An analysis of subunit exchange in the dimeric DNA-binding and DNA-bending protein, TF1

Biochimie (1994) 76, 933-940 © Soci6t6 fran~;aisede biochimie et biologie mol6culaire/ Elsevier, Paris

933

An analysis of subunit exchange in the dimeric DNA-binding and DNA-bending protein, TF1 L Andera*, GJ Schneider**, EP Geiduschek Department of Biology and Centerfor Molecular Genetics. University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA

Summary - - TF! is the Bacillus subtilis bacteriophage-encoded dimeric type II DNA-binding protein. This relative of the eubacterial HU proteins and of the Escherichia coli integration host factor binds preferentially to 5-(hydroxymethyluracil)-containing DNA. We have examined the dynamics of exchange of monomer subunits between molecules of dimeric TFI. The analysis takes advantage of the fact that replacement of phenylalanine with arginine at amino acid 61 in the [l-loop 'arm' of TF1 alters DNA-bending and -binding properties, generating DNA complexes with distinctively different mobilities in gel electrophoresis. New species of DNA-protein complexes were formed by mixtures of wild type and mutant TFI, reflecting the formation of heterodimeric TFI, and making the dynamics of monomer exchange between TFI dimers accessible to a simple gel retardation analysis. Exchange was rapid at high protein concentrations, even at 0°C, and is proposed to be capable of proceeding through an interaction of molecules of TF! dimer rather than exclusively through dissociation into monomer subunits. Evidence suggesting that DNA-bound TFI dimers do not exchange subunits readily is also presented. DNA binding proteins I TFI I subunit-dynamics / gel retardation / phage SPOI / HU

Introduction

The type ll DNA-binding (DBPII) proteins are ubiquitous, small, abundant proteins of the eubacterial and archaebacterial prokaryotes. Proteins of this class include the general Escherichia coil DNA-binding protein, HU, and the moderately site-specific E coil DNA-binding protein, integration host factor (IHF). The E ,.,~1; i-II1 !11 i~ not ~h~ah,tely essential for cell viability, but bacteria that entirely lack HU are defective in growth, plasmid maintenance and gene expression [ 1-9]. IHF, which is almost as abundant an E coil protein as HU according to recent reports [ 10], is also non-essential for viability, but bacteria entirely lacking it are deficient in site-specific recombination and pleiotropically defective in gene regulation (reviewed in [ 10--13]).

*Present address. CNRS-LGME/INSERM-U184, Institut de Chimie Biologique, Facult6 de M6decine, 11, rue Humann, 67085 Strasbourg Cedex, France **Present address: Gen-Probe Inc, 9880 Campus Point Drive, San Diego, CA 92 ! 21, USA

Our interests in this family of proteins have focused on TF1 [14], which is encoded by the Bacillus subtilis lytic phage SPO1, and was the first member of this family of proteins to be identified and purified. In the approximately 140 kbp genome of phage SPOI, 5-(hydroxymethyl)uracil (hmUra), entirely replaces thymine. TF1 has a correspondingly strong preference for hmUra-containing DNA over T-containing DNA, as well as a strong preference for certain sites in SPOI DNA [14-17]. The DBPII are dimeric. The non-specific DNAbinding eubacterial HU proteins have 90- to 92-amino acid subunits; with the exception of the E coil and Salmonella typhimurium HU proteins, they are homodimers. The site-specifically binding IHF and TFI both have C-terminal extensions: TF1 is a homodimer of 99-amino acid subunits; IHF is a heterodimer of 94- and 98-amino acid subunits. The C-terminal extension of TF1 is required for DNA binding [18], and the C-terminal extension of the IHF a subunit is also involved in DNA interaction [19, 20]. IHF and TF1 bind to DNA as dimers, strongly bend DNA, probably to a similar extent [17, 21-24]. They differ from each other in that TF1 forms nested DNA complexes around its preferred core DNA-binding sites, presumably through auxiliary lateral protein-

