From the Department of Biological Chemistry, UCLA
School of Medicine, Los Angeles, California 90095-1737 and the
Molecular Biology Institute, UCLA,
Los Angeles, California 90095
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ABSTRACT |
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The non-histone proteins 6A/B (NHP6A/B) of
Saccharomyces cerevisiae are high mobility group proteins
that bind and severely bend DNA of mixed sequence. They exhibit high
affinity for linear DNA and even higher affinity for microcircular DNA.
The 16-amino acid basic segment located N-terminal to the high mobility
group domain is required for stable complex formation on both linear and microcircular DNA. Although mutants lacking the N terminus are able
to promote microcircle formation and Hin invertasome assembly at high
protein concentrations, they are unable to form stable complexes with
DNA, co-activate transcription, and complement the growth defect of
nhp6a/b mutants. A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch
between residues 8 and 10 is required for the formation of monomeric
complexes with linear DNA. Mutational analysis suggests that proline 18 may direct the path of the N-terminal arm to facilitate DNA binding,
whereas the conserved proline at position 21, tyrosine 28, and
phenylalanine 31 function to maintain the tertiary structure of the
high mobility group domain. Methionine 29, which may intercalate into
DNA, is essential for NHP6A-induced microcircle formation of 75-bp but
not 98-bp fragments in vitro, and for full growth complementation of
nhp6a/b mutants in
vivo.
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INTRODUCTION |
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The high mobility group (HMG)1 proteins are a family of heterogeneous chromatin-associated DNA-binding proteins in eukaryotic cells. They are the most abundant non-histone proteins found in the nucleus and are divided into three classes: HMG-I/Y, HMG14/17, and HMG1/2 (1, 2). These proteins were classically recognized by their high electrophoretic mobility in polyacrylamide gels and acid solubility. The HMG-I/Y class of proteins function as accessory transcription factors, whereas the HMG14/17 class are associated with nucleosomes. The HMG1/2 class of proteins contain the HMG DNA binding domain and are present at a level of about 1 copy/2-3 nucleosomes (3). The HMG domain is a 70-80-amino acid region consisting mainly of hydrophobic and charged residues with a few highly conserved aromatic residues (4).
Members of the HMG1/2 class can be further divided into two subfamilies based upon the number of HMG domains, their DNA sequence specificity, and their evolutionary relationship (5). The sequence-specific HMG1/2 proteins contain a single HMG domain, which are usually restricted by cell type and interact with relatively high affinity to a specific DNA sequence. These proteins include the human sex-determining factor SRY (6, 7), the lymphoid enhancer-binding factor LEF-1 (8), and the T-cell factor TCF-1 (9). The other subfamily of HMG1/2 proteins are ubiquitously expressed and bind to DNA with structural specificity but little or no sequence specificity. One group of the non-sequence-specific DNA-binding proteins contain multiple HMG domains such as the abundant HMG1 and HMG2 proteins (10), human nucleolar transcription factor hUBF (11), mitochondrial transcription factor mTF-1 (12), and yeast ARS-binding protein ABF-2 (13). Other non-sequence-specific HMG1/2 proteins found in yeast (14, 15), plants (16), insects (17, 18), and protozoa (19, 20) contain only a single HMG domain and, often, an accessory basic and/or acidic domain.
The HMG domain of all these proteins probably fold into an L-shaped
region of three -helices, as shown by the NMR structures of rat HMG1
domain B, SRY, LEF-1, HMG1 domain A, HMG-D, and SOX4 (21-27). The
domain contains a primary hydrophobic core at the vertex of the
L-shaped structure formed by conserved aromatic residues from the three
-helices. The NMR structures of the two sequence-specific HMG
proteins, SRY and LEF-1, include their DNA recognition sequence (23,
24). These structures show the DNA to be greatly distorted in the
region of protein contact, with an overall bend of 80° and 120° for
SRY and LEF-1, respectively. The DNA is severely underwound, resulting
in a widened and shallow minor groove and a highly compressed major
groove. In conjunction with the helical underwinding, large positive
roll angles are induced by numerous DNA-protein contacts, which include
a partial intercalation of an amino acid side chain into the minor
groove of the DNA.
The sequence-specific HMG structures provide a basis for understanding how the non-sequence-specific HMG1/2 proteins interact with high specificity to distorted DNA containing bends, cruciforms, or DNA kinked by cisplatin (28-32). Recently, it has been demonstrated that a subgroup of non-sequence-specific HMG1/2 proteins, which contain only one HMG box, can bind with higher affinity to linear DNA than HMG1/2 but exhibit only a modest preference for cruciform DNA (30, 33, 34). It has become increasingly clear that the addition of basic amino acids to a single minimal HMG box enhances the bending capacity and affinity for DNA (24, 35-37). However, despite the fact that some of the non-sequence-specific single HMG box proteins rival the DNA binding affinities of sequence-specific proteins, the exact mode of DNA binding by the non-sequence-specific HMG proteins has not yet been determined.
