SNF was recruited by a transcription activator. SWI/SNF was found by DNA footprinting to contact tightly around one gyre of DNA spanning ϳ50 bp from the nucleosomal entry site to near the dyad axis. The DNA footprint is consistent with nucleosomes binding to an asymmetric trough of SWI/SNF that was revealed by the improved imaging of free SWI/SNF. The DNA site-directed cross-linking revealed that the catalytic subunit Swi2/Snf2 is associated with nucleosomes two helical turns from the dyad axis and that the Snf6 subunit is proximal to the transcription factor recruiting SWI/SNF. The highly conserved Snf5 subunit associates with the histone octamer and not with nucleosomal DNA. The model of the binding trough of SWI/SNF illustrates how nucleosomal DNA can be mobilized while SWI/SNF remains bound.