Download Highly conserved features of DNA binding between two divergent

© 2001 Oxford University Press
Nucleic Acids Research, 2001, Vol. 29, No. 2
527–535
Highly conserved features of DNA binding between two
divergent members of the Myb family of transcription
factors
Benoît Pinson1,2,*, Elen M. Brendeford2, Odd S. Gabrielsen2 and Bertrand Daignan-Fornier1
1Institut
de Biochimie et Génétique Cellulaires, CNRS UMR5095, 1 Rue Camille Saint-Saëns, F-33077 Bordeaux
Cedex, France and 2Department of Biochemistry, University of Oslo, PO Box 1041 Blindern, N-0316 Oslo, Norway
Received August 4, 2000; Revised and Accepted November 7, 2000
ABSTRACT
Bas1p, a divergent yeast member of the Myb family of
transcription factors, shares with the proteins of this
family a highly conserved cysteine residue proposed
to play a role in redox regulation. Substitutions of this
residue in Bas1p (C153) allowed us to establish that,
despite its very high conservation, it is not strictly
required for Bas1p function: its substitution with a
small hydrophobic residue led to a fully functional
protein in vitro and in vivo. C153 was accessible to an
alkylating agent in the free protein but was protected
by prior exposure to DNA. The reactivity of cysteines
in the first and third repeats was much lower than in
the second repeat, suggesting a more accessible
conformation of repeat 2. Proteolysis protection,
fluorescence quenching and circular dichroism
experiments further indicated that DNA binding
induces structural changes making Bas1p less
accessible to modifying agents. Altogether, our
results strongly suggest that the second repeat of the
DNA-binding domain of Bas1p behaves similarly to its
Myb counterpart, i.e. a DNA-induced conformational
change in the second repeat leads to formation of a
full helix–turn–helix-related motif with the cysteine
packed in the hydrophobic core of the repeat.
INTRODUCTION
The Myb transcription factor family contains numerous
members from a wide spectrum of eukaryotic organisms as
phylogenetically distant as yeast and human. All the members
of the family are characterised by a conserved DNA-binding
region located in the N-terminal part of the protein (1,2). This
domain is composed of two or three imperfect tandem repeats
containing highly conserved tryptophan residues. These
repeats are predicted to form helix–turn–helix-related structures
which are critical for DNA binding (3–5). Indeed, several
mutations in these repeats have been obtained, in particular
replacement of tryptophans (6,7) and of residues in the third
‘recognition helix’ in each repeat, and shown to affect DNA
binding in vitro (3,4). A study of the yeast Saccharomyces
cerevisiae transcription factor Bas1p has revealed that mutations
in the tryptophan residues strongly impair function of the
protein both in vitro and in vivo (8). An interesting exception to
this rule is the CDC5 subfamily, which contains the Cef1p
protein from S.cerevisiae (9). Proteins from this subfamily are
involved in pre-mRNA splicing and there is no clear evidence
yet that they can act as transcription factors (10). Single mutations
of the tryptophan residues in the first or second repeat of Cef1p
did not affect function of the protein in vivo, while a combination
of three or more of these mutations resulted in loss of function.
Therefore, the conserved repeats are required for Cef1p function,
but somewhat less stringently than in other Myb-like proteins
which are known to bind DNA.
Besides the highly conserved tryptophan residues, it has
been found that the Myb proteins carry in their second repeat a
critical cysteine residue that is as conserved as the tryptophan
residues (Fig. 1). Reduction of this residue is essential for
c-Myb DNA binding in vitro (21–23) and it has been proposed
to act as a molecular sensor making Myb function sensitive to
redox conditions. A serine replacement of this cysteine in
v-Myb neither transactivated transcription in vivo nor transformed
myeloid cells, probably due to a defect in DNA binding (22).
In contrast, its replacement by a hydrophobic residue allowed
DNA binding in vitro and made the protein insensitive to
SH-specific alkylating agents such as N-ethylmaleimide (23).
This reactive residue has also been used to probe conformational
changes in the second repeat and it was shown that, in the presence of the Myb DNA-binding site, the conserved cysteine
became protected, probably due to a conformational change in
the second repeat (23). The role of this residue was also evaluated in vivo in c-Myb and Cef1p by changing it to a serine.
While this change affected Myb function in vivo (22), no effect
was detected for Cef1p (9). To further evaluate the role of this
highly conserved residue we have randomly mutated it in
another yeast Myb-like protein named Bas1p.
Bas1p is a transcription factor, the binding site of which covers
8–9 bp, including the core consensus sequence 5′-TGACTC-3′, as
judged by mutagenesis and by DNase I, missing base and DMS
footprinting analyses (11,24,25). Together with the homeodomain protein Bas2p (Pho2p), Bas1p activates expression of
genes in the histidine and purine biosynthesis pathways in
response to exogenous adenine (24,26–28). In a gcn4 background, mutations that abolish BAS1 function lead to histidine
auxotrophy and therefore BAS1 function can be easily scored
*To whom correspondence should be addressed at: IBGC CNRS UMR5095, 1 Rue Camille Saint-Saëns, 33077 Bordeaux Cedex, France. Tel: +33 5 56 99 90 19;
Fax: +33 5 56 99 90 59; Email: [email protected]
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Figure 1. Optimal sequence alignment of the second repeat of the DNA-binding domain of Bas1p and repeats in other various Myb family members. Letters on
the right refer to the species from which the sequences used for alignment were extracted. Sc, Saccharomyces cerevisiae; Gg, Gallus gallus; Xl, Xenopus laevis;
Sp, Schizosaccharomyces pombe; Al, Aspergillus nidulans; Dm, Drosophila melanogaster; Hs, Homo sapiens; Dd, Dictyostelium discoideum; At, Arabidopsis
thaliana. The identical and the highly conserved residues in all sequences aligned are marked by the symbols * and ¤, respectively. The conserved cysteine residue
(C153 in Bas1p) is boxed. Numbers on the right refer to the position in the protein sequence.
in vivo (26). Furthermore, expression of the Bas1p target genes
can be monitored, allowing a more quantitative estimation of
Bas1p function. We took advantage of these properties to study
in vivo the conserved cysteine in the Bas1p second repeat.
