Title: Remodeling The Genome With Dna Twists
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Arthur T Knackerbracket has found the following story [sciencemag.org]:
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In complex organisms such as humans, a single genetic blueprint can give rise to a multitude of different cell types, from nerve to liver to muscle. Such cellular diversity relies on restricting which portions of genomic DNA are accessible and therefore can be read by cellular machinery. Ultimately, access to DNA depends on placement of a repetitive, spool-like structure called the nucleosome, the basic packaging unit of chromosomes. The nucleosome occludes two tight loops of DNA and thus represents a fundamentally repressive element. When and where nucleosomes are positioned can affect complex transcriptional programs, and therefore disruptions in the factors responsible for nucleosome positioning often result in cancers and multisystem developmental diseases. Although the mechanism of shifting nucleosomes along DNA has long proved elusive, a recent flurry of structural, biophysical, and biochemical work has revealed a core mechanistic framework explaining how nucleosomes are actively repositioned throughout the genome.
Nucleosomes are the most ubiquitous protein-DNA complexes in all eukaryotic cells. The core of each nucleosome is a symmetric, disk-like structure made of histone proteins that provides a scaffold around which two loops of the DNA helix are snugly wrapped (1 [soylentnews.org]). Histones are often modified through, for example, acetylation, methylation, and phosphorylation, which add an additional layer of information on top of the genetic code. This epigenetic information demarcates functionally distinct regions of the genome—for instance, whether a gene is active or designated to remain silent—for each cell type.
Owing to their extensive protein-DNA interface, nucleosomes are relatively stable structures. Active placement and reorganization of nucleosomes depend on chromatin remodelers. As the gatekeepers of nucleosome packaging, these enzymes participate both in activating and repressing gene expression. Remodelers can assemble, disassemble, and exchange histones within the nucleosome, as well as shift the position of the histone core along DNA. Acting on either face of the nucleosome disk, remodelers can move the histone core back and forth on DNA, changing which parts of DNA are exposed and which are wrapped up in the nucleosome. Increased exposure of DNA occurs when remodelers shift adjacent nucleosomes into each other, resulting in histone ejection (2 [soylentnews.org]).
The ability of remodelers to manipulate nucleosome structure stems from a highly conserved adenosine triphosphatase (ATPase) motor that belongs to a larger superfamily of helicase-like ATPases called superfamily 2 (SF2). On the basis of intense biophysical and biochemical research on SF2 and the related SF1 ATPases, understanding how these enzymes move and interact with DNA and RNA has revealed how remodeler ATPases move and engage DNA (3 [soylentnews.org]). Both SF1 and SF2 ATPases consist of two distinct domains that together form an ATP-binding pocket and a nucleic acid binding surface. Dictated by the occupancy of the nucleotide-binding pocket, the two domains open and close like a clamshell, which in turn alters interactions with the bound nucleic acid. For many SF1 and SF2 enzymes, alternating between open and closed states ratchets the nucleic acid past the ATPase in what is known as an inchworm-type mechanism (3 [soylentnews.org]).
Consistent with an inchworm-type translocation mechanism, nucleosomal DNA is shifted by remodelers with an elementary step of a single base pair (4 [soylentnews.org]). Given the spiral structure of the duplex, nucleosomal DNA must shift around the histone core in a corkscrew fashion. Remodeler ATPases remain in a fixed location on the histone core during DNA translocation, which means that DNA all around the nucleosome must shift in response to localized action at the ATPase binding site. According to the classic interpretation of the inchworm model, DNA on both sides of the ATPase should be shifted simultaneously with each ATP hydrolysis cycle, requiring that the motor be physically coupled to the histone core to generate force. Instead, remodeler ATPases shift nucleosomal DNA discontinuously, with DNA movement resulting from both open and closed states of the ATPase that bypass the strict requirement for a separate histone foothold to push against (5 [soylentnews.org]). In its open (nucleotide-free and adenosine diphosphate–bound) state, the ATPase pulls entry-side DNA toward itself in a corkscrew fashion, creating a DNA bulge at the binding site. When poised for hydrolysis, the ATPase in its closed state eliminates this bulge, corkscrewing DNA toward the nucleosome midpoint (dyad) on the other side. This creation and elimination of a DNA bulge is equivalent to altering DNA twist.
Changes in DNA twist, known as twist defects, have been proposed as a low-energy means of ratcheting DNA past the histone core (6 [soylentnews.org]). Twist defects were observed in the first nucleosome crystal structure, demonstrating the inherent capability of nucleosomal DNA to sample different geometries (1 [soylentnews.org]). When DNA moves, a twist defect arises when two adjacent segments of the same DNA duplex do not undergo a corkscrew shift in unison. The twist defect can be thought of as the junction between a stationary and a mobile stretch of DNA: one side of the junction undergoes a corkscrew shift (mobile), while the other side remains stationary with respect to the histone core. Twist defects therefore reflect a gain or loss of a base pair resulting from the corkscrew shift of DNA. Twist defects dissipate once one side of the junction undergoes a compensatory corkscrew shift, restoring DNA to its canonical conformation on the nucleosome. If the corkscrew shift occurs on the previously stationary DNA segment, another twist defect can be created farther downstream, where the newly mobile DNA segment again runs up against a stationary segment. Through such discontinuous motions, propagation of twist defects all the way around the nucleosome repositions the entire length of nucleosomal DNA relative to the histone core. Spontaneous nucleosome sliding via twist defects has been visualized through molecular simulations, suggesting that twist can be absorbed or buffered at multiple locations on the nucleosome (7 [soylentnews.org]).