934 protein interactions of TF 1 dimers [ 16, 24], while IHF dimers generally bind in isolation to their preferred DNA sites [23, 25, 26]. The stability of the secondary structure of the type II DNA-binding proteins probably is closely connected to dimerization. The monomer-monomer interface of the Bacillus stearothermophilus HU dimer is extensive (approximately 1700 ~2; j Reisman, personal communication), solvent-excluding, and involves numerous hydrophobic interactions. A TFI model constructed according to the coordinates of the B stearothermophilus HU protein [27] and energyminimized has generally the same characteristics [28] and must also have a large, solvent-excluding monomer-monomer interface. Thus, the dynamics of subunit exchange in TFI and the HU proteins might correlate with stability of secondary structure. Direct measurements of subunit exchange in the type ii DNA-binding proteins have not previously been reported. We show here how a variant TFI protein can be used to follow subunit exchange through the appearance of protein-DNA complexes with characteristic electrophoretic properties. Materials a n d m e t h o d s

Escherichia coil strains. TF I, en:ymes and chemicals E coil BL21 (met +, Ion-, ompT-) containing plasmid pR61X

[29] was grown with rapid shaking in L broth with 60 pg/ml ampicillin at 30°C to Asx~ = 2. Overexpression of TFI-R61 was achieved by shifting the temperature to 43°C for 10 min, with continued cultivation at 37°C for 90 min. Cells were harvested by eentrifugation, washed and stored at -70°C. Restriction endonucleases. T4 polynucleotide kinase, calf thymus alkaline phosphatase and [¥-32PlATP (6000 Ci/mmol) were purchased from BRL-Gibco, New England Biolabs, Boehringer-Mannheim or NEN-Dupont. Wild type TFI was previously purified from E coli HBi01 harboring overexpression plasmid pTFI X [29]. The preparation of 3H-labelled wild type TFI has also been described [24]. TFI-R61 was purified from the above referred-to ove~roducing E coil BL21 cells as described for TFI [29] with the following modifications: cells were lysed after 30 min preincubation in lysis buffer containing 20 mM Tris-HCI (pH 8.0), 0.1 M NaCI, 5% (v/v) glycerol, 5 mM Na:EDTA, 5 mM 2-mercaptoethanol, 0. I mM PMSE 20 iag/ml leupeptin, 20 pg/ml pepstatin, and 0.5 mg/ml lysozyme by the addition of Triton X-100 to 0.5% (v/v). Polymin P (BASF) was added dropwise from a 13% (v/v) solution to ,~ final concentration of 0.3%. After centrifugation to remove precipitated protein and nucleic acid, the proteins in the supernatant were fractionated by ammonium sulfate precipitation, and TFI-R61 was further purified as described previously. Purification ¢¢'SPO i DNA probes

DNA fragments RHS00 and RH600 were prepared from the EcoRI-26 fragment of SPOI DNA (I.1 kbp) which contains early gene E5 and part of gene E4 [30], and was obtained from

a 1.8 kbp BstYl SPO I DNA fragment by digestion with EcoRl under star conditions. Both DNA fragments, 1.8 kbp BstYl and EcoRI-26, wei-e purified by preparative agarose gel electrophoresis. Only flanking slices of gels were stained with ethidium bromide and the corresponding fragments were isolated from unstained parts of the gels. Purified EcoRI-26 DNA was dephosphorylated, 5' end-labelled using ['g-32PIATP and T4 polynucleotide kinase, and digested with HinfI. The resulting 5' end-labelled RH500 and RH600 SPOI DNA probes were separated in 4% (w/v) polyacrylamide gel, eluted and further purified on NACS columns (Bethesda Research Laboratories). Alternatively, the RI-26 DNA fragment was first cut with Hinfl and then both strands were 5' end-labelled. Probes were dissolved in 10 mM Tris-HC! (pH 8.0), 0.1 mM Na:EDTA, 5% (v/v) glycerol and stored at 4°C. Gel electrophoresis of protein-DNA complexes and kinetic analysis

Wild type TFI and TFI-R61 were mixed at desired concentrations ~nd temperatures and incubated for various chosen periods of time in binding buffer containing !0 mM Tris-HCI (pH 8.0), 50 mM NaCi, 10 mM MgCI,, 0.1 mM Na,EDTA, 0.1 mM D'vr, 50 pg/m! bovine serum albumin (BSA), and 0.06% (w/v) Brij 58. One-pl aliquots were taken, diluted into 9 pl of ice-cold binding buffer also containing 0.2-0.4 fmol of 5' end-labelled DNA probe and 2.5% (w/v) Ficoli 400, rapidly mixed and the entire samples were immediately loaded, with the power on, into the wells of prerun 4% (w/v) polyacrylamide gels (39:1 acrylamide:bisacrylamide) in 50 mM Tris-borate (pH 8.3), ! mM Na,EDTA. After electrophoresis at 300 V (12.5 V/cm), gels were dried for autoradiography. Autoradiographs were quantified on a laser densitometer using software written by AMBIS. Each experiment was repeated at least three times and averaged values were used for graphical presentation. The specific activity of 13HI-TFI complexes with [Y'PI-DNA was determined as described [241. Results