MATERIALS AND METHODS
Yeast strains and media
Yeast strains used in this study were L3080 (MATa gcn4-2
ura3-52 bas1-2) and Y329 (MATa leu2-3,112 ura3-52 gcn4-2
bas1-2). Yeast were grown in SD (2% w/v glucose, 0.17% w/v
nitrogen base and 0.5% w/v ammonium sulphate), SD-CASA
(SD supplemented with 0.2% w/v casamino acids from Difco
Laboratories) or SC [SD supplemented with amino acids as
described by Sherman et al. (29)].
Oligonucleotides
The following oligonucleotides were used for BAS1 sitedirected mutagenesis: 73, 5′-TGGAGGAACAGAGGATCAANNNGCGAAAAGGTACATTGAA-3′; 94, 5′-CCTGATTGGTGGAGGTGC-3′. For PCR amplification of the ADE5,7
probe used in electrophoretic mobility shift assays (EMSAs)
the following oligonucleotides were used: 125, 5′-CGCCCCGTCGGTAG-3′; 126, 5′-AGTTCAAGCCCATCGC-3′.
The oligonucleotides used for radiolabelled ADE1 and ADE17
probes in northern blot experiments were: 36, 5′-CGCCCCGGGTTAGTGAGACCATTTAGA-3′; 41, 5′-CGCAGATCTTAATGTCAATTACGAAAGA-3′; 53, 5′-GCTGGTTGATGGAAAATA-3′; 54, 5′-TGCATGCACAGCAGGGTG-3′. Oligonucleotides used in protease treatment, fluorescence quenching
and circular dichroism (CD) experiments were 5′-AGTGCCGACTGACTCGTGTCCTGG-3′ and 5′-CCAGGACACGAGTCAGTCGGCACT-3′ for the ADE5,7 specific probe and
5′-GCATTAATAACGGTTTTTTAGCGC-3′ and 5′-GCGCTAAAAAACGCTTATAATGC-3′ for the unrelated probe
(MRE from Myrset et al.; 23).
Site-directed mutagenesis of BAS1
The plasmid used for Bas1p expression in yeast is termed P79.
This centromeric plasmid carrying the URA3 and BAS1 genes
was previously described (8). All the mutants described below
were constructed in the P79 vector and were verified by
sequencing. All the Bas1p C153 mutants were obtained by the
gap repair method (30) as follows. Yeast strain L3080 was
transformed with either plasmid P79 linearised with XhoI or
plasmid P838 (31) linearised with BssHII and with a PCR
fragment obtained using P79 as template with oligonucleotides
73 and 94 (oligonucleotide 73 contained a randomised
sequence at the cysteine codon). C153K (P931) and C153D
(P932) mutant plasmids were extracted by the method of
Robzyk and Kassir (32) from yeast clones unable to grow on
SC medium lacking uracil and histidine, whereas C153S
(P925), C153A (P926) and C153V (P928) mutant plasmids
were extracted from yeast clones able to grow on the same
medium. The C82A (P1014), C206V (P1152), C82AC153V
(P1067) and C82AC153VC206V (P1163) mutants were
obtained as described (31). The remaining double mutants
were obtained in two steps. First, the P1152 BamHI–XhoI
fragment was replaced by the BamHI–XhoI fragment from
pLU12 (33) to give plasmid P1154. The mutants C82AC206V
and C153VC206V were then obtained by replacement of the
P1154 BamHI–XhoI fragment by the BamHI–XhoI fragment
from P1014 and P928, respectively. The resulting plasmids
were named P1161 and P1158, respectively.
GST–HA–Bas1p fusion construction and purification
The plasmid used for the wild-type GST–HA–Bas1p fusion,
termed P841, has been described previously (8). To facilitate
construction of the various GST–HA–Bas1p mutants, a derivative
plasmid of P841 containing a kanamycin resistance-conferring
cassette was used (P870; 8). Plasmids carrying the various
mutant versions of the GST–HA–Bas1p fusion for the C82A,
C153R, C153D, C153K, C153S, C153A and C153V simple
mutants and the C82AC153V double mutant were obtained by
replacement of the BamHI–XhoI fragment of P870 by the
BamHI–XhoI fragment from P1040, P838, P932, P931, P925,
P926, P928 and P1089, respectively. The GST–HA–Bas1p
fusion for the C206V simple mutant and the C82AC206V and
C153VC206V double mutants were obtained by replacement
of the BamHI–BbuI fragment of P870 by the BamHI–BbuI
fragment from P1152, P1161 and P1158, respectively. The
GST–HA–Bas1p fusion for the C82AC153VC206V triple mutant
was constructed as described (31). All these GST–HA–Bas1p
fusions were purified from Escherichia coli on glutathione–
Sepharose 4B resin as described (8).
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Expression and DNA binding analysis of c-Myb proteins
Protein separation and detection
The minimal DNA-binding domain of the chicken c-Myb
protein, R2R3, and its mutant derivatives (23) were expressed
in E.coli using the T7 system (34) and proteins purified as
described (4). DNA binding was monitored by EMSA as
previously described (4), with the following modifications
when performed at defined temperatures. After complex
formation the binding reaction was loaded on a precast polyacrylamide gel (CleanGel; Amersham Pharmacia Biotech)
rehydrated in 0.5× TBE buffer and placed on a flatbed electrophoresis unit (Multiphore; Amersham Pharmacia Biotech)
equipped with a cooling plate connected to a thermostatic
circulator (Multitemp III; Amersham Pharmacia Biotech)
which provided cooling water at defined temperatures. Electrophoretic separation was performed at 200 V for 135 min
before the gel was exposed for autoradiography.
SDS–PAGE, western blot analyses and EMSAs were
performed as previously described (8).
Protease treatment of Bas1p
Protease treatment was performed on a Bas1p fragment
[containing the first 272 amino acid residues, designated
NB1B2B3C55 in Høvring et al. (25)] using a derivative of the
method used for c-Myb (23). Briefly, purified Bas1p protein
(0.25 µg) was incubated in 10 µl of TEN buffer (10 mM Tris–HCl,
pH 8, 1 mM EDTA, 50 mM NaCl) with or without DNA
(DNA:protein ratio 2:1 mol/mol) for 10 min at 37°C. Proteinase K
(0.25–2.5 ng in 10 µl of TEN buffer) was added to the sample
which was then incubated for a further 15 min at 37°C. The
reaction was stopped by addition of SDS loading buffer and
boiling for 2 min before loading on a 17.5% SDS–PAGE gel
(35). Proteins were revealed by a silver staining procedure (36). In
these experiments the specific DNA used was a double-stranded
oligonucleotide corresponding to a sequence in the promoter of
the ADE5,7 gene containing one Bas1p-binding site (37). The
unrelated DNA used was the MRE (23), which does not bind
Bas1p as determined by EMSA experiments (25).
