Amalendra Kumar

Role of the "Helix Clamp" in HIV-1 Reverse Transcriptase Catalytic Cycling as Revealed by Alanine-scanning Mutagenesis

Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the “helix clamp” (Hermann, T., Meier, T., Götte, M., and Heumann, H.(1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two α-helices (αH and αI) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in αH make important interactions with nucleic acid and influence frameshift fidelity. To define the role of αI (residues 278-286) during catalytic cycling, we performed systematic site-directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine. Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized. In contrast to alanine mutants of αH, alanine substitution in αI did not have a significant effect on template•primer (T•P) binding as revealed by a lack of an effect on Km,T−P, Ki for 3′-azido-2′,3′-dideoxythymidine 5′-triphosphate, koff,T−P, and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of αI lowered the apparent activity of every mutant relative to wild-type enzyme. Titration of two mutants exhibiting the lowest activity with T•P (L282A and R284A) demonstrated that these mutant enzymes could bind T•P stoichiometrically and tightly. In contrast, active site concentrations determined from “burst” experiments suggest that the lower activity is due to a smaller population of enzyme bound productively to T•P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent. In contrast, interactions between several residues of αH and the DNA minor groove, 3-5 nucleotides from the 3′-primer terminus, are suggested to be critical for DNA binding and fidelity.

The Basic Amino Acid-Rich DNA-Binding Element of the NF-IL6 Transcription Factor Contains Two Functionally Distinct Subdomains.

Nuclear factor-interleukin-6 (NF-IL6), a human transcription factor of the CCAAT-box/enhancer binding protein (C/EBP) family, is widely implicated as a “master regulator” of hepatic acute-phase response and inflammatory cytokine gene expression. NF-IL6 contains, at its N-terminus, an alanine- and proline-rich transactivation domain, followed at the C-terminus by a basic domain-leucine zipper (bZIP) DNA-binding motif. Understanding how the NF-IL6 transcription factor interacts with DNA is fundamental to its role as a transactivator because sequence-specific DNA-binding is prerequisite to promoter activation. We review our findings on the identification of the minimal “core” DNA-binding domain and the discovery of a novel bZIP element that influences DNA-binding kinetics. Manipulation of this domain allows for design of molecules that can be used as competitive antagonists or targeted delivery of molecules to selected regions of the eukaryotic genome.