Our findings indicated that marrow histologic findings alone do not reliably predict the probability of HLH, and an isolated finding of hemophagocytosis, even when present in a high amount, lacks specificity for HLH.

Four conserved amino acids of type IB topoisomerases (Arg130, Lys167, Arg223, and His265 in vaccinia topoisomerase) catalyze the attack by tyrosine on the scissile phosphodiester to form a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The mechanism entails general acid catalysis (by Lys167 and Arg130) and transition-state stabilization (via contact of His265 with the pro-Sp oxygen). Here we query the function of Arg223, which accelerates transesterification by a factor of 10(5). The requirement for Arg223 is alleviated by a neutral Sp methylphosphonate (MeP) linkage at the cleavage site. Arg223 is not required for the 30,000-fold activation of the latent endonuclease activity of topoisomerase by the Sp MeP. The rate of autohydrolysis by the DNA-(3'-MeP)-topoisomerase intermediate approaches 10% of the rate of religation to a 5'-OH DNA strand. These findings underscore the importance of transition-state electrostatics in determining the composition of the active site and dictating the balance between strand transferase and hydrolase functions.

Vaccinia topoisomerase IB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at its target site 5'-CCCTTp downward arrow in duplex DNA. The contributions of backbone electrostatics and individual phosphate oxygens to the transesterification reaction were probed by introducing 22 single Rp and Sp methylphosphonate diastereomers at 11 positions flanking the cleavage site. Methyl groups at eight positions (four on the scissile strand and four on the nonscissile strand) inhibited the rate of single-turnover cleavage by factors of 50-50,000. Stereospecific interference was observed at several phosphates, thereby distinguishing simple electrostatic contributions from putative specific polar contacts to either the pro-Sp or pro-Rp oxygens. The functionally relevant phosphate oxygens are located on the minor groove face of the helix on which the scissile phosphodiester resides. Our findings, combined with available crystal structures of vaccinia and human topoisomerase IB, show how specific phosphate contacts remote from where chemistry occurs are critical for assembly of the active site.

The RNA triphosphatase component (CaCet1p) of the mRNA capping apparatus of the pathogenic fungus Candida albicans differs mechanistically and structurally from the RNA triphosphatase of mammals. Hence, CaCet1p is an attractive antifungal target. Here we identify a C-terminal catalytic domain of CaCet1p from residue 257 to 520 and characterize a manganese-dependent and cobalt-dependent NTPase activity intrinsic to CaCet1p. The NTPase can be exploited to screen in vitro for inhibitors. The amino acids that comprise the active site of CaCet1p were identified by alanine-scanning mutagenesis, which was guided by the crystal structure of the homologous RNA triphosphatase from Saccharomyces cerevisiae (Cet1p). Thirteen residues required for the phosphohydrolase activity of CaCet1p (Glu287, Glu289, Asp363, Arg379, Lys396, Glu420, Arg441, Lys443, Arg445, Asp458, Glu472, Glu474 and Glu476) are located within the hydrophilic interior of an eight-strand beta barrel of Cet1p. Each of the eight strands contributes at least one essential amino acid. The essential CaCet1p residues include all of the side chains that coordinate manganese and sulfate (i.e., gamma phosphate) in the Cet1p product complex. These results suggest that the active site structure and catalytic mechanism are conserved among fungal RNA triphosphatases.

Type IB topoisomerases cleave and rejoin DNA strands through a stable covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. The stability of the intermediate is a two-edged sword; it preserves genome integrity during supercoil relaxation, but it also reinforces the toxicity of drugs and lesions that interfere with the DNA rejoining step. Here, we identify a key determinant of the stability of the complex by showing that introduction of an Sp or Rp methylphosphonate linkage at the cleavage site transforms topoisomerase IB into a potent endonuclease. The nuclease reaction entails formation and surprisingly rapid hydrolysis of a covalent enzyme-DNA methylphosphonate intermediate. The approximately 30,000-fold acceleration in the rate of hydrolysis of a methylphosphonate versus phosphodiester suggests that repulsion of water by the DNA phosphate anion suppresses the latent nuclease function of topoisomerase IB. These findings expose an Achilles' heel of topoisomerases as guardians of the genome, and they have broad implications for understanding enzymatic phosphoryl transfer.