The biological functions of HMG1/2-like proteins are just beginning to be elucidated. They can function as architectural factors through their ability to strongly distort DNA structures. In this regard, they have been shown to promote assembly of specialized recombination complexes derived from prokaryotic systems (38-40). HMG1/2 proteins and their homologs can substitute for prokaryotic chromatin proteins to condense DNA in vivo (33) and can facilitate nucleosome assembly and disassembly in vitro (42-45). Moreover, efficient activated transcription of certain genes requires HMG proteins, which, in part, may be related to their ability to promote assembly of preinitiation complexes (41, 46). On the other hand, some in vitro studies have found that HMG proteins inhibit transcription (47-49).
The yeast system provides a genetic tool to investigate the biological
roles of HMG proteins and to correlate in vitro activities with in vivo functions. A number of HMG box proteins have
been reported in Saccharomyces cerevisiae including the
non-histone proteins 6A/B (NHP6A/B) (14) and more recently the high
mobility group proteins HMO1/2 (15). NHP6A/B are closely related 11-kDa proteins that contain a single HMG box. The HMG box of NHP6A is 45%
identical to rat HMG1 box B and 80% identical to the HMG box of NHP6B
(Fig. 1A). NHP6A/B are unique in that they both contain a
highly basic amino acid region that precedes the HMG box. These proteins have a higher affinity for and bend DNA more efficiently than
mammalian HMG1/2 (33). In this report, we investigate the importance of
the N-terminal basic segment for high affinity DNA binding. Neither
NHP6A nor NHP6B is essential since nhp6A and
nhp6B mutants grow normally, but the double mutant grows
slowly at 30 °C and is not viable at high temperatures. The
nhp6A/B mutants display a variety of morphological
changes and are defective in activated transcription of a subset of
genes (41, 50). We show in this paper that the N-terminal segment of
NHP6A is critical for its DNA binding activities and functional
properties. NHP6A mutants containing substitutions in other selected
regions have also been analyzed with respect to their in
vitro and in vivo properties.
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EXPERIMENTAL PROCEDURES |
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Construction of NHP6A Mutants--
The NHP6A mutants were
constructed by direct cloning of PCR products using mutant
oligonucleotide primers or by site-directed mutagenesis using the
method of Kunkel (51). The sequence of the oligonucleotides used to
generate the mutations is given in Table I. In several cases (P21A,
M29A, and M29D), mutant PCR products were cloned by a three-way
ligation using internal restriction sites (Table
I). pRJ1228 (pET11a-NHP6A; Ref. 33) was
used as the template for PCR and the vector for reconstructing mutant NHP6A genes. pRJ1340 and pRJ1341 were generated by cloning into pBS
KS+ and pBS KS, respectively, a PCR product
obtained using a 5' NHP6A primer containing an
EcoRI/NdeI site and a 3' NHP6A primer containing a BamHI site. Single-stranded DNA for site-directed
mutagenesis was prepared from pRJ1340 and pRJ1341 using CJ236
(dut ung; Ref. 52). The mutant genes were subsequently
transferred using the NdeI and BamHI sites into
pET11a for protein overexpression. Each mutant gene was sequenced in
its entirety.
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Protein Expression and Purification--
Recombinant proteins
were expressed from the pET11a-derivatives in RJ1878 (BL21 (DE3)
hupA::cm hupB::km; Ref. 33). NHP6A synthesis was induced for 3 h at 37 °C in LB when the cells
reached an A600 = 0.6 by the addition of 1 mM isopropyl-1-thio--D-galactopyranoside. Two liters of cells were disrupted by sonication in 1/10 volume of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 1 mM
phenylmethylsulfonyl fluoride. The extract was clarified by
centrifugation at 30,000 × g, and the NaCl
concentration was increased to 1 M. Polyethyleneimine
(Sigma) was added to 0.3%, and the nucleic acids were removed by
centrifugation at 20,000 × g. Residual
polyethyleneimine was removed by batch chromatography with 20% (v/v)
cellulose phosphate P-11 (Whatman), and the supernatant was dialyzed
overnight against 0.05 M buffer A (20 mM
Tris-HCl (pH 7.5), 1 mM DTT, 1 mM EDTA, 10%
glycerol, plus 0.05 M NaCl). The dialysate was passed
through a 4-ml S-Sepharose (Pharmacia) column equilibrated in the same
buffer, and the NHP6A protein was eluted in a 50-ml linear gradient
from 0.05 M to 1.0 M NaCl in buffer A. Fractions containing the NHP6A protein were pooled and subject to 2%
trichloroacetic acid precipitation at 0 °C for 30 min to remove
contaminating proteins. After centrifugation for 30 min at 30,000 × g, the supernatant was adjusted to 10% trichloroacetic
acid, and the homogeneous NHP6A was recovered by centrifugation as
before. The precipitate was washed with acetone, dried briefly,
resuspended in buffer B (20 mM HEPES (pH 7.5), 0.1 M NaCl, 1 mM DTT, 1 mM EDTA, and
50% glycerol) and dialyzed overnight in the same buffer. Wagner
et al. (55) reported that trichloroacetic acid precipitation
affected DNA binding by HMG1, but we have been unable to detect any
difference between NHP6A purified using trichloroacetic acid
precipitation or under entirely native conditions using multiple
chromatography steps. Proteins concentrations were quantitated by laser
densitometry (Molecular Dynamics) of SDS-polyacrylamide gels stained
with Coomassie Blue using a titration of lysozyme as a standard. Values
obtained were within 1-3% of the amounts determined by quantitative
amino acid analysis (UCLA Protein Microsequencing Facility).