The approximately 500-bp SPOI DNA restriction fragment, RHS00, contains two preferred binding sites for wild type and F61 ---> R mutant T F I protein that are located between the early promoters PE5 and PE6. Each of these two proteins generates a ladder of progressively more retarded complexes (fig l, lanes 2 and 3) by adding T F I dimers, one at a time, to the DNA [24]. (DNA molecules binding 1, 2, 3 etc, molecules of TF 1 dimer are called complexes I, If, Ill, etc, respectively.) The mobility differences of TFI wild type-DNA and TF! R61-DNA complexes arise partly because the two proteins do not bend DNA equally [17]; the two proteins may also differ somewhat in their partitioning between specific and general DNA-binding states [291. Mobility differences accordingly depend at least in part on the sizes of DNA fragments, and on the locations within them of preferred DNA-binding sites. Regardless of the physical basis of these mobility differences, they offer an opportunity to search for the formation of new molecular species in mixtures of wild type T F I and

935 TFI-R61, by allowing these protein mixtures to bind to DNA and looking for new bands on native gels that might reflect the characteristic properties of hybrid protein. New molecular species were detected when mixtures of wild type TFI and mutant TFI-R61 were allowed to bind to DNA and were then examined on native gels (fig 1, lanes 4-9), the implication being that they are formed by binding of TFI wild typeTFI-R61 heterodimers and homodimers in various combinations. The dynamics of formation of these hybrid TFI dimers should closely reflect the properties of monomer interchange in the wild type protein, since the mutation that distinguishes wild type and mutant TFI changes the tip of the DNA-binding arm 122] rather than the monomer-monomer interface. The most rapidly migrating of these new bands, which has an electmphoretic mobility intermediate between that of wild type TFI-DNA complex I and of TFI-R6 ! complex l, should be formed by a molecule of DNA binding a single molecule of TFI heterodimer (ie it should represent heterocomplex I; [24]). A preliminary double label experiment confirmed this conjecture in the following way. Complexes were formed on 32P-labelled DNA fragment RH500 with a preincubated mixture of 3H-labelled wild type TFI and unlabelled TFI-R61 and separated by gel electrophoresis. The ratios of 3H IO 32p in bands corresponding to complex I of 3H-labelled wild type TFI and of the suspected heterodimer complex I were compared as described previously [24]. The putative heterocomplex had 38% of the 3H/32p radioactivity ratio of wild type complex I. This is close to, but less than, the 50% expected lbr a wild type-R61 TFI heterodimer. The difference is readily accounted for by noting the slight tendency for TF1-DNA complexes, particularly complexes with TF!-R61, to dissociate on the gel generating a diffuse background of 32P-labelled DNA ( c f [17]). The detection of only one heterocomplex I in figure 1 suggests, in addition, either that the two distinct orientations of TFI heterorimer on the DNA probes of these experiments generate identical mobilities, or that TFI can change its orientation on DNA (perhaps by dissociating within the gel network, tumbling and reassociating) on the time scale of a gel electrophoresis experiment. The kinetics of formation of TFi wild type-R61 heterodimer at 0, 12, 24 and 36°C were followed by gel retardation using the RH500 restriction fragment as probe ~,~s , a - I, 2A). At each time point, the percent of heterocomplex I relative to total complex I was determined by densitometry of autoradiograms such as figure 1 (see Materials and methods). It should be noted that *,he relative abundance of heterocomplex I in the retardation gel is the product of two contributions: the relative concentration of TF1 heterodimer at the time of sampling, and the relative affinity of the

heterodimer for binding to DNA. The latter quantity has not been independently determined, because it is not required for internal comparisons. The strongly temperature-dependent rate of formation of TFI heterodimers is shown in figure 2A. At the highest temperature, 36"C, appreciable formation of heterocomplex I occurred during the shortest time period available for manually mixing proteins, adding DNA, applying the sample to the gel and allowing the DNA to enter the gel (approximately 2-3 min). At this low protein concentration, the subunit exchange reaction was very slow at 0°C, but given enough time, samples held at 0°C came to the same state as samples equilibrated at 360C (table I). It is worth noting that the secondary structure of TF1 is temperature-dependent