Fluorescence spectroscopy
Fluorescence experiments were carried out as described for
c-Myb (23) with purified Bas1p protein used at a concentration
of 2 µM. The specific and unrelated DNAs used were the
ADE5,7 and MRE double-stranded oligonucleotides described
in the previous section.
Circular dichroism
CD spectra were recorded using a Jasco J-810 spectropolarimeter.
Measurements were performed at 23°C, using a quartz cuvette
with a path length of 0.1 cm and a protein concentration of
0.15 mg/ml in 20 mM sodium phosphate buffer. Samples were
scanned twice at 50 nm/min with a response time of 1 s and
band width of 1 nm over the wavelength range 193–240 nm.
The data were averaged and the spectrum of a protein-free
control sample with or without DNA was subtracted. Thermal
denaturation curves were determined by monitoring the change
in the CD value at 222 nm with a temperature slope of 1°C/min.
The α-helical content of Bas1p[1–272] was calculated after
smoothing (means-movement, convolution width 5) from
mean residual ellipticity at 222 nm ([θ]222) using the formula
ƒH = [θ]222/[–40 000(1–2.5/n)], where ƒH and n represent the
α-helical content and the number of peptide bonds, respectively (38).
Northern blots
Yeast strain Y329 was transformed with either an empty
plasmid as control (B836) or a plasmid carrying a wild-type
(P79) or C153A mutant (P926) BAS1 gene. Cells were grown
in SD-CASA to an OD600 of 0.5. RNA extraction, separation
by electrophoresis, capillary transfer on positively charged
nylon membrane, hybridisation and de-hybridisation were
performed as previously described (28). Each blot was probed
with a radiolabelled fragment specific for the gene studied. The
probes used were as follows. For ADE1 and ADE17 specific
probes were amplified by PCR using yeast genomic DNA as
template and, respectively, the following pairs of synthetic
oligonucleotides: 36 and 41 for ADE1, 55 and 56 for ADE17.
For ACT1 the probe corresponds to the ClaI–ClaI internal
fragment of the gene. Radiolabelling of the probes was done
using the random priming procedure (Ready to go DNA labelling beads; Amersham Pharmacia Biotech).
β-Galactosidase (β-Gal) activity
For β-Gal assays Y329 cells were co-transformed with a
plasmid carrying the lacZ fusion and either a centromeric
plasmid carrying the BAS1 gene (wild-type or C153A mutant)
or a corresponding centromeric empty plasmid. Six clones of
each transformation were grown overnight in SC medium and
then diluted at 0.1 OD600 in the same medium supplemented or
not with 0.15 mM adenine. After 6 h at 30°C β-Gal assays were
performed as described (8).
RESULTS AND DISCUSSION
The conserved C153 residue can be changed to a
hydrophobic residue without impairing Bas1p function
in vivo and in vitro
Random mutagenesis of residue C153 in Bas1p, using gap
repair in yeast (see Materials and Methods for experimental
details), allowed us to isolate six mutants of C153. These
mutants carried on a yeast expression vector were tested for
their ability to complement a bas1 chromosomal deletion. As
mentioned above, disruption of BAS1 in a gcn4 background
leads to a strict histidine requirement, most probably because
the HIS4 gene is not sufficiently expressed when these two
transcription factors are absent (26). The results presented in
Figure 2A show that three of the six mutations (the charged
replacements C153R, C153K and C153D) totally abolished
BAS1 function in vivo. The three remaining mutants (the
neutral replacements C153A, C153S and C153V) fully
complemented the bas1 disruption phenotype. The same six
mutations were introduced in the BAS1 E.coli expression
plasmid and purified mutant proteins were assayed for in vitro
binding to DNA by EMSA (Fig. 2B). While similar amounts of
mutant and wild-type proteins were used in this experiment
(Fig. 2C), the three mutations that impaired complementation
in vivo clearly abolished binding to DNA in vitro. Conversely,
the three mutants that complemented the histidine auxotrophy
bound DNA as efficiently as wild-type Bas1p. Finally, the
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Nucleic Acids Research, 2001, Vol. 29, No. 2
Figure 2. In vivo and in vitro effect of substitutions of the C153 residue of
Bas1p. (A) In vivo effect of mutations in the C153 codon of BAS1. Y329 cells
were transformed with either the B836 control plasmid (vector), the wild-type
BAS1 gene (WT) or the various C153 substitution mutants. Transformants in
exponential growth phase were resuspended in water. Three drops of cellular
suspension corresponding to serial dilutions of the cells (104, 103 and 102 cells)
were spotted on SC medium lacking uracil and supplemented (right) or not
(left) with histidine. Cells were grown for 36 h at 30°C. (B) In vitro DNA-binding
activity of Bas1p mutants. Purified GST–HA (control lane) or GST–HA–Bas1p
wild-type (WT) or C153 mutants (0.5 µg each protein) were incubated for 15 min
at room temperature with 100 fmol radiolabelled ADE5,7 promoter probe and
were then separated by 4% non-denaturing gel electrophoresis. The gel was dried
on paper and radioactivity was revealed by autoradiography. GST–HA–Bas1p/DNA
complexes are indicated by the black arrow. (C) Western blot analysis of wild-type
Bas1p and C153 mutants. Each purified GST–HA–Bas1p (4 µg each protein)
was subjected to 12.5% SDS–PAGE and electroblotting on PVDF membrane.
The blot was incubated with anti-HA antibodies (0.5 µg/ml) followed by
horseradish peroxidase-conjugated IgG (1:2500) as secondary antibodies and,
finally, luminescent substrate before exposure to film. The GST–HA protein
(25 kDa) in the control lane is much smaller than the GST–HA–Bas1p fusion
(115 kDa) and therefore does not appear in the figure. (D) Effect of C153A
substitution in Bas1p on ADE1 and ADE17 transcription. Strain Y329 was
transformed with plasmids B836 (vector), P79 (WT, wild-type BAS1) or P926
(C153A mutant). Transformants were grown in SD-CASA to an OD600 of 0.5.
