Together these results suggest a distance between cross-links perhaps as low as 10–20 kb. Their cutting frequency should be in the 10-kb (5-bp cutters) or 40-kb (6-bp cutters) range, assuming their site accessibility is limited only by the fraction of linker DNA. No effect on chromosome elasticity was observed even after prolonged digestion with several 5- and 6-bp cutters. Based on the 60% reduction in the force constant for stretching, they estimate ≈60% of cross-linked segments would be cut, yielding a mean distance between cross-links of 15 kb, assuming a 25-kb spacing of Cac8 I cutting. Based on a rate of force reduction with cutting 1/10 that observed for the 4-bp cutters, the authors assume this reflects a 10-fold lower number of allowed cutting sites for Cac8 I, implying a cutting frequency of 1/25 kb. 1).įrom these results, the authors conclude the mechanical integrity of chromosomes is largely dependent on DNA, and not on an internal protein network, behaving as a cross-linked network of 30-nm fibers. Further stretching, beyond the elastic regime, produced severe, nonuniform elongation with blob-like chromosome regions separated by thinner fibers that could be digested by further nuclease digestion (Fig. Each cycle produced a progressive drop in the required force. Alternatively, nonstretched chromosomes were exposed to micrococcal nuclease and then stretched through several cycles of extension and relaxation. Additional digestion lead to thinning, severing, and then disappearance of the chromosome. When micrococcal nuclease was used, the tension generated by the elongated chromosome dropped to zero after ≈60-sec digestion, without any obvious chromosome shape changes. Chromosomes were stretched and relaxed to determine their elastic response, then held under a small, constant force while exposed to a spray of nuclease from a third pipette. Controlled movement of the stiffer pipette allowed force/extension measurements. In turn, this led to a speculative model in which chromosome condensation from interphase through metaphase might be driven by similar transitions in scaffold structure from a dispersed interphase network to a contracted mitotic conformation ( 8, 9).Ĭhromosomes were attached at one end to a large stiff pipette, and at the other end to a more flexible pipette whose deflection provides measurement of applied force. Reversible swelling and contraction of isolated scaffolds driven by changes in ionic conditions suggested that similar conformational changes of intact chromosomes might be driven by the salt-induced changes in scaffold structure ( 9). The use of nucleases to isolate the scaffold without its associated DNA halo suggested the scaffold was a structurally independent entity whose integrity was independent of DNA ( 8). The match between the shape and size of the extracted chromosome core and the native, unextracted chromosome led directly to the concept that that this residual structure might reflect the remnants of a nonhistone protein “scaffold.” Stability of the scaffold and DNA halo in 2 M NaCl was interpreted as consistent with the scaffold holding the DNA loops in a compact conformation. Their results prompt serious reconsideration and debate of the standard textbook model for mitotic chromosome condensation. In the second, published in a recent issue of PNAS ( 5), micromechanical force measurements on individual, isolated chromosomes probed changes in chromosome structure produced by nuclease digestion (see Fig. In the first ( 3, 4), the in vivo dynamics of the scaffold protein topoisomerase 2 were examined by using a GFP protein. Two recently published experimental approaches effectively reevaluate these questions of interpretation by testing predictions, rather than assumptions, of the radial loop model. This controversy derives in large part from key assumptions that are difficult to address experimentally but implicit in the interpretation of the original experiments. Yet the radial loop model has remained highly controversial. Striking images of extended DNA “halos” surrounding a linear protein core, roughly the shape of the native chromosome, led to a simple model in which a central protein “scaffold” constrained DNA into a set of loops, ≈50 kb in length. By a neat trick of high-salt or polyanion-driven protein extraction under conditions preserving remnants of mitotic chromosome structure, the difficult issue of higher order and large-scale chromatin folding was effectively bypassed. Nearly all textbooks feature the radial loop model of mitotic chromosome condensation, derived from experiments largely conducted 15–25 years ago.
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