Zuzana Hostomska

The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site.

During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.

2.3 Å crystal structure of the catalytic domain of DNA polymerase β

The crystal structure of the catalytic domain of rat DNA polymerase beta (pol beta) has been determined at 2.3 A resolution and refined to an R factor of 0.22. The mixed alpha/beta protein has three subdomains arranged in an overall U shape reminiscent of other polymerase structures. The folding topology of pol beta, however, is unique. Two divalent metals bind near three aspartic acid residues implicated in the catalytic activity. In the presence of Mn2+ and dTTP, interpretable electron density is seen for two metals and the triphosphate, but not the deoxythymidine moiety. The principal interaction of the triphosphate moiety is with the bound divalent metals.

Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently inEscherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved inde novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed inE. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced inE. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.

11 Ribonucleases H

I. INTRODUCTION Ribonuclease H (RNase H) (Ribonucleate [in RNA:DNA hybrids] 5′-oligonucleotidohydrolase, E.C.3.1.26.4) specifically cleaves the RNA strand in an RNA:DNA hybrid duplex. The designation H (for hybrid) was introduced by Hausen and Stein (1970) to describe a new ribonuclease activity discovered in extracts from calf thymus (Stein and Hausen 1969). All known nucleases with RNase H activity require divalent metal cations for hydrolysis of the RNA phosphodiester bond producing 5′-phosphate and 3′-hydroxyl termini. The presence of the free 3′-hydroxyl terminus makes the resulting RNA fragment suitable as a primer for DNA polymerase. RNases H comprise a family of ubiquitous enzymes, detected in multiple forms in all organisms from bacteria to mammals. However, with the exception of their well-understood function in the life cycle of retroviruses, the biological roles of other cellular RNases H are much less clear. In addition to their presumed involvement in DNA replication (and the practical usefulness of the isolated enzyme as a reagent for in vitro manipulations with nucleic acids), two other factors with possible therapeutic implications have contributed to the recent increased interest in RNases H: (1) the presumed role of endogenous RNase H activity in the mechanism of action of antisense oligonucleotides and (2) the potential importance of the enzyme as an antiviral target, especially in connection with human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). Since the first comprehensive overview of RNases H by Crouch and Dirksen (1982), two more recent reviews have appeared (Crouch 1990; Wintersberger 1990) that deal with...