Total RNA was extracted, separated by electrophoresis, transferred and hybridised with radiolabelled probes. Equal loading of each lane was monitored by
ethidium bromide staining of the gel showing the large (L. rRNA) and small
(S. rRNA) rRNAs and an ACT1-specific probe hybridisation. Each hybridisation
was done independently and assembled for the figure. (E) In vivo effect of
C153A substitution in Bas1p on expression of lacZ reporter fusions. Y329
cells were co-transformed with a plasmid containing the lacZ fusion and with
a centromeric plasmid harboring the BAS1 wild-type or C153A mutant gene. –Ade
and +Ade correspond to cells grown in the absence or presence of adenine,
respectively. The control lane (No Bas1p) corresponds to a transformation with
the B836 plasmid not containing the BAS1 gene. For each lacZ fusion β-Gal units
are expressed as per cent expression measured in the absence of adenine with the
wild-type (WT) BAS1 gene.
effect of the C153A mutation on expression of Bas1p target
genes was tested by northern blot on the ADE1 and ADE17
genes (Fig. 2D) and by measurement of expression of ADE1–lacZ,
ADE17–lacZ and HIS4–lacZ fusions (Fig. 2E). Both
approaches showed that the Bas1p C153A protein is fully
functional in vivo. These data establish that the highly
conserved C153 residue can be changed to small hydrophobic
residues without affecting function of the transcription factor
in vivo. Although our random mutagenesis of the C153 codon
was not exhaustive, it is noteworthy that in the screening
process we obtained three Cys→Ala, three Cys→Ser and four
Cys→Val substitutions, therefore suggesting that most of the
other substitutions might not lead to a functional transcription
factor in vivo. It is intriguing that this cysteine is so highly
conserved throughout evolution despite our demonstration here
that several substitutions of the cysteine are fully functional.
The corresponding cysteine residue in chicken c-Myb
(C130) was previously shown by Gabrielsen and co-workers
(23,39,40) to be in an unfolded region of the protein which, in
the presence of DNA, undergoes a conformational change,
folding into a ‘recognition’ helix with the cysteine moved into
the hydrophobic core of the protein. Our substitution data on
Bas1p C153 are consistent with a similar mechanism for
Bas1p, since mutations that change C153 into a small hydrophobic residue did not affect Bas1p function (binding to DNA
and activation of target genes) while replacing C153 by a
charged residue totally abolished binding of Bas1p to DNA
and its function in vivo. This strong structural and functional
conservation between Bas1p and c-Myb was further validated
by studying a specific feature of the Cys→Ser replacement in
both proteins. We noticed that the C153S mutation in Bas1p
led to only partial complementation of the histidine requirement at
37°C while complementation was total at 30°C (Fig. 3A). The
other two changes (C153A and C153V) were functional at
both temperatures. The same mutants and the wild-type protein
were assayed for DNA binding in vitro. Once again, a clear
correlation was found between DNA binding and complementation, i.e. the C153S mutant could only bind DNA at low
temperature while the other proteins could bind DNA at both
temperatures (Fig. 3B). Strikingly, the equivalent mutant
(C130S) in c-Myb led to a very similar DNA-binding defect.
The mutant was fully active at low temperature (10°C) but
gradually lost DNA binding at higher temperatures (25 and
35°C; Fig. 3C). Although the precise temperature responses of
the Cys→Ser mutants were slightly different, the similar ts
phenotypes of Ser replacements in the two proteins further
substantiates a strong structural and functional conservation
between the c-Myb and Bas1p DNA-binding domains. The
stronger temperature sensitivity of the c-Myb mutant may also
explain why the C65S mutant of v-Myb was without transactivation and transformation activity at 37°C (22), while the C153S
mutant of Bas1p was fully functional at 30°C.
Binding to DNA induces a conformational change in Bas1p
The results obtained in the previous section suggest that the
second repeat of the DNA-binding domain in Bas1p behaves
as in c-Myb, with a conformational change occurring during
DNA binding. To further support this hypothesis, we first used
the irreversible SH-specific alkylating agent N-ethylmaleimide
(NEM) to test the effect of C153 modification on DNA-binding
activity. Increasing concentrations of NEM were added to
Bas1p in EMSA experiments. Wild-type Bas1p binding to the
probe was abolished when 3 mM NEM was added to the
protein prior to addition of the probe, while binding of the
C153A (data not shown) and C153V (Fig. 4A) mutants was
totally insensitive to the alkylating agent. These results show
Nucleic Acids Research, 2001, Vol. 29, No. 2
Figure 3. Temperature sensitivity of Cys→Ser replacement in the second
repeat of the c-Myb and Bas1p DNA-binding domains. (A) In vivo effect of
mutations in the C153 codon of BAS1 at 37°C. Y329 cells were transformed
with either the B836 control plasmid (vector), the wild-type BAS1 gene or the
various C153 substitution mutants and treated as in Figure 2A, but cells were
grown at 37°C. The panel –His 30°C is a copy of the panel from Figure 2A
added to facilitate a comparison of growth between the two temperatures tested.
(B) In vitro DNA-binding activity of Bas1p mutants at different temperatures. Two
microlitres (0.5 µg protein) of purified GST–HA (control lane) or GST–HA–Bas1p
wild-type or C153 mutants were treated and analysed by EMSA as in Figure 2B at
20 and 37°C. (C) In vitro DNA binding activity of c-Myb mutants at different
temperatures. Purified Myb R2R3 proteins (10 fmol both wild-type and mutants
as indicated) were incubated with 10 fmol MRE probe at the indicated temperature
for 10 min. The complex formed was separated by native electrophoresis using a
thermostatic equipment set-up as described in Materials and Methods.