Gel Mobility Shift, Ligase-mediated Circularization, and Hin DNA
Inversion Assays--
Labeled DNA fragments for gel mobility shift and
ligation assays were obtained using PCR reactions containing
[-32P]dATP and pRJ551-44, pRJ551-66, or pRJ551-76
as described (38, 56). After digestion with EcoRI, fragments
of lengths 66, 75, and 98 bp containing EcoRI cohesive ends,
respectively, were purified in 10% polyacrylamide gels. Circular DNA
was formed from reactions containing 8 ng of linear fragment, 40 ng of
NHP6A, and 20 units of T4 ligase that were incubated at
30 °C for 1 h under the conditions described previously (33).
The products were then digested with 100 units of exonuclease III for
60 min, extracted with phenol/chloroform (1:1, v/v) and precipitated
with ethanol resulting in >90% monomer circle. Gel mobility shift and
ligase-mediated circularization assays were performed as described in
Ref. 33 and electrophoresed on 6% polyacrylamide gels (29:1
acrylamide:bisacrylamide). Hin-catalyzed DNA inversion reactions were
performed as described previously using pMS551-83, which contains 83 bp between the centers of hixL1 and the proximal Fis binding
site in the enhancer (56).
Stoichiometry of NHP6A-DNA Complexes--
NHP6A-HMK, containing
the heart muscle kinase recognition sequence (RRASV) fused to the
C-terminal end of NHP6A, behaved identically to the wild-type in DNA
binding assays (data not shown). 10 µg of NHP6A-HMK was labeled by
incubating with 5 units of bovine heart muscle kinase (Sigma) and 50 µCi of [-32P]ATP (>6000 mCi/mmol; Andotek) in 20 mM HEPES (pH 7.5), 100 mM NaCl, and 12 mM MgCl2 for 1 h at 37 °C. The reaction
was quenched with 10 mM EDTA, precipitated with
trichloroacetic acid as described above, and the
32P-labeled NHP6A-HMK was resuspended in buffer B. The
specific activity of the protein under potential gel quenching
conditions was determined by polymerizing a known amount in a 6%
polyacrylamide gel plug (29:1
acrylamide:N,N-bisacryloylcystamine; Sigma). The plug was
incubated with 50 mM
-mercaptoethanol for 30 min at 50 °C to dissolve the disulfide cross-links and counted in
scintillation fluid (EcoSint; National Diagnostics). 98-bp DNA
fragments were prepared as described above. The DNA concentration was
determined by a Hoefer TK100 fluorometer using Hoechst dye 33258 (Sigma) and by quantitation (ImageQuant, Molecular Dynamics) of digital images of ethidium bromide-stained polyacrylamide gels in which an
aliquot of the fragment was electrophoresed together with varying concentrations of pBR322 digested with HaeIII. Both methods
gave the same results. Gel mobility shift assays were performed as described above, except that electrophoresis was in 6% 29:1
acrylamide:N,N-bisacryloylcystamine gels. The gels were
stained with SybrGreen (Molecular Probes) and visualized with a Hitachi
FMBIO II Fluorimager. The first and second complex bands were excised,
the gel slice dissolved with
-mercaptoethanol as described above,
and counted in scintillation fluid. The molar amount of protein in each
complex was calculated from its specific activity; the molar amount of
DNA was determined by comparison to varying amounts of the DNA fragment
electrophoresed on the same gel.
Generation Times, NHP6A Expression Levels, and in Vivo CUP1
Transcription Assays--
The wild-type and NHP6A mutant genes
contained on pRS314 were transformed into RJY6012 (MAT
ura3-52 leu2-3, 112 his3
200 trp1-
201 lys2-801 suc2-
9
gal3 nhp6A:: ura3 nhp6B::LEU2) by the lithium
acetate method (57). The transformants were streaked once on selective
media (SD minus His or Trp; Ref. 57) and inoculated into liquid
selective media. Growth was monitored at A600,
and the doubling time in log phase at 30 °C was determined in
duplicate for two individual transformants.
In Vitro Transcription--
Transcription reactions (25 µl)
were performed using 130 µg of yeast nuclear extracts in the presence
of the acidic activator GAL4-VP4, NHP6A, and 100 ng of
G5E4T as the DNA template. mRNA levels were
detected by primer extension. Reaction conditions, GAL4-
VP4, and
G5E4T have been described previously (41,
59).
