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Figure 1.
Fig. 1. The product distribution of T5 exonuclease
reaction varies with pH. (A) Activity was assayed on the
pseudo-Y substrate, with 10 mM MgCl[2] cofactor. Lanes marked M
show the result of reactions in mixtures that contained no
enzyme, and lanes 1-6 contained 4 nM wild-type enzyme in buffers
at pH 5.5, 6.0, 7.0, 8.0, 9.3, and 11.3, respectively. The sizes
(nucleotides) of the products are as indicated. (B) Time courses
of wild-type 5'-3' exonuclease at pH extremes. Wild-type
exonuclease, at a concentration of 0.8 nM, was incubated with
the labeled pseudo-Y substrate at 37°C for 2.5, 5, or 10 min
at pH 5.5 (lanes 1-3) or pH 9.3 (lanes 4-6) in the presence of
10 mM MgCl[2]. The products of the reaction were separated on a
7 M urea/15% acrylamide gel. Untreated substrate, lane M, and
substrates incubated at 37°C for 10 min at each pH in the
absence of enzyme (lanes 7 and 8) are shown. (C) Phosphoimager
data from A showing the percentage of product plotted against pH
for both exonucleolytic (  ) and
endonucleolytic (  )
cleavage. (D) Graphical representation of the time course at pH
5.5 and 9.3 from data obtained from B. Endonucleolytic product
is shown by dark bars, exonucleolytic product, by light bars.
Differences between C and D in the absolute levels of exo- and
endonuclease activity reflect the concentration of the enzyme
used. In C all of the original substrate has been degraded.
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