again that limited alterations are allowed in location of the C153
residue and suggest that, as shown for c-Myb, the irreversible
modification of the C153 residue caused by NEM could
interfere with the normal conformational change essential to
acquire the DNA-binding structure in Bas1p. Consistent with
this conformational hypothesis, addition of NEM to the
preformed Bas1p–DNA complex resulted in a much greater
resistance of the wild-type protein to the alkylating agent
(Fig. 4A). This DNA-induced protection of Bas1p from alkylation
is most easily explained by assuming a DNA-induced conformational change where C153 becomes inaccessible to the
modifying agent. We reasoned that if this is correct, Bas1p
531
should be in a more compact conformation when bound to
DNA and then could be more resistant to limited proteolysis
treatment. To directly test this, Bas1p, either free or complexed
to DNA, was subjected to partial proteolysis with proteinase K
(Fig. 4B). As expected, binding of Bas1p to a specific DNA
probe resulted in an increased resistance of Bas1p to proteolysis. This resistance was not observed in the presence of an
unrelated control DNA probe that gave a digestion pattern
highly similar to that observed in the absence of DNA. These
results strongly suggest that, as shown for c-Myb, Bas1p might
adopt a much more structured conformation when bound to
DNA. To further substantiate this hypothesis, the effect on
Bas1p structure of binding to DNA was analysed by fluorescence
quenching experiments. In these experiments the effect of
conformational changes in Bas1p during DNA binding was
monitored by measuring the accessibility of the tryptophan
residues to acrylamide, a fluorescence quenching agent
(Fig. 4C and D). The fluorescence quenching observed for
native free Bas1p (Fig. 4C, closed square) was found to be
significantly less than that observed for both guanidinium
chloride-denatured Bas1p (Fig. 4C, open circle) or free
tryptophan (Fig. 4C, open diamond), used here as a reference
for free accessibility. Addition of the ADE5,7 specific DNA
led to a further decrease in fluorescence quenching of Bas1p
protein (Fig. 4C, open triangle), while addition of the same
DNA had no significant effect on quenching of free tryptophan
(data not shown). Finally, addition of an unrelated DNA
(Fig. 4C, closed triangle) gave a quenching intermediate
between those observed for free Bas1p and Bas1p complexed
to the ADE5,7 probe. This intermediate behaviour could be due
to non-specific binding of Bas1p to the unrelated DNA probe
that would partially protect tryptophan residues against
acrylamide, thus leading to an intermediate level of quenching.
The same set of quenching experiments was done with Bas1p
treated with NEM at 5 mM, a condition abolishing DNA
binding (Fig. 4A). Under these conditions fluorescence
quenching was the same both in the presence and absence of
the specific DNA probe (Fig. 4D). Altogether, these results
strongly support the idea of a DNA-induced conformational
change in Bas1p. This was finally verified by CD analyses,
showing that DNA binding was coupled to a structural change
in Bas1p. CD spectra (Fig. 4E) of Bas1p[1–272] indicated
typical α-helical characteristics similar to what has been
reported for c-Myb R123 (41). Our finding of a 40% α-helical
content in Bas1p and an increase to 45% when the protein is
bound to specific DNA is also in good agreement with the
results obtained for c-Myb (41). No change in mean residual
ellipticity at 222 nm was observed upon complex formation
with the unrelated DNA (MRE) and the α-helix content
measured in this condition was 40% (spectrum not shown).
Finally, Figure 4F shows the thermal denaturation of Bas1p[1–272]
and Bas1p in complex with specific DNA measured by CD. It
is clear from this experiment that the stability of the protein–
DNA complex is higher compared to Bas1p alone. The melting
temperature (Tm = temperature at 50% unfolding) was 54°C for
Bas1p in complex with specific DNA, compared to 46°C
obtained for Bas1p alone. This increased α-helical content and
enhanced thermal stability strongly supports a structural
change in Bas1p occurring during DNA binding, as previously
described for the c-Myb oncoprotein.
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Nucleic Acids Research, 2001, Vol. 29, No. 2
The C153 residue in the second repeat is the most
accessible of the three cysteine residues in Bas1p,
suggesting a conserved difference in thermal stability
between the three repeats
Figure 4. A DNA-induced conformational change in Bas1p. (A) Sensitivity of
wild-type Bas1p and the C153A mutant to alkylation by NEM. (Lower) Two
microlitres (0.5 µg protein) of purified wild-type GST–HA–Bas1p were incubated
for 15 min at room temperature with increasing concentrations of NEM either
before (1.DNA, left of panel) or after (3.DNA, right) incubation with 100 fmol
radiolabelled ADE5,7 promoter probe. Samples were then analysed by EMSA
as in Figure 2B. (Upper) As above, but carried out with the GST–HA–Bas1p
C153V mutant. (B) Partial digestion of Bas1p, either free or complexed to
DNA, with proteinase K. Bas1p[1–272] (0.25 µg) free (–DNA), in complex
with ADE5,7 DNA (+ADE5,7 DNA) or in complex with unrelated DNA
(+MRE DNA) was treated with increasing amounts of proteinase K. Proteins were
then separated by 17.5% SDS–PAGE and revealed by silver staining. (C and
D) Fluorescence quenching by acrylamide of free and DNA-bound Bas1p. The
fluorescence emission of 2 µM Bas1p was quenched by successive addition of
acrylamide to Bas1p treated (D) or not (C) with 5 mM NEM. In each condition
fluorescence was measured at the wavelength corresponding to the emission
maximum for Bas1p and the results are presented as the ratio between the
unquenched fluorescence (Fo) and the fluorescence measured after acrylamide
addition (F). Results correspond to the average of between six and nine
independent measurements. Inter-assay variation was found to be <15 %.
Closed square, free Bas1p; open triangle, Bas1p bound to ADE5,7 specific
DNA; closed triangle, Bas1p bound to unrelated DNA; open circle, Bas1p
denatured for 60 min in 6 M guanidinium chloride; open diamond, 10 µM free
tryptophan solution used as a reference. (E) CD spectrum of Bas1p[1–272]
either alone (thick line) or in complex with specific DNA (ADE5,7 DNA, thin
line). The protein concentration was 0.15 mg/ml in phosphate buffer and equimolar
concentrations of DNA were added. The α-helical content of Bas1p[1–272]
alone and in complex with specific DNA was calculated as described in Materials
and Methods and found to be 40 and 45%, respectively. (F) Thermal denaturation
curves of Bas1p[1–272] either alone (thick line) or in complex with specific
DNA (thin line). The apparent fraction of folded protein, obtained by monitoring
the CD value at 222 nm, is shown as a function of temperature.
Although the three homologous tandem repeats in c-Myb have
a high sequence similarity, they are not structurally equivalent.
