(Received for publication, November 22, 1994; and in revised form, February 3, 1995)
From the
Recently, DNA ring closure assays showed that high mobility
group protein HMG-1 and its close homolog HMG-2 mediate
sequence-independent DNA flexion. This DNA-bending activity appears to
be central to at least some of the recently elucidated functions of
HMG-1/2, such as the enhancement of progesterone receptor DNA binding.
Here we show that standard purification procedures utilizing perchloric
and trichloroacetic acid can produce HMG-1 significantly deficient in
its abilities to bind and bend double-stranded DNA, while
acid-independent methods purify HMG-1 that is superior in these
respects. Significant losses of DNA ring closure activity were seen
upon limited 2-5-h exposures of non-acid-purified HMG-1/2 to
perchloric acid and/or trichloroacetic acid. Measurements of the
apparent DNA dissociation binding constant (K) of acid-extracted
preparations of HMG-1 gave a wide range of values, and only those
preparations demonstrating little DNA ring closure activity had K
values near the previously published
value (
10
M). The highest ring closure
activities and lowest K
(<3
10
M) were obtained
for HMG-1 purified without acids. These combined results support the
use of alternative, non-acid purification procedures for preserving the
DNA-bending activity of HMG-1/2 and suggest that past procedures
utilizing acids have led to an underestimation of the affinity of HMG-1
for DNA.
The high mobility group protein HMG()-1 was first
identified over two decades ago (1) and has been indirectly
linked to a number of eukaryotic cellular processes, such as
transcription(2, 3, 4) ,
recombination(5) , and cellular dedifferentiation (6) ,
yet its precise role in these processes is only just beginning to be
elucidated. In this regard, several laboratories recently described a
hitherto unknown activity of HMG-1, the ability to induce
sequence-independent DNA flexion, or
bending(7, 8, 9) . More significantly, there
is evidence that this ability to alter DNA structure by bending may be
important to at least some of the cellular functions of the protein.
Paull et al.(7) , for example, have demonstrated
that HMG-1 (or the closely related HMG-2) can functionally substitute
for the bacterial DNA-bending protein HU in vitro in a
recombinational DNA inversion reaction requiring a sharp bending and
looping of the DNA, and it is speculated that HMG-1 may function in
analogous recombination reactions in eukaryotic systems.
Oñate and colleagues (9) have recently
demonstrated that HMG-1 serves as an accessory factor in facilitating
the binding of the human progesterone receptor to its target DNA
sequences in vitro, with evidence for a mechanism based on DNA
bending. Studies in our laboratory ()also showed a similar
stimulatory effect of HMG-1 on transcription factor NF-
B, with
additional evidence for an effect mediated through DNA bending. The
combined results suggest that HMG-1, by altering DNA structure, may be
functioning to cooperatively assist the bindings and interactions of
transcription factors and other proteins that require energetically
unfavorable DNA distortions, such as loops or bends.
During our recent experiments using HMG-1 purified by standard denaturing/oxidizing methods involving perchloric/trichloroacetic acid extractions, we assayed many preparations of the protein that possessed suboptimal DNA-associated activities, i.e. higher dissociation constants, lower DNA-bending activities, and reductions in ability to stimulate binding of transcription factors. Here, we demonstrate that these activities of HMG-1 are improved when HMG-1 is prepared by alternative methods that avoid acids. Our results also indicate that past purification techniques have led to an underestimation of the affinity of HMG-1 for double-stranded DNA. These combined results support the use of alternative non-acid procedures in purifying the protein.
Following essentially the ``non-acid'' method of Adachi et al.(10) , but without prior dialysis of the
extract, the 0.35 M boiled supernatant was chromatographed on
a PBE 94 column equilibrated with 20 mM Tris, pH 7.5, 0.35 M NaCl, 5 mM DTT and using approximately 1.5 ml of
PBE 94 resin per starting rat liver. HMG-2 and HMG-1 eluted at about
the 0.6 M and 0.75 M points, respectively, in a
0.35-1 M NaCl gradient. These steps purified HMG-1 and
HMG-2 to >90% homogeneity with 33% yield of starting ring closure
activity (see Table 1). Purified HMG-1 and HMG-2 were desalted on
Centriprep 10 devices (Amicon) versus 5 mM Hepes, pH
7.6, 5% glycerol, 0.1% Tween 20, and concentrated to 100 ng/µl
by volume reduction on the Centripreps. Fresh DTT was added to 5
mM, and the proteins were frozen in 10-µl aliquots at
-70 °C. Occasionally, a significant contaminant of about 40
kDa was retained with HMG-1 after the PBE column. Carboxymethyl
cellulose was found to not bind this contaminant, but to avidly retain
HMG-1, indicating that this resin may be useful for achieving further
purity of HMG-1, if needed.
