THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 14, Issue of May 15, pp. 10546-10552,1393 Printed in U.S.A.

EBP-80, a Transcription Factor Closely Resembling the Human Autoantigen Ku, Recognizes Single- to Double-Strand Transitions in DNA*

(Received for publication, December 2, 1992)

Miriam FalzonS, Joseph W. Fewell, and Edward L. Kuff From the Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892

We have previously reported the purification and characterization of the transcription factor EBP-80 (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chern. 264, 21915-21922). EBP-80 mediates the DNA methyla- tion effect on transcription from an endogenous pro- viral long terminal repeat. Here we show that EBP-80 is very similar if not identical to the Ku autoantigen, a heterodimeric nuclear protein first detected by anti- bodies from autoimmune patients (Mimori, T., Aki- zuki, M., Yamagata, H., Inada, S., Yoshida, S., and Homma, M. (1981) J. CZin. Invest. 68, 611-620). A number of laboratories have shown that the Ku protein complex binds to free double-stranded DNA ends. In this study, we have examined the binding properties of EBP-80. EBP-80 binds single-stranded DNA with low affinity. Binding to random sequence double-stranded DNA depends on the length of the duplex and is optimal with oligomers of 30 and 32 base pairs; the protein complexes formed with these oligomers have Kd values of 15-20 p ~ . It binds with comparable high affinities to blunt-ended duplex DNA, to duplex DNA ending in hairpin loops, and to constructs in which an internal segment of duplex DNA is flanked by single-strand extensions. EBP-80 also interacts effectively with cir- cular duplex molecules containing a 30-nucleotide sin- gle-stranded region (gap) or a double-stranded seg- ment of nonhomology (bubble), but only weakly with the corresponding closed circular construct made up entirely of duplex DNA. EBP-80 prefers A/T to G/C ends. The binding properties of EBP-80 are consistent with the hypothesis that it recognizes single- to double- strand transitions in DNA. A model is presented for the interaction of EBP-80 with its target sequence in the proviral long terminal repeat.

In previous studies we reported the purification and char- acterization of the transcription factor EBP-80 (1-3). EBP- 80 is a protein fraction isolated from HeLa and 293 cells by affinity chromatography on an oligonucleotide representing a regulatory sequence in intracisternal A-particle (IAP)’ long terminal repeats (LTRs). The EBP-80 fraction consists of equimolar amounts of 85- and 70-kDa proteins (1). It mediates

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence should be addressed Laboratory of Biochemistry, National Cancer Inst., Bldg. 37, Rm. 4C03, Bethesda, MD 20892. Tel.: 301-496-6855; Fax: 301-496-0260.

The abbreviations used are: IAP, intracisternal A-particle; LTRs, long terminal repeats; HPLC, high-performance liquid chromatogra- phy; bp, base pair(s); nt, nucleotide(s).

in part the methylation response of this LTR (3). In the presence of a large excess of nonspecific competitor, EBP-80 binds preferentially to sequences with enhancer core homol- ogy (1). EBP-80 enhances transcription in vitro from the IAP LTR. Enhancement is methylation-sensitive providing the template is presented in closed circular form (2, 3). In this study, we show that EBP-80 is indistinguishable by several criteria from the previously described human autoantigen Ku.

Ku is a heterodimeric nuclear protein first detected by antibodies from certain autoimmune patients (4, reviewed in Ref. 5). Human Ku preparations contain equimolar amounts of 69- and 83-kDa polypeptides; the cDNAs of both subunits have been cloned and sequenced (6-9). Ku binds to ends of double-stranded DNA (10). A study of Ku polypeptides syn- thesized in vitro indicated that it is the 70/85-kDa complex rather than the individual subunits that possess the DNA- binding properties (11). However, Chou et al. (12) found that fusion products of the 70-kDa subunit with TrpE protein, as well as the cellular 70-kDa form itself when carefully rena- tured, could bind DNA as efficiently as the heterodimeric complex. A minimal DNA binding site was localized on the 70-kDa subunit (12).