Biophysical studies have shown that the second repeat has a
much lower thermal stability than the first and third repeats
(42). To determine whether this structural feature is conserved
during evolution, we took advantage of the specific cysteine
distribution along the Bas1p protein, having one cysteine in
each repeat of the Myb-like DNA-binding domain (8). We
reasoned that the use of oxidative modification combined with
systematic site-directed mutagenesis of these cysteine residues
should provide information on the accessibility and structural
stability of each of the repeats. First, the C82 and C206 residues,
in the first and third repeats, respectively, were mutated
in vitro. The resulting mutations were combined with the
C153V mutation to generate all combinations of single, double
and triple substitutions. These mutants were all shown to
complement the bas1 disruption (Fig. 5A), suggesting that the
cysteine residues in the first and third repeats are not essential
for Bas1p function, as shown for the C153 residue in the
second repeat in the previous sections. The corresponding purified
GST–HA–Bas1p proteins were then assayed by EMSA for
their ability to bind DNA in the presence of diamide, a thiol
oxidising agent that can be used to probe the function of
cysteine residues. The results presented in Figure 5B show that
mutations C82A and C206V, either alone or combined,
resulted in a wild-type sensitivity to diamide. C153V was
partially resistant to diamide up to 2 mM. Interestingly, the
C153V mutation combined with C82A or C206V showed
some DNA binding at high concentrations of diamide (up to
50 mM) (Fig. 5B). Finally, the triple mutant was fully resistant
to diamide, as expected if diamide treatment specifically
affects cysteine residues. A western blot of the various mutant
proteins (Fig. 5C) showed that similar amounts of proteins
were used in this experiment. The results presented in Figure 5
were interpreted as follows: when the C153 residue is present,
it confers on Bas1p wild-type sensitivity to diamide, while in
the absence of C153 the two other cysteine residues confer a
weak sensitivity to diamide. This reduced sensitivity to
diamide treatment of the cysteine residues in the first and third
repeats could be interpreted in different ways: (i) the cysteine
residues are on the surface, but in an environment where bulky
hydrophobic residues mask them from diamide treatment;
(ii) the domains corresponding to the first and third repeats are
in a more compact structure with the cysteine residues hidden
in the hydrophobic core of the protein. In both cases the
reduced sensitivity to diamide treatment could be explained by
a reduced accessibility of the cysteine residues to the oxidative
reagent. Thus a partial denaturation of Bas1p carrying only the
C82 or C206 residues should lead to a sensitivity to diamide
treatment equivalent to that of the wild-type protein. We therefore assayed the resistance of two double mutants carrying
only C82 or C206 after preincubation with 2 mM diamide at
45°C (instead of 25°C as in previous assays) to partially
denature Bas1p, as shown in Figure 4F. After oxidation the
proteins were slowly brought back to room temperature and
used for EMSA. The results presented in Figure 6A show that
Nucleic Acids Research, 2001, Vol. 29, No. 2
533
Figure 6. Effect of a partial denaturation on accessibility of the cysteine
residues in Bas1p. (A) Effect of increased temperature on oxidation of C82 and
C206 in Bas1p. Purified wild-type and mutant GST–HA–Bas1p proteins were
incubated with or without 1.5 mM diamide at the indicated temperature (either
25 or 45°C) and immediately allowed to slowly return to room temperature.
Samples were then incubated for 15 min with 100 fmol radiolabelled ADE5,7
promoter probe and analysed by EMSA as in Figure 2B. (B) Increased
temperature has no major effect on diamide reactivity. Purified wild-type
GST–HA–Bas1p was incubated with increasing concentrations of diamide at
either 25 or 45°C and immediately allowed to slowly return to room temperature.
Samples were then treated as in (A).
Figure 5. Comparison of the sensitivity to oxidation of the three cysteine
residues in Bas1p. (A) In vivo effect of mutations of the three Cys residues in
Bas1p. Y329 cells were transformed with either the B836 control plasmid
(vector), the wild-type BAS1 gene or the various Cys substitution mutants and
treated as in Figure 2A. (B) In vitro sensitivity to diamide of the different
cysteine mutants of Bas1p. Purified wild-type GST–HA–Bas1p or cysteine
mutants were incubated for 15 min at room temperature with increasing
concentrations of diamide. Samples were then incubated with radiolabelled
ADE5,7 promoter probe and analysed by EMSA as in Figure 2B. (C) Western
blot analysis of purified wild-type and mutant GST–HA–Bas1p. Purified wildtype GST–HA–Bas1p and mutants fusions (10 µl) were separated by 12.5%
SDS–PAGE and electrotransferred to PVDF membrane. GST–HA–Bas1p proteins
were revealed by western blot analysis as in Figure 2C. Lane 1, GST–HA control;
lanes 2–9, wild-type Bas1p and the C82A C153V C206V, C153V C206V,
C82A C206V, C82A C153V, C206V, C153V and C82A Bas1p mutants,
respectively.
the two mutants were insensitive to diamide at 25°C (as previously shown in Fig. 5A) but their binding to DNA was totally
abolished after preincubation with diamide at 45°C. The same
proteins preincubated at 45°C in the absence of diamide were
able to bind DNA normally, thus showing that the 45°C
preincubation did not alter the capacity of Bas1p to bind DNA.
As expected, the wild-type protein was sensitive to 2 mM
diamide at both 25 and 45°C and the triple mutant was totally
resistant under both temperature conditions (Fig. 6A). Finally,
we show (Fig. 6B) that increasing the temperature has no effect
on C153 oxidation, thus ruling out the possibility that diamide
would be a more efficient oxidant at 45°C. These results show
that the C153 residue in the second repeat is the most
accessible of the three cysteine residues in Bas1p and further
confirm a location of this residue in a more exposed region
than the other two cysteine residues. This strongly suggests
that the lowered thermal stability and the more open conformation of the second repeat described for c-Myb could be a
feature of the Myb-type DNA-binding domain important
enough to have been conserved during evolution from yeast to
vertebrates. Taken together, all these results demonstrate a
high structural and functional homology between the DNAbinding domain of both Bas1p and c-Myb oncoprotein.
