Genes VII

Some insights into the structure of nucleosomal DNA emerge when we compare predictions for supercoiling in the path that DNA follows with actual measurements of supercoiling of nucleosomal DNA. Much work on the structure of sets of nucleosomes has been carried out with the virus SV40. The DNA of SV40 is a circular molecule of 5200 bp, with a contour length ~1500 nm. In both the virion and infected nucleus, it is packaged into a series of nucleosomes, called a minichromosome.

As usually isolated, the contour length of the minichromosome is ~210 nm, corresponding to a packing ratio of ~7 (essentially the same as the ~6 of the nucleosome itself). Changes in the salt concentration can convert it to a flexible string of beads with a much lower overall packing ratio. This emphasizes the point that nucleosome strings can take more than one form in vitro, depending on the conditions.

The degree of supercoiling on the individual nucleosomes of the minichromosome can be measured as illustrated in Figure 19.16. First, the free supercoils of the minichromosome itself are relaxed, so that the nucleosomes form a circular string with a superhelical density of 0. Then the histone octamers are extracted. This releases the DNA to Then the histone octamers are extracted. This releases the DNA to follow a free path. Every supercoil that was present but restrained in the minichromosome will appear in the deproteinized DNA as V1 turn. So now the total number of supercoils in the SV40 DNA is measured.

The observed value is close to the number of nucleosomes. The reverse result is seen when nucleosomes are assembled in vitro on to a supercoiled SV40 DNA: the formation of each nucleosome removes ~1 negative supercoil.

So the DNA follows a path on the nucleosomal surface that generates ~1 negative supercoiled turn when the restraining protein is removed. But the path that DNA follows on the nucleosome corresponds to V1.65 superhelical turns (see Figure 19.4). This discrepancy is sometimes called the linking number paradox.

The discrepancy is explained by the difference between the 10.2 average bp/turn of nucleosomal DNA and the 10.5 bp/turn of free DNA. In a nucleosome of 200 bp, there are 200/10.2 19.6 turns. When DNA is released from the nucleosome, it now has 200/10.5 19.0 turns. The path of the more tightly wound DNA on the nucleosome absorbs V0.6 turns, and this explains the discrepancy between the physical path of V1.65 and the measurement of V1.0 superhelical turns. In effect, some of the torsional strain in nucleosomal DNA goes into increasing the number of bp/turn; only the rest is left to be measured as a supercoil (for review see Travers and Klug, 1987).