The cellular function of Ku is not known. The end-binding property of Ku has led to suggestions that it could have a role in recombination or DNA repair. On the other hand, protein preparations designated TREF and PSE-1 that are similar to or identical with Ku have been identified as sequence-specific transcription factors for the transferrin (13) and U1 snRNA (14) genes, respectively. A very similar protein has been isolated from a human B-cell line and shown to bind to the eukaryotic octamer motif as well as to DNA ends (15). This preparation, designated Ku-2, is a heterodimer of 72- and 83- kDa subunits which show strong sequence similarities to the corresponding subunits of Ku but are not identical. A protein fraction from HeLa cell nuclei, originally termed NFIV and subsequently identified as Ku (16, 17), was shown to bind to the ends of adenovirus-2 DNA and translocate inward to form a DNA/multimeric protein complex (16). Some of these pro- teins, as well as EBP-80, may be sequence variants encoded by separate members of a recently demonstrated family of Ku-homologous genes (18).

There are conflicting data on the interaction of Ku with single-stranded DNA. Ku bound relatively weakly to a heat- denatured 300-bp fragment in a filter-binding assay (lo), and TREF binding was not competed by single-stranded DNA (13). However, circular single-stranded M13 DNA competed efficiently for NFIV/Ku in a gel shift system using a 94-bp adenovirus-4 origin-containing probe (16). The 70-kDa com- ponent of Ku, expressed from a baculovirus construct, bound tightly to both single- and double-stranded DNA columns (19).

10546

EBP-80 Recognizes Single to Double-strand Transitions i n DNA 10547

T h e present study was designed to compare the DNA- binding properties of EBP-80 with those of Ku and t o show how these properties might be related to the interaction of EBP-80 with its target sequence in the IAP LTR.

MATERIALS AND METHODS

Purification of EBP-80 and Amino Acid Sequencing of Tryptic Peptides-EBP-80 was purified from nuclear extract of the adenovi- rus type-5 transformed 293 cell line (20) as previously described (l), with one modification. Following passage through the heparin-Seph- arose column, the EBP-80-containing fraction was passed through a MonoQ column (Pharmacia) before being applied (twice) to the oligonucleotide affinity column (1). The 70- and 85-kDa EBP-80 components, which elute together from the affinity column, were separated from one another on a 10% SDS-polyacrylamide gel. Pro- tein was electroeluted from the gel slices using an Amicon centrilutor. Following ethanol precipitation, the pellets from the two separated EBP-80 components were dried in uucuo and redissolved in 10 pl of 8 M urea, 0.4 M ammonium bicarbonate (pH 8.1). The protein yield was roughly estimated by running 1 pl on a 10% SDS-polyacrylamide gel and staining with silver nitrate. Approximately 6 pg of each protein was digested with trypsin essentially as described by Stone et al. (21). The tryptic digests were diluted with an equal volume of 10% acetonitrile, 0.1% trifluoroacetic acid and loaded onto a 2.1 by 150- mm Vydac CIS reversed-phase HPLC column (The Nest Group) on an Applied Biosystems Model 130A separations system. Peptides were eluted with a gradient of 8-50% of acetonitrile in 0.1% trifluo- roacetic acid, and individual peaks were collected onto glass fiber filters. The filters were dried in uacw and subjected to amino acid sequence analyses on an Applied Biosystems 477A protein sequencer coupled to a 120A analyzer.

Probe and Competitor Preparation-Random sequence oligonucle- otides were synthesized as self-complementary single-stranded mol- ecules and annealed prior to use as probes and competitors. They were 5’-end-labeled with [y-32P]ATP as previously described (22). Oligonucleotides containing hairpin structures (Fig. 1, i-l) were syn- thesized as single-stranded molecules which were then self-annealed. The two dumbbell-shaped oligonucleotides (Fig. 1, rn and n) were synthesized as single-stranded 83-mers. After self-annealing, the 5’- gapped ends were 32P-labeled, filled-in using Klenow polymerase and dATP, and ligated. Fill-in and ligation were assessed by treating part of the final products with calf intestinal phosphatase (Boehringer Mannheim). Both ligated preparations contained at least ten times more phosphatase-resistant radioactivity than their unligated precur- sors.

