Nucleic Acids Research, Vol 25, Issue 12 2336-2343, Copyright © 1997 by Oxford University Press
NV Kumar and U Varshney
Excision of uracil from tetraloop hairpins and single stranded
('unstructured') oligodeoxyribonucleotides by Escherichia coli uracil DNA
glycosylase has been investigated. We show that, compared with a single
stranded reference substrate, uracil from the first, second, third and the
fourth positions of the loops is excised with highly variable efficiencies
of 3.21, 0.37, 5.9 and 66.8%, respectively. More importantly, inclusion of
E.coli single stranded DNA binding protein (SSB) in the reactions resulted
in approximately 7-140-fold increase in the efficiency of uracil excision
from the first, second or the third position in the loop but showed no
significant effect on its excision from the fourth position. In contrast,
the presence of SSB decreased uracil excision from the single stranded
('unstructured') substrates approximately 2-3-fold. The kinetic studies
show that the increased efficiency of uracil release from the first, second
and the third positions of the tetraloops is due to a combination of both
the improved substrate binding and a large increase in the catalytic rates.
On the other hand, the decreased efficiency of uracil release from the
single stranded substrates ('unstructured') is mostly due to the lowering
of the catalytic rates. Chemical probing with KMnO4showed that the presence
of SSB resulted in the reduction of cleavage of the nucleotides in the
vicinity of dUMP residue in single stranded substrates but their increased
susceptibility in the hairpin substrates. We discuss these results to
propose that excision of uracil from DNA-SSB complexes by uracil DNA
glycosylase involves base flipping. The use of SSB in the various
applications of uracil DNA glycosylase is also discussed.
ARTICLES
Contrasting effects of single stranded DNA binding protein on the activity of uracil DNA glycosylase from Escherichia coli towards different DNA substrates
Centre for Genetic Engineering, Indian Institute of Science, Bangalore 560 012, India.
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