W0247

T4 RNase H, a 5’ – 3’ exonuclease encoded by the bacteriophage T4, is a member of the RAD2 family of enzymes. This family includes the prokaryotic polymerase associated 5’ to 3’ nucleases and the eukaryotic flap endonucleases (FEN-1s). The T4 RNase H plays a vital role in bacteriophage T4 DNA replication by removing the RNA primers of Okazaki fragments on the lagging strand. (Bhagwat and Nossal, J. Biol. Chem. 2001) We have previously reported the 2.06 Å crystal structure of the full-length native enzyme with two fully hydrated magnesium ions present in the active site (Mueser et al., Cell, 1996). Sequence alignment and mutational analysis clearly define the catalytically important residues. The majority of the highly conserved residues are acidic residues clustered around the active site metals. We present here the crystal structures of the metal free native enzyme at 1.8Å resolution and a metal free active site mutant at 1.55Å resolution. The new crystal form was discovered when the metal content of native crystallization conditions was eliminated. Most crystals grown in the absence of metal diffract poorly, but approximately 1 crystal in 50 tested display the high resolution diffraction. Contrary to the anticipated result, the metal free structures have increased intrinsic order. The active site appears more compact in the absence of the metals with basic residues repositioned to replace the stabilizing magnesium counter ions. The bridge residues positioned above the active site, present in structures of related proteins but disordered in native T4 RNase H, is clearly seen in the new metal free structures. A comparison between metal bound and metal free enzyme structures and with related enzymes will be presented.