TFI

w t R61

time (rain)

0.5

1

2

wt:R61-1:l

0.5 0.5

3

4

$

5

I0

6

20

40

•

: D

7

8

80

9

Fig 1. Formation of new DNA-protein complexes by mixtures of wild type TFI and TFI-R61. The two proteins were mixed (4.6 nM of each) at 36°C for the time intervals shown above lanes 4-9, then diluted 10-fold into binding buffer containing 32p-labelled RH500 DNA and analyzed as described in Materials and methods. DNA complexes of wild type TFI (lane 2) and TFI-R61 (lane 3) were run alongside. Open circles mark DNA-TFI complexes I (one molecule of TFI dimer per molecule of DNA) of the wild type ai~d R61 proteins. The closed circle marks the most rapidly migrating of tile ,~w protein-DNA cc.,'ni~lexes that formed when the RH500 DNA probe was added to a preincubated mix of the two proteins. The arrow at the side marks a minor contaminant band of DNA.

936 <.--. Fig 2. Dependence of the rate of formation of TFI heterodimers on temperature and protein concentrations. Equimolar mixtures of TFI wild type and TFI-R61 were incubated for the times shown, diluted I O-fold into binding buffer and analyzed as specified in Materials and methods and the legend to figure 1 to quantify the fraction of all DNA complexes 1 constituted by the TF1 heterodimer. A. Rate of heterodimer formation in a mixture containing 4.6 nM TFI wild type and 4.6 nM TFIR6I. The 'percentage of heterodimer" refers to DNA heterocomplex I and was determined as described in Materials and methods. B. Dependence of the formation DNA heterocomplex I on protein concentration. Equimolar mixtures of TFI wild type and TFI-R61 were incubated at 0°C for 10 min, then rapidly diluted at 0°C with a final dilution into 32p-labelled DNA and analyzed for formation of DNA heterocomplex I. The total TFI concentration of the original protein mixture is plotted on the abscissa.

40 36oC

240C

i ,° OoC

'i

Table 1. Subunit exchange at 0 and 36°C. The conditions of the experiment were as specified in the legend to figure 2A.

0.5

2'0

4'0

6'0

8'0

100

Time of incubation (min)

Time [mini

0.5 20 80 300 1500

40'

i

i

30'

o ,,G

nc

20'

10'

0

. . . . . . . .

10 °

,

101

. . . . . . . .

,

101

. . . . . . . .

,

10 s

. . . . . . . .

,

. . . . . . .

1 O*

Concentration (nM)

in the investigated range of these experiments [31, 39], and that a correlation between secondary structure stability and rate of subunit exchange has been noted [39]. The concentration-dependence of the rate of heterodimer formation was briefly explored in the experiment shown in figure 2B. Proteins were mixed at 0°C

wt-R61 heterodimer (%) O°C

36°C

3.5 7.8 19.9 16.2 39.5

8.5 31.4 34.6 40.8 41.1

for 10 min at I:1 molar ratio, and at various total concentrations, then serially diluted at 0°C with a final dilution into binding buffer with DNA, also at O°C, that generated the same final protein concentration for all samples (0.46 nM). Analysis by gel electrophoresis was at room temperature. (The convenience of room temperature electrophoresis can only have generated negligible perturbations in the data of figure 2B, for the following reasons: 1) very little exchange of monomers occurred at 24°C in samples loaded on the gel immediately upon mixing (fig 2A); 2) all samples were submitted to the same regimen.) At 3 pM T F I , the heterodimer and homodimers came to equilibrium within 10 min of mixing. The formation of heterocomplexes depended on the relative concentrations of wild type T F I and TFI-R61 in the expected manner, as shown in figure 3. The relative concentration of heterocomplex I (open circle) was greatest when the proteins were mixed in 1:1 molar ratios. With wild type protein in excess, heterodimer I was more abundant than TFI-R61 complex I (bottom, closed circle), while in excess TFI-R61 (bottom, closed circle) the heterodimer was more abuadan~ than wild type TFI complex 1 (top, closed circle). The changing relative abundances of