Minicircle DNA was prepared from the plasmid pSA508, provided by H. E. Choy and S. Adhya (Laboratory of Molecular Biology,

PROBE (pM) Kd

- 14 8. - >Em

16 b. = 60

c. - 30

d. = 18 - 24

25-30

e. - 1520

f. 40

33.32

9. 18 18

18 2530

PROBE (DM) Kd

140)

-

i. 105115 (115)

_.

m. C

n. 30

4 3 0 4 25-30 1 2 5 )

FIG. 1. Diagrammatic representation of oligonucleotide constructs and their dissociation constants ( K d ) for binding to EBP-80. Numerals on the diagrams indicate numbers of base pairs or nucleotide residues. Sequences of the 5’-oligonucleotides used for making double-stranded probes are shown under “Materials and Methods.” Kd values were calculated as described under “Materials and Methods.” One standard deviation of the mean (67% probability) corresponds approximately to f 5 pM; two standard deviations (98% probability) compares to k 10 PM. Values in parentheses were derived from competition assays.

National Cancer Institute, National Institutes of Health). pSA508 contains a DNA segment with multiple cloning sites and a transcrip- tion termination sequence sandwiched between the X phage attach- ment site attP’OP and the corresponding bacterial site, attB’OB. In the presence of X Int and host IHF proteins, this plasmid undergoes the phage site-specific recombination upon induction at 42 “ c , giving rise to an approximately 350-bp DNA circle containing the multiple cloning site? The circle was linearized with EcoRI and Sal1 and cloned into pUC19 in order to obtain large quantities of the linear form. Gel-purified fragment was used as a source of DNA for prepar- ing circular constructs with single- to double-strand DNA transitions. We prepared constructs shown in Fig. 7, containing (a) a 30-bp region of single-stranded DNA (gap), (b) a 30-bp region of nonhomology (bubble), and (c) complete homologous strands. The oligonucleotides used to prepare these constructs were synthesized as single-stranded molecules, annealed, 5’-end phosphorylated and ligated to the linear EcoRI/SalI fragment described above. The circular products were gel- purified for use as competitors in gel retardation and filter-binding assays. The closed circular nature of each construct was verified by treatment with calf intestinal alkaline phosphatase followed by 5’ end labeling with [y-32P]ATP. Incorporation of radiolabel by the circular constructs was only 5-6% that of the linear form.

Sequences of the oligonucleotides used for making double-stranded probes were: a, GATCCACAGTGCAT (14-mer); b, TC-a (16-mer); C,

f, e-TCGATCCACAGTGCATGGCTCTGGTTGCTATG (64-mer). b-GG (18-mer); d, c-CTCTGG (24-mer); e, d-TTGCTATG (32-mer);

These were annealed with their complementary strands and end- labeled with [y-32P]ATP. Top strand of g, the 18-bp duplex with 5’ 18-nt overhangs was: 5’-tcgatccacagtgcatggCTCTGGTTGCTATGT CGA(g2). Bottom strand of g was: 5’-agagccatgcactgtgga-complement g2. Lower-case letters indicate unpaired nucleotide residues in the annealed forms.

Sequences of the oligonucleotides used for making h, the 18-bp duplex with four 21-nt single-strand extensions were 5”tgcaaggtccct tcaactatg(hl)-CCATGCACTGTGGATCGA(h2)-tcgtagcaccatgcatggt at(h3), and 3’-hl-h2complement-h3. Structure i was obtained by self-annealing the oligonucleotide 5’-CTCTGGTTgctatgtcgAACCA G A G . Structure j was 5”tcgatccacagtgcatgg-i; k was 5”i-tgca- aggtgccttcagctatg. Structure l contained i plus the unpaired strands of both j and k . The dumbbell form m, with 4-nucleotide loops, was made by self-annealing, filling-in, and ligating the 84-mer CTGTGG ATCGACGTaaatACGTCGATCCACAGTGGCATGCGTCTTGGT T C G T A T C G T a a a t A C G A T A C G A A C C A G A C G C A T G C C . A dumbbell form with 22 bp of central duplex and 20-nt loops (n) was made in similar fashion.

