Search

Research has been conducted on the ribonucleoside analogue 1-beta-D-ribofuranosyl-3-nitropyrrole. The results of the analogue's chemical synthesis, biochemical analysis, antiviral evaluation and structural characterization are presented.

Results reveal that the virus helicases displace streptavidin only from the 5'-ends of oligonucleotides and this feature of the enzymes with the ability to displace only from one end of biotin-labeled oligonucleotides accounts for the directional bias in translocation on single-stranded DNA.

Investigations of dihydrofolate reductase (DHFR) from Escherichia coli have shown that glycine-121 has large-amplitude backbone motions on the nanosecond time scale. The dynamic-function relationships of this residue were studied by the formation of a mutant enzyme in which glycine was replaced by valine. The Val-121 mutant retained wild-type binding characteristics with respect to dihydrofolate and tetrahydrofolate. Results indicated the existence of dynamic coupling of a remote residue to kinetic activity at the active site of DHFR.

The endonuclease and directional processing activity of the wild-type ribonuclease H (RNase H) is similar to both Mg2+ and Mn2+, but the RNase H activity is seen only in the presence of Mn2+. Substitution of the residue Glu478 with Gln makes the enzyme inactive towards Mg2+ and this inactivity can be removed with the addition of Mn2+. This mutant enzyme is capable to catalyze the removal of the tRNA replication primer but its efficiency is less than that of the wild-type protein.

The single-stranded oligonucleotide substrates containing a fluorescent probe on the 5' end was used to examine the DNA translocase, DNA unwinding, and protein displacement activity of nonstructural protein helicase (NS3h) encoded by hepatitis C virus. The NS3h was found to be translocated on ssDNA as a monomer, but the translocase activity does not correspond to comparable DNA unwinding activity or protein displacement activity under identical conditions.

The stoichiometry of the newly developed NS3-NS5B oligomeric protein complex which could be used for treatment of liver diseases caused by Hepatitis C virus (HCV) is described.

A kinetic and thermodynamic analysis of 3D(super pol) derivatives containing substitutions in the ribose-binding pocket with ATP analogues containing correct and incorrect sugar configurations is performed. It is found that Asp-238, a residue in structural motif A that is conserved in all RNA-dependent RNA polymerases, is a key determinant of polymerase fidelity.

The kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3D(super pol) in the presence of Mn(super 2+) are presented. The tools to characterize the complete kinetic mechanism of single nucleotide incorporation catalyzed by 3D(super pol) are developed.

We have solved the complete kinetic mechanism for correct nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3Dpol. The phosphoryl-transfer step is flanked by two isomerization steps. The first conformational change may be related to reorientation of the triphosphate moiety of the bound nucleotide, and the second conformational change may be translocation of the enzyme into position for the next round of nucleotide incorporation. The observed rate constant for nucleotide incorporation by 3Dpol (86 s-1) is dictated by the rate constants for both the first conformational change (300 s-1) and phosphoryl transfer (520 s-1). Changes in the stability of the "activated" ternary complex correlate best with changes in the observed rate constant for incorporation resulting from modification of the nucleotide. With the exception of UTP, the Kd values for nucleotides are at least 10-fold lower than the cellular concentration of the corresponding nucleotide. Our data predict that transition mutations should occur at a frequency of 1/15000, transversion mutations should occur at a frequency of less than 1/150000, and incorporation of a 2'-deoxyribonucleotide with a correct base should occur at a frequency 1/7500. Together, these data support the conclusion that 3Dpol is actually as faithful as an exonuclease-deficient, replicative DNA polymerase. We discuss the implications of this work on the development of RNA-dependent RNA polymerase inhibitors for use as antiviral agents. [ABSTRACT FROM AUTHOR]

The use of Mn2+ as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg2+ often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3Dpol) in the presence of Mn2+. The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg2+ and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase- primer/template-nucleotide complex. In the presence of Mn2+, the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn2+, it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nupleotide-selection process in the presence of Mg2+, highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn2+ to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg2+, this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn2+ may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg2+. [ABSTRACT FROM AUTHOR]