Scheme 1. The model in (A) illustrates a polymerase (solid color) bound to a template-primer complex. The template, which is longer than the primer, has a single-stranded region. The 3′ end of the primer contains the photoreagent (R) covalently attached to the terminal base. The diagram also illustrates a photosensitizer (S) bound in the polymerase active site. Excitation of the photosensitizer with 365 nm UV light results in activation of the photoreagent, as shown in (B), and crosslinking to the polymerase (C). A DNA binding protein unable to bind the photosensitizer is not crosslinked by 365 nm UV light exposure (D). The 5′-³²P radiolabel is illustrated by an asterisk.