Measurement of Helical Pitch

Measurement of Helical Pitch

It is not straightforward to determine the helical repeat of DNA under in vivo conditions. Such measurements have been made, and will bedescribed later. Here we shall consider measuring the helical pitch invitro of linear DNA not bound to any proteins.

Klug and co-workers found that DNA can bind tightly to the flat surface of mica or calcium phosphate crystals. While bound to such surfaces, only a portion of the cylindrical DNA is susceptible to cleavage by DNAse I, an enzyme that hydrolyzes the phosphodiester backbone of DNA (Fig. 2.8). Consider the consequences of: 1. utilizing an homoge-nous population of DNA molecules, 2. radioactively labeling each mole-cule on one end with ³²PO₄, 3. rotationally orientating all the DNA molecules similarly, i.e. the 5’ end of the labeled strand begins in contact with the solid support, 4. performing a partial digestion with DNAse I, so that on average, each DNA molecule is cleaved only once.

In the population, the labeled strand will be cleaved more frequently at those positions where it is on the part of the helix up away from the support, and thus cleavages will be concentrated at positions ¹⁄2, 1¹⁄2, 2¹⁄2 etc. helical turns from the labeled end. A similar population of labeled DNA digested while in solution will possess some molecules

Figure 2.8 Determination of the helical pitch of DNA while bound to a solidsupport. While bound to the support, and while free in solution, the DNA is lightly digested with DNAse, the denatured fragments are separated according to size by electrophoresis and an autoradiograph is made of the gel. When the DNA is on the solid support, DNAse has only limited access.

cleaved at every position. Electrophoretic separation of the populations yields the pattern shown (Fig. 2.8). In the sample cleaved while on the flat support most cleavages, and hence the darkest bands, are separated by 10 or 11 base pairs. The DNA cleaved in solution shows all sizes of DNA fragments.