The heating step in the procedure, although providing a manyfold purification of HMG-1/2 and inhibiting proteolysis (see ``Materials and Methods''), may also be somewhat harmful to the proteins, as about 25% of the starting ring closure activity is lost on boiling the 0.35 M NaCl extract 3 min (Table 1). Some of this loss is probably due to trapping of HMG-1/2 protein within the heat-sensitive proteins as they are precipitated, but some may also be due to inactivation. Ideally, a future method might substitute a milder procedure for this heating step. Adachi et al.(10) reported that HMG-1 and 2 can be purified directly from 0.35 M NaCl extracts of porcine thymus by chromatography on PBE 94. Our experience indicates that this is probably insufficient in purifying HMG-1/2 from rat liver.
Figure 1: Effects of PCA and trichloroacetic acid on the ring closure activity of the HMG-1/2 proteins. Standard ring closure incubations contained, in addition to 10 ng of cohesively ended DNA and 40 units of T4 DNA ligase, the following: lane 1, an aliquot of partially pure HMG-1/2 prepared without acids; lane 2, an aliquot identical to the lane 1 material but exposed to PCA for 4 h on ice; lane 3, no additional proteins; lane 4, an untreated aliquot (approximately 5 ng) of non-acid-purified HMG-1/2 proteins; lane 5, an aliquot identical to the lane 4 material but exposed to trichloroacetic acid for 2 h on ice; lane 6, an aliquot identical to the lane 4 material but exposed to PCA for 5 h then trichloroacetic acid for 2 h on ice. It was previously demonstrated that the DNA bands migrating at the indicated positions MC and ML are the monomer circle and monomer linear DNA, respectively(21) . Lanes 1 and 2 utilized an 88-bp DNA fragment, lanes 3-6 a 94-bp fragment.
The above ring closure assays used amounts of HMG-1/2 less than required to convert all the DNA to monomer circles in the reactions. Under these conditions, there is a linear relationship between the amount of DNA cyclized and the amount of active HMG-1 in standard ring closure reactions (data not shown), suggesting that an observed fractional decrease in ring closure activity after acid treatment directly reflects the losses incurred.
Figure 2:
Gel mobility shift assays titrating the
binding of HMG-1 to linear DNA under different conditions. Lanes
1-4, incubations and electrophoresis on 8% polyacrylamide
performed under standard conditions. Mixtures containing constant
concentrations (0.06 nM) of P-labeled 207-bp
linear DNA were incubated 15 min at 25 °C with the varied nanomolar
concentrations of HMG-1 protein shown above the lanes. Lanes 5-10, incubations and electrophoresis performed
under the conditions of Pil and Lippard (18) (see also
``Materials and Methods''). Mixtures containing constant
concentrations (0.07 nM) of
P-labeled 207-bp
linear DNA were incubated with the varied nanomolar concentrations of
HMG-1 shown above the lanes, before electrophoresis on a 10%
polyacrylamide gel. Bands corresponding to the free and complexed DNA
are indicated by Df and Dc,
respectively.
Acid-extracted HMG-1
preparations showed much variability in their K values, sometimes giving a K
of
10
M or greater. The DNA
binding data for several different preparations of HMG-1, including the
corresponding K
of each
preparation is given in Fig. 3. Also indicated (Fig. 3B) is the ring closure activity of each
preparation, estimated by titrations of each in ring closure assays
(data not shown). There was a consistent relationship between the K
of an HMG-1 preparation and its ring closure
activity: those with the lowest K
values
correspondingly had the highest specific ring closure activity
(units/µg of HMG-1 protein). It is noteworthy that the preparation
with the highest affinity for DNA (and also the most ring closure
activity) was the preparation prepared by the non-acid procedure. The
discrepancy between the low K
values determined
here for active HMG-1 and earlier reports that the K
for HMG-1 was about 10
M in
buffer/salt mixtures similar to buffer B (18) (see also (22) ), may be attributable to purification using acids,
non-ideal storage conditions, and/or use of recombinant HMG-1. Indeed,
the acid-extracted preparation designated as prep 2 shows a K
approximating the previously
reported values (Fig. 3).
Figure 3:
DNA-associated activities of several
different HMG-1 preparations. Panel A, binding titrations of
three representative HMG-1 preparations to linear DNA. The approximate K values obtained from
the data for these different preparations are as follows:
non-acid-purified HMG-1, 2.5 nM; acid-extracted HMG-1 (prep 1), 32 nM; acid-extracted HMG-1 (prep
2), 650 nM. The sizes of the linear DNAs used in these
titrations are 94, 144, and 207 bp, respectively, for the
non-acid-extracted preparation, acid-extracted prep 1, and
acid-extracted prep 2. Panel B, the corresponding specific DNA
ring closure activity (units/µg of HMG-1 protein) of the three
HMG-1 preparations described in Panel A. Units of ring closure
activity are as described for Table 1.