Gel Retardation and Filter Binding Assays-Gel shift binding and competition assays were carried out as previously described (1, 22). EBP-80 concentration in the stock preparation was determined by a protein-gold assay (Integrated Separation Systems) that gives linear optical density readings over a range of 5 to 200 ng of protein. The reaction was calibrated with bovine serum albumin. EBP-80 is as- sumed to bind as the 155-kDa heterodimer (5,10,23) for the purpose of calculating molar concentrations. Affinity constants (Kd values) for binding of EBP-80 to duplex oligonucleotides were calculated from filter-binding data. Binding of complexes to nitrocellulose filters (Millipore; HAWP-0025, 0.45 Nm) was carried out at room tempera- ture (20-23 “C) in a buffer consisting of 20 mM Tris hydrochloride, pH 7.8,lOO mM KC1,2 mM EDTA, 2 mM dithiothreitol, bovine serum albumin at 100 pg/ml, and 10% glycerol. The reaction volume was 25 pl. For each experiment, a set of tubes was prepared with a constant amount of 32P-labeled oligonucleotide probe (nominally 9 fmol), and a standard set of EBP-80 levels ranging from 0.55 to 110 fmol. Tubes were allowed to stand 35-40 min, a time established in preliminary experiments as sufficient to permit equilibration for all ratios of EBP- 80 to probe. The mixtures were then applied to filter membranes. The retained radioactivities, representing the EBP-BO-bound probe, were expressed as fractions of the counts bound at the highest (saturating) EBP-80 levels. In practice, the absolute amounts of probe were not known because of indeterminate losses during purification of the radiolabeled oligonucleotides and variations in the efficiency of both end-labeling and annealing from one preparation to another. Accordingly, it was necessary to treat both Kd and total probe con- centration [P,] as variables in calculating a set of binding curves that could be tested for best fit to the experimental data. For this purpose, the equation for a bimolecular reaction at equilibrium, Kd = [P,][Ef] /[&.&,I, where p, and E, represent free oligonucleotide probe and

H. E. Choy and S. Adhya, personal communication.

10548 EBP-80 Recognizes Single to Double-strand Transitions in DNA

free EBP-80, respectively, and Pb.Eb is a one:one complex of bound probe and EBP-80, was rearranged to give [Et] /xZ - ( [ P , ] + [Et] + & ) / X - [P,] = 0, where E, and P, represent total EBP-80 and probe, respectively, and x = [pb]/[Pt] . Using a computer program, the quadratic equation was solved for a number of combinations of K d

and [P,], generating a series of binding curves, x us. [E,] , which could be tested against the experimental data. For each combination of K d and [P,], the program determined the arithmetic sum of the differ- ences between the computed and experimental values of x over a standard set of EBP-80 levels, the statistical mean of these differ- ences, and the standard deviation of the mean. The combination(s) giving a sum of differences near zero and the least mean difference was taken to represent the best estimate of K d and Pt. K d values were calculated at intervals of 5 p ~ . Frequently two successive K d S gave equally favorable fits, in which case both values are shown in Fig. 1. Unless otherwise noted, the curves connecting data points in the figures were calculated using the best fit values of K d and [P t ] .

RESULTS

Relatedness of EBP-80 and Ku-Preparations of EBP-80 contain equimolar amounts of 70- and 85-kDa components (l), as does Ku. Two peptides from each species of EBP-80 were isolated and microsequenced as described under “Mate- rials and Methods.” Unambiguous sequences were obtained (Table I), which upon comparison with sequences in the GenBank and EMBO Data Bank, perfectly matched residues within the 70-kDa (amino acids 39-46 and 207-218) and 80- kDa (amino acids 906-916 and 1984-1989) subunits of Ku. Both components of EBP-80 reacted on Western blots with a human antibody preparation (24) that had been affinity- purified on authentic Ku (data not shown). EBP-80 is clearly related to Ku; however, proof of identity would require se- quencing of the entire protein.

Oligonucleotides Tested for Binding of EBP-80-A variety of synthetic oligonucleotides with conventional or atypical structures were studied. These are diagrammatically illus- trated in Fig. 1, together with the dissociation constants ( K d )

for binding of EBP-80. All of the sequences are intended to be nonspecific in terms of known preferences for EBP-80 or Ku.