Physical Properties-- Circular dichroism spectra from 190 to 240 nm was measured with an AVIV 62ADS circular dichroism spectrophotometer with 20 iterations at 25 °C. Proteins were dialyzed into 10 mM sodium phosphate buffer (pH 7.4) and protein concentrations determined as above. Secondary structure characteristics were calculated by the self-consistent method using DICROPROT (60).2 Thermal denaturation curves were obtained under the same conditions at 222 nm. The temperature was varied from 10-90 °C with an incubation time of 5 min for every 5 °C.
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RESULTS |
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DNA Binding Properties of Wild-type NHP6A
NHP6A forms stable complexes on DNA of varying sequences as revealed by polyacrylamide gel electrophoresis (Fig. 2A; Ref. 33). With increasing concentrations of NHP6A, a discrete set of higher order complexes are obtained. 32P-Labeled NHP6A was used to determine the stoichiometry of binding in the first and second complexes formed on a 98-bp DNA fragment as described under "Experimental Procedures." Quantitation of four to eight individual NHP6A-DNA complexes from separate experiments gave average molar ratios of 0.92 ± 0.2 for the first retarded complex and 2.2 ± 0.4 for the second complex. Therefore, NHP6A binds to DNA as a monomer in a stepwise manner. The binding constant (KD) for the initial complex is 100 nM, a value that is constant on DNA of different sequences and lengths (23-300 bp).
The ability of NHP6A to bend DNA is most directly demonstrated by
ligase-mediated circularization assays where the wild-type protein can
form up to 70% monomer circles on DNA substrates as short as 66 bp
(33). By measuring the amount of wild-type NHP6A required to generate
the half-maximum yield of 98-bp circles, we calculate a
Kcircle of 1.5 × 108
M (Fig. 3B). NHP6A binds much more tightly to
curved DNA of mixed sequence than to linear DNA. This is shown in Fig.
4A where binding to a 98-bp microcircle was assayed by
polyacrylamide gel electrophoresis. Two discrete complexes are formed
at low concentrations of NHP6A, followed by the formation of higher
order species with increasing protein concentrations. Gel mobility
shift assays on 75-bp microcircles also generates two high affinity
complexes, but three high affinity complexes are formed on 66-bp
microcircles (Fig. 4, F and G). The number of
high affinity complexes were identical regardless of whether NHP6A was
added to purified microcircles or whether the products of the NHP6A + DNA ligase reactions used to generate the microcircles were directly
analyzed by native gel electrophoresis (data not shown). The binding
constant for these curved DNA molecules is ~1.5 nM.
Binding to the pre-bent substrates is extremely stable, as revealed by
the resistance of NHP6A-microcircle DNA complexes to added competitor
DNA (Fig. 4H). The addition of 2.5 mg/ml salmon sperm DNA
(corresponding to a 2,500,000:1 w/w ratio of competitor to
microcircular DNA) was unable to remove NHP6A from the two high
affinity sites on the 98-bp microcircle, but 5.0 µg/ml (500:1 w/w
ratio of competitor to linear DNA) was sufficient to dissociate most of
the prebound NHP6A from a linear substrate.
The N Terminus Is Necessary for Efficient Binding and Bending of DNA
The 94-amino acid NHP6A protein contains a 16-amino acid region
located N-terminal to the minimal HMG domain. This segment contains two
blocks of basic residues: KKR between residues 8 and 10 and RKKK
between 13 and 16 (Fig. 1). To determine
the importance of these amino acids for DNA binding and NHP6A function,
three different truncations of NHP6A were constructed: a deletion of the entire N terminus (2-16), a deletion that removed the first block of basic residues
(2-12), and a deletion that retains both blocks of basic residues
(2-7).
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Deletion of the entire N terminus of NHP6A abolished the high affinity
of the protein for linear DNA with no distinct DNA-protein complexes
being formed (Fig. 2D). We
estimate that this mutant binds approximately 600-fold poorer than
wild-type protein by gel mobility shift assays. This resembles the low
affinity association of HMG1 to linear DNA observed under similar assay
conditions (29, 33, 61). NHP6A (2-16) also bound poorly to
microcircles (Fig. 4C). This unstable DNA association by the
minimal HMG box of NHP6A represents a functional HMG interaction and
not a nonspecific association since
(2-16) was able to induce
microcircle formation. As shown in Fig.
3A, NHP6A
(2-16) converted
40% of a 98-bp DNA fragment into monomer circles; however, the amount
of protein required was about 300-fold higher than for the wild-type
protein (Table II). These results
demonstrate that the basic N-terminal tail of NHP6A performs a critical
function in stable DNA binding, although a weak functional association
with DNA is possible with only the minimal HMG box.
