CRYSTAL-STRUCTURES OF HUMAN DNA-POLYMERASE-BETA COMPLEXED WITH DNA - IMPLICATIONS FOR CATALYTIC MECHANISM, PROCESSIVITY, AND FIDELITY

Mammalian DNA palymerase beta (pol beta) is a small (39 kDa) DNA gap-f illing enzyme that comprises an amino-terminal 8-kDa domain and a carb oxy-terminal 31-kDa domain. In the work reported here, crystal structu res of human poi beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pal beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexe d with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kuma r, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume onl y a small number of stable conformations. The thumb subdomain is in a more open position in the human poi beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb up on nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. In termolecular contacts between the DNA and the 8-kDa domain of a symmet ry-related pol beta molecule reveal a plausible binding site on the 8- kDa domain far the downstream oligonucleotide of a gapped-DNA substrat e; in addition to a lysine-rich binding pocket that accommodates a 5'- PO4 end group, the 8-kDa domain also contains a newly discovered helix -hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa doma in. DNA binding by both HhH motifs is facilitated by a metal ion, In t hat HhH motifs have been identified in other DNA repair enzymes and DN A polymerases, the HhH-DNA interactions observed in pol beta may be ap plicable to a broad range of DNA binding proteins. The sequence simila rity between the HhH motif of endonuclease III from Escherichia coli a nd the HhH motif of the 8-kDa domain of pol beta is particularly strik ing in that all of the conserved residues are clustered in one short s equence segment, LPGVGXK, where LPGV corresponds to a type II beta-tur n (the hairpin turn), and GXK corresponds to a part of the HhH motif t hat is proposed to be critical for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa dom ain of pol beta may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities . A model for productive binding of pol beta to a gapped-DNA substrate requires a 90 degrees bend in the single-stranded template, which cou ld enhance nucleotide selectivity during DNA repair or replication.