In conclusion, our study of the conserved C153 residue in the
Bas1p DNA-binding domain shows that in this Myb-like
transcription factor C153 is more accessible to oxidising and
alkylating agents than the other two cysteines. This most
probably reflects a conserved structural feature in the three
tandem Myb repeats, with the second repeat being in a more
open disordered conformation than the other two and undergoing a change to a more ordered conformation upon binding
to DNA. Indeed, CD experiments show an increase in α-helical
content from 40 to 45%, corresponding to the expected folding
of one additional α-helix in B2, as previously stated for the c-Myb
oncoprotein (41). Our data establish that, despite its very strong
534
Nucleic Acids Research, 2001, Vol. 29, No. 2
conservation during evolution, the cysteine residue in the
second repeat of Myb proteins can be replaced by small hydrophobic amino acids without altering its function in vivo under
laboratory conditions. This leads to the conclusion that a
cysteine residue is not strictly required at this position in the
sequence and raises the question of why this residue should be
so highly conserved. We do not have any experimental
evidence for a selective advantage due to the presence of the
cysteine residue in repeat 2 of Bas1p, but sequence comparisons
of Myb proteins strongly suggest that the presence of this
residue conferring redox regulation is important under some
physiological conditions that remain to be identified. We
indeed observed redox regulation of expression of the Bas1p
target genes (31) but a Bas1p mutant protein deprived of
cysteine residues was unable to make transcription of these
target genes resistant to oxidative stress (31). Another puzzling
observation is the conservation of this cysteine residue among
the CDC5 members of the Myb family (see Fig. 1; 9). These
CDC5 proteins are known to be involved in pre-mRNA
splicing and may not be transcription factors. If this were the
case the role of this cysteine residue would not be restricted to
DNA binding. This residue has been changed to a serine in
Cef1p without any clear effect in vivo (9), again showing that a
cysteine is not strictly required at this position.
Our results also point to very strong conservation between
distant members of the Myb family, such as c-Myb and Bas1p.
Although sequence homology between the two transcription
factors is limited to the DNA-binding domain (11) and despite
the fact that the two proteins bind to different DNA sequences,
the mechanism of DNA binding appears highly conserved,
involving a disorder-to-order transition in the second repeat
moving the critical cysteine from an exposed to a hidden position
in the protein. Our results thus validate the yeast transcription
factor Bas1p as a model for the study of in vivo DNA binding of
Myb-like proteins.
ACKNOWLEDGEMENTS
We thank F. Borne for help with mutant constructs and Dimitris
Mantzilas and Anne Bostad Hegvold for their assistance with
CD and one EMSA experiment, respectively. This work was
supported by grants from the Conseil Régional d’Aquitaine,
CNRS (UMR5095), The Norwegian Research Council, The
Norwegian Cancer Society and the Anders Jahres Foundation.
B.P. was supported by a FEBS post-doctoral long-term fellowship.
REFERENCES
1. Ness,S.A. (1996) The Myb oncoprotein: regulating a regulator. Biochim.
Biophys. Acta, 1288, 123–139.
2. Oh,I.H. and Reddy,E.P. (1999) The myb gene family in cell growth,
differentiation and apoptosis. Oncogene, 18, 3017–3033.
3. Frampton,J., Gibson,T.J., Ness,S.A., Doderlein,G. and Graf,T. (1991)
Proposed structure for the DNA-binding domain of the Myb oncoprotein
based on model building and mutational analysis. Protein Eng., 4, 891–901.
4. Gabrielsen,O.S., Sentenac,A. and Fromageot,P. (1991) Specific DNA
binding by c-Myb: evidence for a double helix-turn-helix-related motif.
Science, 253, 1140–1143.
5. Ogata,K., Morikawa,S., Nakamura,H., Sekikawa,A., Inoue,T., Kanai,H.,
Sarai,A., Ishii,S. and Nishimura,Y. (1994) Solution structure of a specific
DNA complex of the Myb DNA-binding domain with cooperative
recognition helices. Cell, 79, 639–648.
6. Saikumar,P., Murali,R. and Reddy,E.P. (1990) Role of tryptophan repeats
and flanking amino acids in Myb-DNA interactions. Proc. Natl Acad. Sci.
USA, 87, 8452–8456.
7. Kanei-Ishii,C., Sarai,A., Sawazaki,T., Nakagoshi,H., He,D.N., Ogata,K.,
Nishimura,Y. and Ishii,S. (1990) The tryptophan cluster: a hypothetical
structure of the DNA-binding domain of the myb protooncogene product.
J. Biol. Chem., 265, 19990–19995.
8. Pinson,B., Sagot,I., Borne,F., Gabrielsen,O.S. and Daignan-Fornier,B.
(1998) Mutations in the yeast Myb-like protein Bas1p resulting in
discrimination between promoters in vivo but not in vitro.
Nucleic Acids Res., 26, 3977–3985.
9. Ohi,R., Feoktistova,A., McCann,S., Valentine,V., Look,A.T., Lipsick,J.S.
and Gould,K.L. (1998) Myb-related Schizosaccharomyces pombe cdc5p
is structurally and functionally conserved in eukaryotes. Mol. Cell. Biol.,
18, 4097–4108.
10. Burns,C.G., Ohi,R., Krainer,A.R. and Gould,K.L. (1999) Evidence that
Myb-related CDC5 proteins are required for pre-mRNA splicing.
Proc. Natl Acad. Sci. USA, 96, 13789–13794.
11. Tice-Baldwin,K., Fink,G.R. and Arndt,K.T. (1989) BAS1 has a Myb motif
and activates HIS4 transcription only in combination with BAS2. Science,
246, 931–935.
12. Gerondakis,S. and Bishop,J.M. (1986) Structure of the protein encoded by
the chicken proto-oncogene c-myb. Mol. Cell. Biol., 6, 3677–3684.
13. Sleeman,J.P. (1993) Xenopus A-myb is expressed during early
spermatogenesis. Oncogene, 8, 1931–1941.
14. Nomura,N., Takahashi,M., Matsui,M., Ishii,S., Date,T., Sasamoto,S. and
Ishizaki,R. (1988) Isolation of human cDNA clones of myb-related genes,
A-myb and B-myb. Nucleic Acids Res., 16, 11075–11089.
15. Ohi,R., McCollum,D., Hirani,B., Den Haese,G.J., Zhang,X., Burke,J.D.,
Turner,K. and Gould,K.L. (1994) The Schizosaccharomyces pombe
cdc5+ gene encodes an essential protein with homology to c-Myb. EMBO
J., 13, 471–483.