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In contrast to the properties of the deletion of the entire 16-amino
acid N terminus, a deletion of the first 12 amino acids resulted in a
mutant that retained relatively high affinity binding, although it was
not capable of producing discrete DNA complexes on polyacrylamide gels
(Fig. 2C and Table II). Therefore, we conclude that the RKKK
motif between residues 13 and 16, which are retained in this mutant, is
the critical determinant within the N terminus for mediating high
affinity DNA association. Although NHP6A (2-12) did not form
individual complexes on linear DNA, it bound to 98-bp microcircles with
similar affinity as the wild-type (Fig.
4B). In addition, the ability
of
(2-12) to form 98-bp microcircles was indistinguishable from
wild-type (Fig. 3B).
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NHP6A (2-7) retains both blocks of basic amino acids in the N
terminus and exhibited completely normal DNA binding, including the
formation of stable complexes on linear DNA (Fig. 2B), and the formation and binding of DNA microcircles (Table II). Therefore, no
role in DNA binding is detectable for residues 2-6. The difference in
binding properties on linear DNA between
(2-7) and
(2-12) suggest that the KKR motif between residues 8 and 10 functions to
stabilize binding of individual protomers of NHP6A to DNA, but it is
clearly not critical for productive DNA interactions.
Circular dichroism was used to assess the folding of wild-type NHP6A as
compared with the N-terminal deletion mutant (Fig. 5A). According to the
self-consistent method of Sreerama and Woody (62), wild-type NHP6A was
predicted to contain 49% -helix, <1%
-sheet, 22% turn, and
19% other structure. These values are slightly lower than the amount
of
-helical structure predicted by circular dichroism data on HMG1
box B (31, 37, 63), although the C-terminal extension of box B was not
included in their spectra. The spectrum of the NHP6A
2-16 mutant
(Fig. 5A) showed that it was highly structured with
approximately 67%
-helix, <1%
-sheet, 13% turn, and 11%
other structure. These values are similar to the 75% estimate of
-helical content derived from NMR data on the minimal HMG1 box B
(25, 26) and the HMG-D box (22). The increase in
-helical content in
the NHP6A N-terminal deletion mutant as compared with wild-type is
consistent with the N-terminal 16 amino acids of NHP6A being
unstructured in solution. In addition, the Tm of
wild-type and NHP6A
2-16 were both found to be 39 °C (Fig.
5D), further indicating that the N terminus does not play a
role in stabilizing the overall structure.
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Importance of Prolines at Position 18 and 21 for DNA Binding by NHP6A
NHP6A contains two prolines (positions 18 and 21) that are located within the N-terminal end of the HMG domain. The proline at position 21 of NHP6A, which shows remarkable conservation among all non-sequence-specific HMG1/2 proteins (4, 5), stabilizes a secondary hydrophobic core that associates the N- and C-terminal ends of the HMG domain (22). In LEF-1, proline 66, located at the C-terminal end of the HMG domain, appears to be important in directing its C-terminal basic extension that is essential for high affinity LEF-1-DNA binding (24). To determine whether the N-terminal prolines in the NHP6A HMG domain may be performing analogous roles, we created alanine substitutions at these positions and analyzed their DNA binding and folding properties.
NHP6A P18A displayed a 2.5-fold reduction in affinity for linear DNA whereas NHP6A P21A showed 4-fold reduced affinity (Fig. 2, E and F). The behavior of the double mutant P18A/P21A was identical to that of P21A (Fig. 2G). Binding to microcircular DNA was similarly reduced with a KD of 4 nM for P18A and 20 nM for both P21A and P18A/P21A (Fig. 4D). All three mutants were able to convert 70% of 98-bp linear DNA fragments into circles, although P18A required 2 times more protein than wild-type and both P21A and P18A/P21A required 4-fold more protein (Fig. 3B, Table II). Thus, the prolines at position 18 and 21 play a modest role in DNA binding.
CD analysis of the proline mutants indicated that P18A is folded
identically to wild-type, whereas the P21A and the double mutant
P18A/P21A displays a small (<10%) reduction in -helical content
compared with wild-type (Fig. 5). Proline 21, therefore, contributes
to the folding of the NHP6A HMG domain probably via hydrophobic
interactions with residues of
-helix III (see
Discussion). Proline 18 may facilitate positioning of the
basic N-terminal segment upon DNA binding as also elaborated in the
Discussion.
Importance of an Intercalating Side Chain for DNA Binding by NHP6A
DNA binding and bending by either LEF-1 or SRY is facilitated by a hydrophobic amino acid located near the N terminus of helix 1 that intercalates between base pairs via the minor groove (23, 24). Alignment of amino acid sequences as in Fig. 1 suggests that methionine 29 of NHP6A would be in the correct position to function as an intercalating side chain. To test whether methionine 29 of NHP6A is important for DNA binding, this residue was mutated to alanine. Surprisingly, M29A did not show any difference in its affinity to linear DNA as compared with wild-type (Fig. 2). Even changing methionine 29 into a negatively charged aspartic acid resulted in less than a two-fold reduction affinity to linear DNA. Both M29A and M29D bound to 98-bp microcircles with a KD of 2.5 nM and formed 98-bp circles with similar efficiency as wild-type NHP6A (Fig. 3).
