Evidence for DNA Translocation by the ISWI Chromatin-Remodeling Enzyme

Abstract

The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5-or 10-bp gaps in the 3-5 strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3-5-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.A general feature of eukaryotes is that their genomic DNA associates with proteins to form chromatin. In addition to providing a means of packaging DNA within nuclei, chromatin provides an additional level at which gene expression can be regulated. Active regions of the genome tend to be maintained in a more accessible state than inactive regions, and the manipulation of chromatin structure has been found to play an important role in the regulation of many genes. In order to be able to regulate gene expression at this level, eukaryotes have devised a number of strategies by which they can manipulate chromatin structure. These include the covalent modification of chromatin structure by posttranslational modification, the manipulation of the protein content of chromatin through the association of variant histone and nonhistone proteins, and the noncovalent alteration of chromatin structure by ATP-dependent chromatin-remodeling activities (2,7,38,67).It now appears likely that all eukaryotes encode multiple yet distinct ATPases with homology to the yeast SNF2 protein (Snf2p). These include the Sth1p, Chd1p, Isw1p, Isw2p, and Ino80p proteins in budding yeast and a spectrum of related proteins in higher eukaryotes (2, 66). Among these, the ISWI proteins represent a discrete class of ATPase involved in chromatin remodeling. The founding member of the ISWI group was identified in Drosophila melanogaster due to its similarity to Snf2p. As the homology is restricted to the helicase-like domain, it was named imitation SWI/SNF (ISWI) (21). Biochemical characterization of ISWI proteins began with the identification of ISWI as the catalytic subunit of three distinct complexes, nucleosome-remodeling factor (NuRF), chromatin accessibility complex (ChrAC), and the ATP-utilizing chromatin assembly and modifying factor (ACF) isolated from Drosophila embryo extracts (36,60,64). Subsequently, ISWI-related proteins have been found to be components of chromatin-remodeling complexes in organisms ranging from yeast to humans, suggesting that these proteins fulfill important functions conserved throughout evolution (5,42,43,49,61). Within each of these complexes, the ISWI protein is associated with a...

Chromatin remodeling: insights and intrigue from single-molecule studies

“…One consistent feature of SF2 translocases is their tracking along the phosphate backbone of one of the two DNA strands, with limited interaction with the bases themselves 39,40 . Consistent with the notion of tracking, the results of triplex-displacement assays suggest that SWI/SNF, ISWI and Rad54 track in a 3′-to-5′ direction along one strand of the DNA duplex and are impeded if the tracking strand bears a gap in the phosphodiester backbone 4,36,38,41 . Furthermore, a series of studies on chromatin assembly by the ACF remodeler (ISWI family) reveal that chromatin is assembled in a processive manner that involves DNA template commitment, consistent with the use of a DNA-tracking mode for the assembly process 42 .…”

“…These models were intuitive and provided important initial concepts. However, all remodeler ATPases are classified as members of the SF2 family of DEAD/H-box helicase/translocases, and a significant advance was the demonstration that remodelers are indeed ATP-dependent DNA translocases 4,5,[35][36][37][38] . This indicated a possible motor force for breaking histone-DNA contacts and moving DNA on the octamer surface.…”

Chromatin remodeling by ATP‐dependent molecular machines

“…4. This model is supported by the ability of ACF and ISWI to translocate along DNA (31,92) as well as the general propensity of ISWI subfamily complexes to assemble nucleosomes rather than to disassemble nucleosomes. (28,29,32,33,51) This model (with a small loop) is also consistent with the inability of ISWI subfamily complexes to catalyze the transfer of histones from one template to another template or to form dinucleosome-like structures from mononucleosomes, although such activities have been observed with SNF2 subfamily complexes.…”

“…(35,60,91) (7) RSC complex, ISWI and Rad54 have been found to exhibit triplex DNA displacement activity and thus appear to translocate along the DNA. (65,92) [In the triplex DNA displacement assay, a short oligonucleotide that binds in the major groove of a pyrimidine-rich target sequence is displaced by motor proteins that translocate through the sequence.] Furthermore, the ACF complex exhibits template commitment during chromatin assembly, which suggests that it functions as a DNA-translocating enzyme.…”

“…8,15,20,31,35,65,68,77,78,82,83,85,[91][92][93][94]. Most of the proposed mechanisms involve the generation of a DNA loop or bulge (of variable size) that is propagated across the surface of the histone octamer.…”