16. Wieser,J. and Adams,T.H. (1995) flbD encodes a Myb-like DNA-binding
protein that coordinates initiation of Aspergillus nidulans conidiophore
development. Genes Dev., 9, 491–502.
17. Peters,C.W., Sippel,A.E., Vingron,M. and Klempnauer,K.H. (1987)
Drosophila and vertebrate myb proteins share two conserved regions, one
of which functions as a DNA-binding domain. EMBO J., 6, 3085–3090.
18. Wong,M.W., Henry,R.W., Ma,B., Kobayashi,R., Klages,N., Matthias,P.,
Strubin,M. and Hernandez,N. (1998) The large subunit of basal
transcription factor SNAPc is a Myb domain protein that interacts with
Oct-1. Mol. Cell. Biol., 18, 368–377.
19. Stober-Grasser,U., Brydolf,B., Bin,X., Grasser,F., Firtel,R.A. and
Lipsick,J.S. (1992) The Myb DNA-binding domain is highly conserved in
Dictyostelium discoideum. Oncogene, 7, 589–596.
20. Kirik,V. and Baumlein,H. (1996) A novel leaf-specific myb-related
protein with a single binding repeat. Gene, 183, 109–113.
21. Guehmann,S., Vorbrueggen,G., Kalkbrenner,F. and Moelling,K. (1992)
Reduction of a conserved Cys is essential for Myb DNA-binding.
Nucleic Acids Res., 20, 2279–2286.
22. Grasser,F.A., LaMontagne,K., Whittaker,L., Stohr,S. and Lipsick,J.S.
(1992) A highly conserved cysteine in the v-Myb DNA-binding domain is
essential for transformation and transcriptional trans-activation.
Oncogene, 7, 1005–1009.
23. Myrset,A.H., Bostad,A., Jamin,N., Lirsac,P.-N., Toma,F. and
Gabrielsen,O.S. (1993) DNA and redox state induced conformational
changes in the DNA-binding domain of the Myb oncoprotein. EMBO J.,
12, 4625–4633.
24. Daignan-Fornier,B. and Fink,G.R. (1992) Coregulation of purine and
histidine biosynthesis by the transcriptional activators BAS1 and BAS2.
Proc. Natl Acad. Sci. USA, 89, 6746–6750.
25. Høvring,P.I., Bostad,A., Ording,E., Myrset,A.H. and Gabrielsen,O.S.
(1994) DNA-binding domain and recognition sequence of the yeast BAS1
protein, a divergent member of the Myb family of transcription factors.
J. Biol. Chem., 269, 17663–17669.
26. Arndt,K.T., Styles,C. and Fink,G.R. (1987) Multiple global regulators
control HIS4 transcription in yeast. Science, 237, 874–880.
27. Springer,C., Künzler,M., Balmelli,T. and Braus,G.H. (1996) Amino acid
and adenine cross-pathway regulation act through the same 5′-TGACTC-3′
motif in the yeast HIS7 promoter. J. Biol. Chem., 271, 29637–29643.
28. Denis,V., Boucherie,H., Monribot,C. and Daignan-Fornier,B. (1998) Role
of the myb-like protein Bas1p in Saccharomyces cerevisiae: a proteome
analysis. Mol. Microbiol., 30, 557–566.
Nucleic Acids Research, 2001, Vol. 29, No. 2
29. Sherman,F., Fink,G.R. and Hicks,J.B. (1986) Methods in Yeast Genetics.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
30. Ma,H., Kunes,S., Schatz,P. and Botstein,D. (1987) Plasmid construction
by homologous recombination in yeast. Gene, 58, 201–216.
31. Pinson,B., Gabrielsen,O.S. and Daignan-Fornier,B. (2000) Redox
regulation of AMP synthesis in yeast: a role of the Bas1p and Bas2p
transcription factors. Mol. Microbiol., 36, 1460–1469.
32. Robzyk,K. and Kassir,Y. (1992) A simple and highly efficient procedure for
rescuing autonomous plasmids from yeast. Nucleic Acids Res., 20, 3790.
33. Cross,F. (1997) ‘Marker swap’ plasmids: convenient tools for budding
yeast molecular genetics. Yeast, 13, 647–653.
34. Studier,F.W., Rosenberg,A.H., Dunn,J.J. and Dubendorff,J.W. (1990)
Use of T7 RNA polymerase to direct expression of cloned genes.
Methods Enzymol., 185, 60–89.
35. Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature, 227, 680–685.
36. Wray,W., Boulikas,T., Wray,V.P. and Hancock,R. (1981) Silver staining
of proteins in polyacrylamide gels. Anal. Biochem., 118, 197–203.
37. Rolfes,R.J., Zhang,F. and Hinnebusch,A.G. (1997) The transcriptional
activators BAS1, BAS2 and ABF1 bind positive regulatory sites as the
38.
39.
40.
41.
42.
535
critical elements for adenine regulation of ADE5,7. J. Biol. Chem., 272,
13343–13354.
Scholtz,J.M., Qian,H., York,E.J., Stewart,J.M. and Baldwin,R.L. (1991)
Parameters of helix-coil transition theory for alanine-based peptides of
varying chain lengths in water. Biopolymers, 31, 1463–1470.
Jamin,N., Gabrielsen,O.S., Gilles,N., Lirsac,P.N. and Toma,F. (1993)
Secondary structure of the DNA-binding domain of the c-Myb
oncoprotein in solution. A multidimensional double and triple
heteronuclear NMR study. Eur. J. Biochem., 216, 147–154.
Zargarian,L., Le Tilly,V., Jamin,N., Chaffotte,A., Gabrielsen,O.S.,
Toma,F. and Alpert,B. (1999) Myb-DNA recognition: role of tryptophan
residues and structural changes of the minimal DNA binding domain of
c-Myb. Biochemistry, 38, 1921–1929.
Ebneth,A., Schweers,O., Thole H., Fagin,U., Urbanke,C., Maass,G. and
Wolfes,H. (1994) Biophysical characterization of the c-Myb DNAbinding domain. Biochemistry, 33, 14586–14593.
Sarai,A., Uedaira,H., Morii,H., Yasukawa,T., Ogata,K., Nishimura,Y. and
Ishii,S. (1993) Thermal stability of the DNA-binding domain of the Myb
oncoprotein. Biochemistry, 32, 7759–7764.