The relatively modest effect on DNA binding by substitutions at position 29 led us to test whether adjacent amino acids could function in this capacity. Tyr-28, Phe-30, and Phe-31 were changed to valine or aspartic acid. F30V exhibited very little difference in affinity for linear DNA and 98-bp microcircles compared with wild-type and did not differ in its DNA bending properties. Y28D and F31D were strongly defective in binding to linear and circular DNA, but we show below that they are unfolded in solution. F31V was able to bind poorly to both linear DNA and microcircles (Figs. 2 & 4E); however, the ability to form microcircles was greatly reduced with a maximum of only 4% of the input DNA ligated into 98-bp circles when very high amounts of protein were added (Fig. 3).
CD analysis demonstrated that Y28D and F31V were largely unfolded in solution, whereas M29A showed only a slight change in CD profile (Fig. 5C). The CD data, combined with the known HMG box structures, suggest that the Phe-31 and Tyr-28 side chains are oriented such that they are contributing to the primary hydrophobic core that stabilizes the HMG fold (22, 64). Since F31V forms complexes with DNA microcircles, interaction with the pre-bent DNA ligand is presumably stabilizing its folded structure. The HMG box structure in F31D, however, is probably completely disrupted since it is not capable of forming complexes with curved DNA.
The lack of any clear effect on DNA interactions by M29A, M29D, or F30V revealed by the previous assays led us to test these mutants for their ability to circularize 75-bp fragments. The formation of 75-bp microcircles are predicted to require the greatest amount of DNA bending per bound NHP6A protomer (see "Discussion"). In this assay, a clear difference between wild-type and M29A or M29D was observed (Fig. 3C). Neither M29A nor M29D was able to form 75-bp microcircles, even at very high concentrations of added protein. F30V was fully functional for 75-bp microcircle formation. Taken together, we conclude that Met-29 does play a role in NHP6A-induced DNA bending, but this is only revealed by assays that demand DNA bending near the maximum possible extent possible for wild-type NHP6A.
Ability of the NHP6A Mutants to Function in Biological Reactions
Growth Phenotypes--
Using Saccharomyces cerevisiae,
we are able to correlate the in vitro properties of DNA
binding by NHP6A mutants to their biological functions. The double
knockout mutant (nhp6a/b-RJY6012) exhibits a slow growth
phenotype forming colonies of heterogeneous size, which are
temperature- and cold-sensitive at 38 °C and 23 °C, respectively
(33, 50). A yeast CEN shuttle vector containing the endogenous NHP6A
promoter was used to express NHP6A and various mutants in the
nhp6a/b cells. Western blotting confirmed that episomal
expression of NHP6A and most NHP6A mutants were similar to endogenous
levels of chromosomal expression (Table
III). The exceptions were Y28D and F31V,
which are unfolded in solution and were present at <10% of wild-type
levels. Interestingly,
(2-12) was present at 70% the level of
wild-type whereas
(2-16) was present at <20% of the wild-type
level despite normal in vitro folding. The presence of
episomal NHP6A and all of the above mutants except for
(2-16), Y28D
and F31V were able to reverse the temperature and cold sensitivity of
nhp6a/b cells (data not shown).
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Activated Transcription at the CUP1 Locus in Vivo--
The NHP6A
mutants were also tested for their ability to specifically enhance
activated transcription of the CUP1 promoter, one of a subset of genes
whose activated expression is facilitated by the NHP6A/B proteins (41).
Following 2 h of exposure to 1 mM CuSO4 in
minimal media, -galactosidase activity from a CUP1-lacZ reporter
construct was induced 40-fold in
nhp6a/b cells expressing NHP6A from pRJ1342, similar to the 47-fold induction measured in
wild-type cells (Fig. 6A). The
induced level of CUP1-LacZ expression in the
nhp6a/b
mutant cells was only 8-fold above basal level. Activated transcription
of the CUP1 promoter by the different NHP6A mutants largely paralleled
their effect on growth.
(2-16) had no activity, whereas
(2-12)
displayed essentially wild-type CUP1 expression levels. CUP1
transcription in the presence of M29A was also notably reduced.
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Stimulation of in Vitro Transcription--
The
G5E4T template containing five GAL4 binding
sites upstream of the adenoviral E4T promoter was used as a
model substrate to test the ability of the mutants to facilitate
activated transcription in vitro. Transcription in yeast
nuclear extracts activated by GAL4-VP4 was stimulated 2-5-fold by
increasing amounts of exogenous NHP6A (Fig. 6B). A similar
increase in transcript levels was observed by the addition of bovine
HMG1 or HMG2 (Fig. 6C), but HU, an unrelated Escherichia coli DNA bending protein, had no effect on this
reaction (data not shown). NHP6A (Fig. 6B) or HMG1/2 (data
not shown) had no effect on basal transcription from this promoter,
similar to that observed for the GAL1 promoter (41).
