Mutational analysis ofsimian virus 40 large T antigen DNA binding ...

K.AJones, R.M.Myers and R.Tjian
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Fig. 1. DNase I footprint protection of wild-type SV40 origin sequences by D2 protein. A DNA fragment containing the EcoRII-G fragment of the SV40
origin was 5' end-labelled at either the EcoRI site (late strand) or at the HindlII site (early strand), mixed with various concentrations of D2 protein and
treated with DNase. The resulting DNA fragments were subjected to denaturing polyacrylamide gel electrophoresis and analyzed by autoradiography. The
fragment labelled on the late strand (left panel) was incubated with D2 protein at the following concentrations: 0 nM (lane 1), 6.25 nM (lane 2), 12.5 nM
(lane 3), 18.75 nM (lane 4), 25 nM (lane 5) and 37.5 nM (lane 6). The fragment end-labelled at the early strand (right panel) was incubated with D2 protein at
the following concentrations: 0 nM (lane 1), 12.5 nM (lane 3) and 25 nM (lane 4); lane 2 displays guanine sequence markers for this strand. A schematic
representation of the SV40 origin fragment is presented below the panels.
ing sites contain two hexanucleotide contact sequences
(G)GCCTC, located 6 bp apart in site I. Within site II, one
hexanucleotide sequence is in an inverted orientation and two
turns of the helix away from the site I contacts (assuming a
B-DNA helical conformation), and a second set of weaker
contacts is located at an additional distance of 5 bp and
follows the same orientation as the site I contacts. The effects
of binding site sequence alterations on the recognition by T
antigen have now been evaluated using DNase footprint and
filter binding techniques (Galas and Schmidt, 1978; Myers
and Tjian, 1980). The mutant binding site templates used in
this study are illustrated in Figure 2. These mutations fall into
four classes: deletions that extend into site I sequences (dl 2,
3, 4); deletions extending into site II sequences from both early
and late directions (dl 22, 18, 23, A, 12) and an external site
II deletion (dl 1); internal site II substitutions (X 8, pIN 4);
and insertion mutants that separate binding sites I and II (pIN
43, 41).
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Binding to site I: effect of binding site deletions
The interaction between T antigen and residual site I sequences
in deletion mutants dl 2, dl 3 and dl 4 was examined
using both the nitrocellulose filter binding and DNase I footprinting
techniques. A template lacking early site I sequences
to base pair 5192 was found to bind T antigen with an efficiency
of 95% that of a wild-type template (containing all
three binding sites) in the nitrocellulose filter assay (mutant
dl 4, Figure 3). In contrast, deletion from the late side of site I
to base pair 5207 or 5195 reduced binding in this assay (dl 2,
dl 3; Figure 3). Mutant dl 2 removes only one guanine contact
from site I sequences, whereas the more severely affected mutant
dl 3 removes one complete hexanucleotide contact sequence
plus intervening nucleotides between the two site I
hexanucleotides. These results were confirmed by DNase
footprinting shown in Figure 4. The binding of T antigen to
site I in mutant dl 4, which retains all contact nucleotides,
confers protection of the 9 bp of bacterial DNA replacing the
deleted SV40 site I sequences. Binding to the extensively
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