(A) Mutagenesis in the Walker A motifs
of the p36, p37, and p40 subunits. The amino acid sequences of the
Walker A motifs in the p36, p37, and p40 subunits are aligned. The
conserved lysine residue (boxed) in each subunit was changed to alanine
by site-directed mutagenesis. (B) Purification of wt and
mutant p40·p37·p36 complexes. The purified wt and mutant
p40·p37·p36 complexes (described in Materials and
Methods) were analyzed by SDS/9% polyacrylamide gels. Lane
1, 3 μg of wt p40·p37·p36 complex; lanes 2–4, 3 μg of the
mutant p40·p37·p36 complex with a mutation in p36 (p36K66A), p37
(p37K84A), and p40 (p40K82A), respectively. (C) ATPase
activity of wt and mutant p40·p37·p36 complexes. ATPase
measurements were carried out as described in Materials and
Methods. Reaction mixtures contained wt and mutant
p40·p37·p36 complexes in amounts as indicated, in the presence of
12.5 μM (as nucleotides) φX174 ssc DNA.