Hin-catalyzed Site-specific DNA Inversion--
When the
recombinational enhancer is located within 100 bp of a recombination
site, HU or an HMG1/2 protein is needed to assemble the catalytically
competent invertasome (33, 38, 56). In this reaction, the auxiliary DNA
bending protein is believed to function strictly as a DNA architectural
factor to facilitate DNA looping of the enhancer segment. In Fig.
7A, the amount of NHP6A mutant
added to the DNA inversion reaction was adjusted to compensate for
defects in DNA binding. Under these conditions, all the mutants are
able to stimulate invertasome assembly with the exception of the
unfolded proteins Y28D and F31V, which bind and bend DNA poorly at all
tested concentrations. NHP6A (2-16) displayed surprisingly high
activity in this assay (Fig. 7B), particularly in comparison
to its complete inactivity in stimulating transcription in
vitro. The proficiency of
(2-16) in promoting invertasome
formation at moderately high concentrations of protein probably
reflects its ability to promote microcircle formation at very high
protein concentrations.
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DISCUSSION |
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DNA Binding and Bending by Wild-type NHP6A-- NHP6A is a member of a subclass of HMG1/2 proteins that contain a single HMG box and bind nonspecifically to linear DNA with relatively high affinity. NHP6A binds equivalently to DNA varying in sequence and length and forms distinct complexes upon polyacrylamide electrophoresis with a KD of 100 nM. We show in this report that each complex represents a monomer of NHP6A bound to DNA. Although the complexes formed on linear DNA are stable during long term electrophoresis in a gel matrix, they readily dissociate in the presence of excess DNA. NHP6A binds with >50-fold higher affinity to microcircular DNA. The complexes formed on microcircular DNA are extremely stable; NHP6A remains bound to the microcircles in the presence of 1000-fold excess linear DNA. Preferential binding to microcircular DNA has also been reported for HMG1 and HMG-D (30, 65). Even though NHP6A binding is presumably targeted to the distorted structures present on microcircular DNA, it does not bind preferentially to four-way junctions (data not shown). High affinity binding to four-way junctions is a feature of many HMG proteins, although the related HMG-D and cHMG1a proteins also do not display this property (17, 66).
The formation of stable complexes of defined numbers of NHP6A protomers on microcircular DNA of varying lengths can be used to estimate the degree of DNA bending introduced upon NHP6A binding. The measured persistence length of DNA (67) gives an average intrinsic flexibility of about 2.4°/bp (360°/150 bp). Thus, DNA lengths of 66, 75, and 98 bp can generate 158°, 180°, and 235° of curvature. Based on the number of NHP6A protomers bound to preformed microcircles (Fig. 3) or upon formation of microcircles (data not shown) of lengths 98 bp (two complexes), a 75 bp (two complexes), or 66 bp (three complexes), the minimal amount of protein-induced bending can be calculated. To create a 98-bp microcircle, each of the two NHP6A-induced bends required to complete the DNA circle would be approximately 60°, which corresponds to a 120° DNA bending angle by each NHP6A protomer relative to linear DNA. A 75-bp microcircle requires a 45° bend in the DNA from each of the two bound NHP6A protomers resulting in a 135° bending angle. Since the 66-bp microcircle has three bound NHP6A protomers, an equilateral triangle can be used to estimate the induced bending of the DNA to be 60°, which corresponds to a bending angle of 120°. Therefore, from these experiments we estimate NHP6A induces bend angles between 120 and 135°. These values are in the range of the angles observed with LEF-1 by NMR (120°; Ref. 24) and cHMG1a by fluorescence resonance energy transfer (150°; Ref. 68). However, they are greater than the 80° observed in the SRY-DNA complex by NMR (23), or the 60° estimated for DNA binding by HMG-D by a ligase-mediated circularization assay (30).High Affinity DNA Binding by NHP6A Requires Its Unique N Terminus-- The 16-amino acid segment located N-terminal of the minimal HMG domain of NHP6A is essential for its unusually stable DNA interaction. Removal of this region abolishes the ability of the protein to form discrete complexes on both linear and microcircular DNA and eliminates most of its biological activities. The remaining minimal HMG box is capable of poorly binding to DNA in vitro, as evidenced by its ability to form microcircles, promote Hin invertasome formation, and induce DNA supercoiling in the presence of topoisomerase I (data not shown) at high protein concentrations. These activities provide strong evidence that the minimal HMG box remains capable of an authentic, albeit weak, HMG-DNA interaction. Circular dichroism and thermal stability data indicate that the N-terminal segment is unstructured in solution and has no effect on the integrity of the folded HMG domain. Upon NHP6A-DNA interaction, the basic N-terminal arm presumably associates with the DNA to anchor the complex. We imagine that the N-terminal arm may cross over the phosphate backbone and protrude into the major groove (Fig. 8A), as does the C-terminal arm of LEF-1 (24), or possibly continue along the minor groove as schematically drawn in Fig. 8B.