Through its ability to bind and bend double stranded DNA in a
sequence-independent manner, protein HMG-1 appears to be serving, at
least in one of its roles, as a general accessory factor that
facilitates the binding and interactions of other DNA-binding proteins (7, 9, 19, 23) . In this
regard, the results of the present report are of significance in that
they demonstrated that the most common purification protocols utilizing
perchloric and trichloroacetic acid extraction can lead to inactivation
of HMG-1 as measured by two important parameters, its ability to bind
DNA with high affinity and its ability to flex double stranded DNA as
measured by DNA ring closure assays.
Short exposures to both PCA and
trichloroacetic acid were shown to have significant detrimental effects
on the ring closure activity of different non-acid-extracted
preparations of HMG-1/2 (Fig. 1). These results are in agreement
with several previous studies, undertaken before the DNA-bending
activity of HMG-1/2 was known, which showed that these acids could
affect other properties of HMG-1/2. Cockerill et
al.(24) , for example, saw a significant difference in the
circular dichroism spectrum of perchloric acid-extracted HMG-1 versus HMG-1 prepared under nondenaturing conditions, which
was interpreted as a decrease of 24% in the relative -helical
content of the acid extracted HMG-1. Their results are particularly
interesting in light of the recent NMR structural determination of one
of the two conserved DNA-binding domains (or boxes) of
HMG-1(25, 26) , which show it to be composed chiefly
of three
-helices arranged in a V-shape, possibly providing the
contacts that are responsible for constraining the DNA in a bent form.
It seems possible that some of the losses in DNA-bending (and
DNA-binding) activities observed in the present report for
acid-extracted HMG-1 could be due to perturbations in the
-helices
of these DNA binding domains.
Oxidation of cysteine sulfhydryl groups, to form intramolecular disulfide bridges, has been reported to occur rapidly during purification of the HMG-1/2 proteins in the absence of reducing agents or EDTA (27, 28) and to adversely affect the interaction of HMG-1/2 with histone H1(29) . Although not specifically examined in the present report, oxidation of cysteines, 3 of which lie within the two DNA binding domains of mammalian HMG-1/2(30, 31) , could be contributing to the adverse effects of the oxidizing PCA (20) and/or nonreducing environments seen here. The non-acid purification procedure outlined under ``Materials and Methods'' included 5 mM DTT in all buffers to minimize these effects.
The studies of HMG-1 binding to double-stranded DNA
indicated a K in the range of
2-8 nM when the interaction utilized protein prepared in
the absence of acids and/or demonstrating high levels of ring closure
activity (>200 units/µg HMG-1). It was noted that this K
differed by several orders of magnitude from
published values(18, 22) . Experiments using different
DNA fragments of unrelated sequence yielded similar K
values for the same HMG-1 preparations (results not shown),
indicating that DNA sequence was not responsible for the discrepancy.
The finding that the lowest K
values were obtained
for non-acid-extracted HMG-1, while only acid-extracted preparations
demonstrated values in the published range, suggests that past
purification procedures utilizing acid extractions and other harsh
treatments have led to an underestimation of the affinity of HMG-1 for
DNA. Variations in the time of exposure to the acids or variations in
the effects of the acids on different HMG-1 preparations may explain
the wide range of binding affinities determined here for different acid
extracted HMG-1 preparations and may be responsible for some of the
conflicting reports in the literature about the properties of HMG-1/2.
There was a consistent correspondence between the ring closure
activity and K for individual
preparations of HMG-1. Such a correlation is expected since the HMG-1/2
protein must first bind the DNA before it can bend it. Previous studies
suggest that the DNA binding domains themselves may be primarily
responsible for the DNA-bending activities of HMG-1/2 since each domain
alone mediates DNA cyclization(9) . Titrations show, also as
expected, that maximal ring closure activities occur at concentrations
of HMG-1 above its K
and in molar excess of the
DNA fragment in the ring closure incubation (data not shown).
The alternative procedure described here for purifying HMG-1/2 avoids acids and is as convenient to use as the conventional methods based on acid precipitations. In addition to producing HMG-1 and HMG-2 in high yields, purity, and activity, the early heating step in this procedure seems to minimize the proteolytic cleavage of HMG-1/2 that commonly occurs at early stages in rat liver extracts (5) using conventional protocols. It is anticipated that this approach will facilitate future studies of the HMG-1/2 proteins.