CRYSTAL-STRUCTURES OF HUMAN DNA-POLYMERASE-BETA COMPLEXED WITH GAPPEDAND NICKED DNA - EVIDENCE FOR AN INDUCED FIT MECHANISM

DNA polymerase beta (pol beta) fills single nucleotide (nt) gaps in DN A produced by the base excision repair pathway of mammalian cells. Cry stal structures have been determined representing intermediates in the 1 nt gap-filling reaction of pol beta: the binary complex with a gapp ed DNA substrate (2.4 Angstrom resolution), the ternary complex includ ing ddCTP (2.2 Angstrom), and the binary product complex containing on ly nicked DNA (2.6 Angstrom). Upon binding ddCTP to the binary gap com plex, the thumb subdomain rotates into the closed conformation to cont act the otherwise solvent-exposed ddCTP-template base pair. Thumb move ment triggers further conformational changes which poise catalytic res idue Asp192, dNTP, and template for nucleotidyl transfer, effectively assembling the active site. In the product nicked DNA complex, the thu mb returns to the open conformation as in the gapped binary DNA comple x, facilitating dissociation of the product. These findings suggest th at pol beta may enhance fidelity by an induced fit mechanism in which correct base pairing between template and incoming dNTP induces alignm ent of catalytic groups for catalysis (via thumb closure), but incorre ct base pairing will not. The structures also reveal that pol beta bin ds both gapped and nicked DNA with a 90 degrees kink occurring precise ly at the 5'-phosphodiester linkage of the templating residue. If the DNA were not kinked in this way, contact between the thumb and dNTP-te mplate base pair, presumably important for the checking mechanism, wou ld be impossible, especially when the gap is but a single nucleotide. Such a 90 degrees kink may be a mechanistic feature employed by any po lymerase involved in filling gaps to completion.