S

right-handed

left-handed

Box 6-1 DNA Has 10.5 Base Pairs per Turn of the Helix in Solution: The Mica Experiment

This value of 10 base pairs per turn vanes somewhat under different conditions. A classic experiment that was earned out in the 1970s demonstrated that DNA absorbed on a surface has somewhat greater than 10 base pairs per turn. Short segments of DNA were allowed to btnd to a mica surface. The presence of 5'-terminal phosphates on the DNAs field them in a fixed orientation on the mica. The mica-bound DNAs were then exposed to DNAse I, an enzyme (a rieoxyrifconude-ase) that deaves the phosphodiester bonds in the DNA backbone. Because the enzyme is bulky, it t5 onfy able to deave phosphodiester bonds on the DNA surface furthest from the mica (think of the DNA as a cylinder lying down on a flat surface) due to the stenc difficulty of reaching the sides or bottom, surface of the DNA. As a result, the length of the resulting fragments should reflect the periodicity of the DNA, the number Of base pairs per turn.

After the mica-bound DNA was exposed to DNAse the resulting fragments were separated by electrophoresis in a polyacrylamide gel, a jelly-like matrix (Box 6 1 f igure 1, see also Chapter 20 for an explanation of gel electrophoresis) Because DNA is negatively charged, it migrates through the gel toward the positive pole of the electric field. The gel matrix impedes movement of the fragments in a manner that is proportional to their length such thai larger fragments migrate more slowly than smaller fragments. When the experiment is carried out, we see clusters of DNA fragments of average sizes 10 and II, 21, 31, and 32 base pairs and so forth, that is, in multiples of 10.5, which is the number of base pairs per turn. This vatue of 10.5 base pairs per turn is dose to that of DNA in solution as inferred by other methods (see the section titled The Double Helix Exists in Multiple Conformations, below). The strategy of using DNAse to probe the structure of DNA is now used to analyze the interaction of DNA with proteins (see Chapter 17)

mica mica

BOX 6-1 FIGURE 1 The mica experiment.

thymine) and a bulky hydrophobic surface (the methyl group on C5 of thymine},, Similarly, the edge of a G:C base pair displays the following groups in the major groove: a hydrogen hond acceptor (at N7 of guanine), a hydrogen bond acceptor (the carbonyl on C6 of guanine), a hydrogen bond donor (the exocyclic amino group on C4 of cytosine), a small nonpolar hydrogen (the hydrogen at C5 of cytosine).

major groove major groove

Was this article helpful?

0 0

Post a comment