This chapter describes a primer-template system in which the position of a cross-linkable aryl azide can be moved stepwise through a 30-base region of the primer by selective elongation of the primers with different combinations of dNTPs. Using this system, it is shown that T4 DNA polymerase and the T4 genes 44/62 and 45 polymerase accessory proteins assemble into different complexes in the presence of ATP and ATPγS, and have mapped primer-protein contacts in each complex. In all of the cross-linking experiments with the T4 replication complex the reactions are carried out with an excess of the polymerase and accessory proteins, with 32 protein sufficient to cover about 50% of the template, which is the optimal ratio of 32 protein to DNA. The major advantages of cross-linking to DNA with the aryl azide are that the photoactivatable residue is located at a specific position in the primer sequence within the major groove of the helix, and is activated at a wavelength (302 nm) above that absorbed by protein and unmodified DNA. The formation of a covalent cross-link between a replication protein and DNA should allow determination of the amino acid linked to the primer; it has been demonstrated for Escherichia coli polymerase I with a primer containing the aryl azide, 6 and for other proteins cross-linked to unmodified DNA, or to DNA with BrU replacing T at specific positions.
All Science Journal Classification (ASJC) codes
- Molecular Biology