Key stages in the nucleosome sliding cycle have been captured by cryo–electron microscopy, adding essential mechanistic insight into how repositioning is achieved. In the initial stage, the open state of the ATPase bound to nucleosomal DNA creates a bulge in only the tracking strand of the DNA duplex but not the complementary guide strand (8 [soylentnews.org]) (half twist defect; step 1 in the figure). A consequence of pulling only one of the two DNA strands is base tilting, which is necessary to maintain base pairing between strands. Although a shift of only one DNA strand was unexpected, a similar tilting of base pairs was observed in the DNA-RNA hybrid cradled by RNA polymerase during transcription (9 [soylentnews.org]) and may create strain that allows the duplex to translocate more easily. For remodelers, strain from base pair tilting would prime the duplex for the next stage, when a compensatory shift of the other strand creates a full twist defect. Such a remodeler-bound state has recently been captured on a nucleosome bound to SWR1 (SWI/SNF-related ) (10 [soylentnews.org]), where both DNA strands bulge, accommodating an additional base pair and restoring the canonical base stacking outside the remodeler binding site (step 2 in the figure). After remodelers have successfully shifted the twist defect toward the dyad, the DNA at the remodeler binding site should return to its canonical structure on the nucleosome (step 3 in the figure). Such a post-shifted state with the bound DNA in a canonical geometry has been visualized with remodeler ATPases trapped in hydrolysis-competent states (5 [soylentnews.org], 8 [soylentnews.org], 11 [soylentnews.org], 12 [soylentnews.org]). Because nucleosomal DNA with a canonical twist aligns with the transition state of the ATPase (5 [soylentnews.org]), elimination of the twist defect may be sufficient to trigger ATP hydrolysis, which would initiate another round of sliding and enforce directionality of twist diffusion around the nucleosome.
A model of how twist defects, stimulated by the remodeler adenosine triphosphatase (ATPase), shift DNA around the nucleosome (shifted DNA is depicted in red). After a full cycle (steps 1 to 3), creation and passage of additional twist defects from subsequent cycles push DNA out of the nucleosome exit side.
These snapshots of remodelers altering DNA twist are complemented by the observation of active movement of nucleosomal DNA by remodelers during three-color single-molecule FRET (Förster resonance energy transfer) experiments, where DNA movements were coupled to ATP hydrolysis (13 [soylentnews.org]). As demonstrated using FRET reporters on both sides of the nucleosome, remodelers pull DNA onto the entry side of the nucleosome before shifting DNA off the exit side. Observed as a time delay, nucleosomes absorbed one or more base pairs of DNA pulled on by the remodeler, which would take the form of twist defects. Future studies should determine the capacity and energetic cost of storing twist defects during remodeling and how buffering twist defects may be influenced by DNA sequence. In addition, because a single nucleosome can simultaneously bind a remodeler on each side, it will also be of interest to determine whether such DNA buffering and changes in twist enable communication between pairs of remodelers vying to shift the histone core in opposite directions (13 [soylentnews.org]).
With the core mechanism of nucleosome sliding finally coming into focus, this recently developed framework provides a launching point for future research. In addition to DNA geometry and energetics, to what extent is twist diffusion dependent on other characteristics of the nucleosome? The histone core has dynamic properties (14 [soylentnews.org], 15 [soylentnews.org]), and it will be interesting to see the degree to which plasticity of histone structure may affect formation and propagation of twist defects within the nucleosome. Histone proteins come in a variety of flavors, and an important goal is identifying whether distinct biophysical properties of histone variants and other epigenetic signatures can determine or bias the outcomes of remodeling reactions.
The ability of remodeler-type ATPases to create twist defects is used for more than just sliding nucleosomes. The SWR1 subclass specializes in histone exchange, swapping out canonical histone H2A-H2B dimers for variant H2A.Z-H2B dimers, a universal epigenetic mark in gene promoters. A key issue for this system will be uncovering the connection between twist defects and histone dimer exchange.
Remodeler-type ATPases encompass enzymes that specialize in non-nucleosomal substrates. This class of enzymes includes factors that are essential for DNA recombination and repair, as well as the recycling of transcriptional machinery in both eukaryotes and bacteria. Despite having distinct targets, these remodeler-type ATPases likely catalyze physical changes in their substrates by distorting the DNA duplex, creating high-energy intermediates analogous to nucleosomal twist defects. Just as insights for SF1 and SF2 ATPases have facilitated thinking about nucleosome sliding, the discovery that remodelers create and eliminate twist defects on the nucleosome will likely advance our understanding of these related yet functionally distinct enzymes, which all wrestle the double helix to transiently blaze a trail through the energetic landscape of duplex DNA.
This is an article distributed under the terms of the Science Journals Default License [sciencemag.org].
Vol 366, Issue 6461
04 October 2019
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By Gregory D. Bowman, Sebastian Deindl
Intricate protein machines repackage DNA to turn genes on and off
By Gregory D. Bowman, Sebastian Deindl
Intricate protein machines repackage DNA to turn genes on and off
Vol 366, Issue 6461
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