W0246

The Crenarchaeal Aeropyrum pernix Flap Endonuclease–1 (FEN-1) DNA Repair Enzyme at 1.4Å Resolution. Steven J. Tomanichek, Brandon K. Collins, and Timothy C. Mueser, Dept. of Chemistry, The Univ. of Toledo, Toledo, OH 43606.

The flap endonuclease-1 (FEN-1) enzymes are structure specific 5’ to 3’ endonucleases that recognize and act on three stranded DNA substrates (flap DNA). FEN-1 enzymes are involved in the processing of Okazaki fragment primers during lagging-strand DNA synthesis and in processing strands displaced during DNA synthesis associated with repair. Previous X-ray crystallographic studies on this family of enzymes have yet to determine the substrate recognition and the role of divalent metal ions in the catalytic mechanism of these enzymes. We have crystallized the native metal free Aeropyrum pernix (Ape) flap endonuclease enzyme (FEN-1) (see B. Collins et al poster for details). An initial 1.9 Å X-ray diffraction data set was solved using the Pyrococcus furiosus (Pfu) flap endonuclease-1 (1B43, 2.00 Å resolution). The Pfu FEN-1 model, positioned in the Ape FEN-1 crystal lattice using AMORE, was transformed into the Ape FEN-1 sequence using the threading program LOOK and then refined using CNS to generate an initial composite omit electron density map. The model is currently under refinement with 85% of the model built. We have recently been able to collect high-resolution (1.4 Å) data and refinement will be extended to this higher resolution. The Ape FEN-1 structure is the first FEN-1 enzyme solved from a Crenarchaeal organisms and is the highest resolution (1.4 Å) structure of a FEN-1 enzyme solved to date. The crystal structure of related enzyme, the bacteriophage T4 RNase H (see Mueser et al poster for details), has two divalent metals bound in the active site. The metal free Ape FEN-1 crystals have large solvent channels that create open access to the active site. The metal free crystals have been soaked in several different divalent metals and data has been collected. Analysis of the metal binding is pending; difference maps will be generated upon completion of the native structure. These results and the comparison of the Ape FEN-1 structure to other members of the RAD2/RAD27 family of enzymes will be presented.