2 DNA fragmentation

The contents of each tube are then subjected to electrophoresis m separate lanes on the same sheet of polyacrylamide gel and the DNAs located by autoradiography A typical electrophoresis gel of a DNA fragment containing 50 nucleotides will exhibit a pattern of 50 bands distributed among the four lanes with no overlaps Each band cor responds to a polynucleotide that is one nucleotide longer than the one that precedes it (which may be m a different lane) One then simply reads the nucleotide sequence according to the lane m which each succeeding band appears... 

DNA fragment formed under conditions of experiment terminates m indicated dideoxynucleoside... 

Determination of DNA Sequence Information. Almost all DNA sequence is determined by enzymatic methods (12) which exploit the properties of the enzyme DNA polymerase. Whereas a chemical method for DNA sequencing exists, its use has been supplanted for the most part in the initial deterrnination of a sequence. Chemical or Maxam-Gilbett sequencing (13) is mote often used for mapping functional sites on DNA fragments of known sequence. 

Theory of Electrophoretic Motion. The study of the mechanics of electrophoresis focuses on the basis of electric potential on the surface of an object, and the relation of the electric potential to the velocity of the particle. Whereas research has been generally limited to nonmolecular particles of weU-defined geometry and is not strictly apphcable to molecules such as proteins and DNA fragments, this work is useful for understanding the physics of electrophoretic motion. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Wang, A.H.-J., et al. Molecular structure of a left-handed DNA fragment at atomic resolution. Nature 282 680-686, 1979. 

Figure 8.11 The DNA-binding domain of 434 repressor. It is a dimer in its complexes with DNA fragments. Each subunit (green and brown) folds into a bundle of four a helices (1-4) that have a structure similar to the corresponding region of the lambda repressor (see Figure 8.7) including the helix-turn-helix motif (blue and red). A fifth a helix (5) is involved in the subunit interactions, details of which are different from those of the lambda repressor fragment. The structure of the 434 Cro dimer is very similar to the 434 repressor shown here.

The lac repressor monomer, a chain of 360 amino acids, associates into a functionally active homotetramer. It is the classic member of a large family of bacterial repressors with homologous amino acid sequences. PurR, which functions as the master regulator of purine biosynthesis, is another member of this family. In contrast to the lac repressor, the functional state of PurR is a dimer. The crystal structures of these two members of the Lac I family, in their complexes with DNA fragments, are known. The structure of the tetrameric lac repressor-DNA complex was determined by the group of Mitchell Lewis, University of Pennsylvania, Philadelphia, and the dimeric PurR-DNA complex by the group of Richard Brennan, Oregon Health Sciences University, Portland. 

Figure 8.22 The lac repressor molecule is a V-shaped tetramer in which each arm is a dimer containing a DNA-hinding site. The helix-tum-helix motifs (red) of each dimer bind in two successive major grooves and the hinge helices (purple) bind adjacent to each other in the minor groove between the two major groove binding sites. The four subunits of the tetramer are held together by the four C-terminal helices (yellow) which form a four helix bundle. The bound DNA fragments are bent. (Adapted from M. Lewis et al., Science 271 1247-1254, 1996.)...

Figure 9.3 Comparison of the consensus nucleotide sequence of the TATA box (a) and the sequences of the DNA fragments used in the crystal structure determinations of the TATA box-binding proteins from yeast (b) and the plant Arabidopsis thaliana (c).

Figure 9.S Schematic diagram illustrating the structure of the complex between TBP and a DNA fragment containing the TATA box. Both the stirmps and the underside of the saddle are in contact with the DNA. (Adapted from V. Kim et al., Nature 365 514-520, 1993.)...

Figure 9.6 The DNA fragment is sharply bent in the TATA box region (orange) so that the DNA helices on both sides form an angle of about 110° to each other instead of 180° for undistorted B-DNA. The view of the DNA is rotated about 90° compared with that of Figure 9.5.

Figure 9.19 Nucleotide sequence of the 21-base pair DNA fragment cocrystalUzed with the DNA-binding domain of p53. The p53 binds in a sequence-specific manner to the shaded region.

Interactions that are not sequence specific are also an Important part of the binding and occur between the sugar and phosphate residues of the DNA and the side-chain and main-chain atoms of the protein. In the crystals the DNA fragment retains the B-DNA structure with only minor distortions. 

---