937 higher order, more slowly migrating complexes also gave hints about their compositions. However, the RH500 fragment is not ideal for resolving these complexes, due to the particular relationships of their electrophoretic mobilities (eg wild type complex I has a mobility that is intermediate between the mobilities of TFI-R61 complexes H and Ill). Another DNA fragment, RH600, whose preferred TFI binding site overlaps the early promoter P~5 [17], proved to be useful for examining these higher complexes, particularly complexes whose mobilities were intermediate between those of wild type and R61 complex H (fig 4). On the basis of their relative electrophoretic mobilities, these complexes are expected to be heterocomplexes II, that is, to contain two molecules of TFI dimers bound to one molecule of DNA, with individual hybrid bands containing the two kinds of TFI monomers in relative molar

wt:R6 I

TFI

wt

R61

2

3

3:1

1:1

1:3

wt R61

4

5

6

7

wt:R61 i

TFI

wt R61

1:1 3:1

3:1

wt

R6!

8

Fig 4. Higher order complexes. Resolution of multiple species of higher-order DNA complexes on DNA probe RH600. Proteins were mixed as specified in the legend to figure, 4. Bands corresponding to wild type TFI and TFIR61 homocomplexes II are marked with filled circles and four resolved heterocomplexes !I are marked by the bracket.

•

1

•

2

3'

• •

4

......

ii~ ' ;

5

i

i !i~ ~i

6

ii ~i~ ~ i i

7

8

Fig 3. Dependence of the abundance distribution of complexes I on the relative proportions of TFI wild type and TFI-R61. Proteins (4.6 nM total) were mixed in 3:1,1:1 and 1:3 molar ratios, incubated at 24°C for 30 min and analyzed for formation of complexes with RH500 DNA. (O): wild type and TFI-R61 homocomplexes I; (0): heterocomplex I; (A): unresolved multiple heterocomplexes.

proportions of 3:1, 2:2, and 1:3. In fact, four bands (bracketed in lanes 4 and 5) with mobilities intermediate between those of the homodimer complexes II were detected. When R61 protein was in three-fold excess, formation of wild type homodimer complex II was barely detectable (too weak to be seen in the reproduced figure 3A, lane 6); TF1-R61 homodimer complex II was similarly only barely detectable in the presence of a three-fold excess of wild type protein (lane 4). Higher complexes gave wide bands, presumably due to inferior resolution associated with the presence of a large number of distinct molecular species with small mobi:'ty differences. However, the general properties of these broad bands were as expected, For example, complexes III shifted their centers to lower mobility for excess wild type protein and to higher mobility for excess TF1-R61 protein, respectively; when either wild type TF1 or TFI-R61 was in excess, the minority homocomplex III was too rare to be detectable.

938

Discussion TFl is a homodimer whose general form and monomer-monomer interface has been conjectured to approximate that of the B stearothermophilus HU protein, the sole DBPII whose detailed structure is known at case accumulating through the ongoing analysis of the three-dimensional structure of TFl in solution by multidimensional NMR spectroscopy, using a selective deuteriation strategy [28, 33, 341. An early result of this analysis shows two aromatic amino acids, F28 and F47, in close vicinity in the core of the TFl dimer and defines the relative orientation of side chains, consistent with a structural model of TFl [28, 351 that is based on the 2.1 A resolution B stearothermophifus HU crystal coordinates [27] in a water surround and has been energy-minimized with distance constraints based on NMR (NOE)-derived information for specific aromatic amino acid side chains. A subsequent nearly complete set of assignments of the proton-NMR spectrum of TFl [33] also shows that the polypeptide chains of TFl and B steurothermophifus HU are globally folded in similar ways and contribute similarly configured a-helix and P-strand domains to the cores of the respective protein dimers. We have analyzed the dynamic stability of the TFl dimer by making use of the fact that an amino acid change that is well removed from the monomermonomer interface alters the electrophoretic mobility of TFl-DNA complexes without greatly changing the overall DNA affinity. The formation of new species of TFl-DNA complexes when variant and

