ATP-Independent Cooperative Binding of Yeast Isw1a to Bare and Nucleosomal DNA

Abstract

Among chromatin remodeling factors, the ISWI family displays a nucleosome-enhanced ATPase activity coupled to DNA translocation. While these enzymes are known to bind to DNA, their activity has not been fully characterized. Here we use TEM imaging and single molecule manipulation to investigate the interaction between DNA and yeast Isw1a. We show that Isw1a displays a highly cooperative ATP-independent binding to and bridging between DNA segments. Under appropriate tension, rare single nucleation events can sometimes be observed and loop DNA with a regular step. These nucleation events are often followed by binding of successive complexes bridging between nearby DNA segments in a zipper-like fashion, as confirmed by TEM observations. On nucleosomal substrates, we show that the specific ATP-dependent remodeling activity occurs in the context of cooperative Isw1a complexes bridging extranucleosomal DNA. Our results are interpreted in the context of the recently published partial structure of Isw1a and support its acting as a “protein ruler” (with possibly more than one tick).

A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: From nucleocapsid-condensed RNA to Vpr-bridged DNA

“…They are however strikingly similar to the nucleofilaments formed between DNA and the family of histone-like nucleoid structural proteins such as E.Coli H-NS or StpA (Dame et al, 2006; Lim et al, 2011; Liu et al, 2010). These structures are also comparable to those formed between DNA and the eukaryotic chromatin remodeling factor, ISW1a (De Cian et al, 2012). These stable, bridged oligomers formed in solution led us to speculate that Vpr may assemble with vDNA in an equivalent manner leading to a yet undiscovered function as a vDNA-condensing agent within the PIC.…”

Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells

“…Samples were prepared from 5 μL of sepiolite or Sep/DNA solution deposited for 1 min on a 600-mesh copper grid covered with a thin carbon film activated by glow-discharge in the presence of pentylamine43. Grids were washed with aqueous uranyl acetate 2% (w/v), dried with ashless filter paper and observed in dark-field mode with a tilted illumination, using a Zeiss 912AB transmission electron microscope.…”

Centromere Structure and Function

“…Chromatin remodeling proteins have also been shown to be loop extruding motors (De Cian et al 2012; Sun et al 2013), and may impart this function at the centromere. The centromere is enriched in several members of various chromatin remodeling protein families.…”

“…The yeast Isw1 complex (Imitation switch) is of particular interest as it has been shown in vitro to function as an inchworm that will form and propagate intra-stand DNA looping (Fig. 4) (De Cian et al 2012). Isw1 is a member of the SWI/SNF ATPase complex and interacts with the centromere DNA-binding factor CBF1 (Moreau et al 2003).…”

“…Isw1a, a member of the SWI/SNF chromatin remodeling family functions as a loop extruding enzyme in vitro, adapted from (De Cian et al 2012). Isw1 interacts with the yeast Cbf1 factor where it functions to maintain nucleosome-free regions at promoters.…”