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Role of Prolines at the N Terminus of the NHP6A HMG Domain-- NHP6A contains two prolines at positions 18 and 21, which are predicted to be located near the top of the L-shaped HMG domain adjacent to the N-terminal arm (see Fig. 8) based upon the structures of box A and B from HMG1 (21, 22, 25, 26). When proline 18 in NHP6A was changed to an alanine, a 2-3-fold decrease in DNA binding to linear and microcircular DNA was measured. CD analysis did not reveal a significant difference between wild-type NHP6A and NHP6A P18A. We postulate that proline 18 may direct the peptide backbone to facilitate positioning of the N-terminal arm. In the absence of this proline, the N terminus is still able to interact with DNA, although not quite as effectively. In all in vitro reactions tested, NHP6A P18A is very active, provided additional protein is added to compensate for the modest effect on binding affinity. This role for proline 18 of NHP6A is analogous to the function of proline 66 of LEF-1, which directs its C-terminal arm into the major groove where extensive DNA contacts are made (24). A proline to alanine substitution at the same relative position in HMG1 box A as our NHP6A P18A mutant has also been analyzed, but it was not shown to have any effect on DNA binding (64).
The proline at position 21 of NHP6A is conserved among all non-sequence-specific HMG1/2 proteins but corresponds to a valine or isoleucine in sequence-specific HMG proteins. The importance of this residue for NHP6A is shown by a 4- to over 10-fold reduction in binding affinity of P21A to linear and microcircular DNA, respectively. NHP6A P21A is partially defective in its ability to complementThe Hydrophobic Core of NHP6A-- Mutations at Tyr-28 and Phe-31 lead to an unfolded protein, as determined by CD analysis in vitro and an unstable protein in vivo. The aromatic side chains of these conserved amino acids are predicted to be directed into the primary hydrophobic core that stabilizes the three-helix fold (Fig. 8), as observed for other HMG box proteins (5). Interestingly, the presence of DNA appears to stabilize the structure of F31V since it is able to form complexes with microcircular and linear DNA with modest affinity. However, F31D is completely defective in all activities measured.
Methionine 29 Is Required for Maximal NHP6A-induced DNA Bending-- The structures of DNA complexes formed with the sequence-specific HMG proteins LEF-1 and SRY show that a hydrophobic amino acid located in the N terminus of helix 1 protrudes into the DNA from the minor groove, leading to a pronounced base pair unstacking. The critical role of the intercalating isoleucine in SRY-DNA interaction is demonstrated by the sex reversing mutation I68V, which causes a severe defect in DNA binding and bending (23, 74, 75). Methionine 29 is located at the analogous position in NHP6A by sequence alignment (Figs. 1A and 8). However, NHP6A M29A and even M29D binding to linear DNA is nearly indistinguishable from wild-type, and the Met-29 mutants are proficient in Hin inversion, and inducing DNA supercoiling (data not shown) in vitro. On the other hand, NHP6A M29A only restored about 50% of the growth rate defect that results from deleting NHP6A/B. Moreover, activated transcription of CUP1 in the presence of M29A in vivo is significantly reduced, as well as activated transcription of G5E4T in vitro in the presence of M29D (and M29A to a small extent). Thus, Met-29 appears to be important for some activities, including co-activation of transcription, but not other HMG1-promoted functions. The different behavior of Met-29 mutants in these reactions may be related to their ability to form microcircles of varying lengths. Although M29A and M29D were unaltered in their ability to form 98-bp DNA microcircles, they were completely defective in 75-bp microcircle formation. Thus, Met-29 is not required for productive DNA binding but is essential to induce maximum DNA bending.
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ACKNOWLEDGEMENTS |
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We thank Tanya Paull for yeast transcription
extracts and advice in the early part of this work, Michael Carey for
pG5E4T, GAL4-VP4 and helpful discussions,
James Bowie for assistance with circular dichroism, and Frederick
Allain for useful discussions and comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM38509.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by National Institutes of Health NIGMS Training Grant GM08042, the Medical Scientist Training Program, and the Aesculapians Fund of the UCLA School of Medicine.
¶ Supported in part by United States Public Health Service National Research Service Award Grant GM07185.
** Recipient of an American Cancer Society Faculty Research Award. To whom correspondence should be addressed: Dept. of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095-1737. Tel.: 310-825-7800; Fax: 310-206-5272; E-mail: rjohnson{at}biochem.medsch.ucla.edu.
1 The abbreviations used are: HMG, high mobility group; bp, base pair(s); PCR, polymerase chain reaction; DTT, dithiothreitol; NLS, nuclear localization signal.
2 DICROPROT software may be obtained via FTP (ftp.ibcp.fr).
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REFERENCES |
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