A

B

C

D

0 TFl-wild type

TFl-FM1

Fig 5. The potentially distinguishable heterocomplexes 11 that two different TFl monomers can form with a single site in non-symmetric DNA.

wild type TFl proteins are mixed has enabled us to follow the exchange of monomers between molecules of TFl dimer. The rate of this exchange is protein concentration- and temperature-dependent (fig 2). During phage SPOl infection of B subtilis, TFl accumulates to a high concentration roughly estimated as 5 x 104 dimers per cell [36]. Its relatives, the heterodimeric E coli HU and II-IF proteins are reported to be present as 2 to 6 x 104 dimers per cell [ lo] corresponding to -26 x 10-5 M. At these concentrations, the subunit exchange of free TFI is extremely rapid, even at O°C (fig 2B). However, the bulk of TFl is likely to be nucleic acid-bound in viva, and we suggest, on the basis of the experiment shown in figure 4, that DNA-bound TFl dimers may not undergo subunit exchange. The concentration dependence of subunit exchange has been briefly examined; the available information (fige 2B) specifies that the reaction rate is proportional to a higher-than-first power of the protein concentration. This is incompatible with a subunit exchange reaction that proceeds exclusively by way of dissociation into monomer units, regardless of protein concentration. At higher TFl concentrations, tit least, direct exchange of subunits between dimers may make significant contributions to the overall rate of the exchange process. Thus, although the temperature dependence of subunit exchange (fig 2A) and other evidence [39] imply that loosening of structure favors more rapid monomer interchange, complete dissociation into subunits is not likely to be exclusively involved. Although subunit exchange in E coli HU and IHF has not been explicitly analyzed, it cannot be comparably facile, otherwise E coli HU would end up, in standard methods of preparation, as the 1: 1:2 mixture of the a, and $ homodimers and the af3heterodimer. respectively, and that is not the case [37]. It has also been concluded in the past that IHF subunits would not readily exchange, because overexpression-production of the individual IHF monomers yields unstable or insoluble material; only co-overexpression of both the himD (hip) and himA genes, coding for the I) and a subunits of IHF, yields a soluble and functional product [38]. The formation of only one heterocomplex I (fig 1) is consistent with the fact that complex I contains a single dimer of TFl bound to a molecule of DNA. Finding only one such band specifies, in addition, either that: 1) the two distinct orientations of TFl heterodimer on these DNA probes generate complexes with identical mobilities; or that 2) TFl can reorient on a DNA site (for example by dissociating within the gel network, rotating and reassociating) on the time scale of a gel electrophoresis experiment. Multiple species of higher order TFl heterocomplexes were

939 detected (figs 4, 5). Among these, the diversity of resolvable complexes H is instructive. Four of these heterocomplexes were resolved on the RH600 DNA fragment (fig 4). We interpret the resolution of these four bands of heterocomplex II in the following way (fig 5). Each band corresponds to a class of DNA complexes containing, respectively, one TFI wild type homodimer and one heterodimer (3 monomers:l monomer of wild type:R61); one TFI-R61 homodimer and one heterodimer (1:3 wild type:R61); two hetemdimers (2:2 wild type:R61); one of each homodimer (2:2 wild type:R61). Each of these classes of complexes is, in turn, composed of several isomers (fig 5). We suspect that the individual isomers of each class do not yield resolvable bands either because they have identical mobilities, or because they are interconvertible during a gel run. Thus, for example, a combination of transient dissociation in the gel network as already referred to above, sliding of the remaining bound TFI dimer to the just-vacated DNA site, rotation of the free protein and reassociation, generates each class A isomer from every other one. We interpret the existence of four rather than three heterocomplex I1 bands as suggesting that class B and C complexes do not isomenze when bound to DNA and, accordingly, that subunit exchange is a property of free TFI, but not of DNA-bound TFI. That is consistent with what is thought to be known about the structure of the TFI dimer-DNA complex, in which DNA bends sharply as it wraps around the protein [17]. DNA-wrapped TFI might well be impeded in undergoing subunit exchange.

Acknowledgments We are grateful to J Kyte for a rigorous kinetic analysis and advice, J Reisman, DR Kearns, J Parello and C Spongier for helpful discussions, and C Spangler for comments on the manuscript. Our research was supported by a grant from the NIGMS.

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