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., Gotte, M., and Heumann, H. (1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two alpha-helices (alpha H and alpha I) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in alpha H make important interactions with nucleic acid and influence frameshift fidelity. To define the role of alpha I (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 alpha H, alanine substitution in alpha I 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 alpha I 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 populations 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 alpha H and the DNA minor groove, 3-5 nucleotides from the 3'-primer terminus, are suggested to be critical for DNA binding and fidelity.
Beard
W A
WA
Sealy Center for Molecular Sicence, University of Texas Medical Branch, Galveston 77555-1068, USA.
Minnick
D T
DT
Wade
C L
CL
Prasad
R
R
Won
R L
RL
Kumar
A
A
Kunkel
T A
TA
Wilson
S H
SH
D000409:Alanine / Q000235:genetics*; D001483:Base Sequence; D002384:Catalysis; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism
D013942:Thymine Nucleotides; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D000409:Alanine; C024157:thymidine 5'-triphosphate