Binding of EBP-80 to Double-Stranded Oligonucleotides of Varying Length-The binding of EBP-80 to radiolabeled du- plex oligonucleotide probes 14-64 bp in length was examined by gel shift assay (Fig. 2). The 14-bp oligomer did not form a stable complex in this system. The others gave major bands of similar mobility. We assume that the 18- and 24-mer, because of their small size, form the minimal reaction product with EBP-80, i.e. one heterodimeric protein molecule per molecule of oligonucleotide. The 32-bp oligomer corresponds in size to the length of DNA occupied by one bound molecule of Ku (16, 17). The slower moving minor band which appears on the gel shift with this probe (Fig. 2c) presumably represents a weak secondary reaction. The 64-bp oligomer is theoretically large enough to accommodate two molecules of EBP-80, and in fact, the gel pattern displays a prominent second band (Fig. 2d).

TABLE I Identical amino acid sequences in EBPdO and human Ku

The sequences of tryptic peptides derived from the 70- and 85-kDa subunits of EBP-80 are identical to those found at the indicated amino acid positions in the corresponding subunits of Ku (6-9).

Protein subunit seauence EBP-80 peptides Identical Ku

kDa 70 IFLVDASK 39-46

KPGGFDISLFYR 207-218 85 LNDDDETEVLK 906-916

FNNFLK 1984-1989

a b c d e

FIG. 2. Gel shift analysis of EBP-80 binding to various oligomers. In lunes a-d, the probes were linear double-stranded oligonucleotides with lengths of 18, 24, 30, and 64 bp, respectively; the binding mixtures were run simultaneously on the same gel. In lune e, the probe was the dumbbell-shaped form with 38 bp of central duplex flanked by 4-nt hairpin turns (Fig. lm) . This probe was run separately from the others but the mobility of the complex was similar to that of the blunt-ended forms.

I

1 .oo

.80

.60

.40

.20

n ” .22 .44 .66 .6a 1.10

EBP-80 (nM)

FIG. 3. Binding of EBP-80 to duplex oligonucleotide probes with lengths of A, 14 bp; A, 16 bp; 0, 18 bp; 0 , 2 4 bp; V, 32 bp. Points were determined by filter binding assay, and continuous curves were calculated from best fit values of K d (see Fig. 1) and total probes. Points connected by broken lines show binding of EBP-80 to single-stranded probes 32 (0) and 64 (M) nt in length.

The affinity of EBP-80 for the same series of oligonucleo- tides was examined by filter binding (Fig. 3). The solid curves in Fig. 3 were calculated using the best fit values of Kd and total probe concentration ( Pt) as described under “Materials and Methods.” Dissociation constants were related to the length of the oligonucleotide (Fig. 1). The 14-bp oligonucleo- tide bound weakly, with a K d in excess of 800 pM. Addition of 2 bp in length (16 bp) increased the affinity more than 10- fold. Additional increments in length gave a further small increase in affinity with K d values estimated at 15-20 f 5 pM for the 30- and 32-bp oligomers. The binding data for the 64- bp oligomer could be closely fitted using a K d of 40 pM (binding curve not shown). This is an approximation, since the binding equation does not apply when the ligand binds significantly more than one molecule of protein (Fig. 2).

Fig. 3 also shows the weak interactions between EBP-80 and single-stranded oligonucleotides 32 and 64 nts in length.

Participation of Single-Stranded Ends in the Binding Re- action-The binding of Ku to restriction enzyme fragments was reported to be independent of whether the DNA ends were blunt or had 5’ or 3‘ overhangs of up to 4 nt (10). To determine whether longer single-stranded extensions affect binding, we made a construct containing 18 bp of duplex DNA with 5’ single-stranded overhangs 18 nt in length (Fig. lg). This construct gave a single major band on gel shift (data not

EBP-80 Recognizes Single to Double-strand Transitions in DNA 10549

shown) and in the filter-binding assay, bound EBP-80 with an apparent K d of about 25 PM. Evidently the long single- stranded extension did not interfere with access of EBP-80 to the binding site.

The effects of single-stranded DNA on binding of EBP-80 were next tested using a construct in which all four strand ends of the 18-bp duplex segment were extended with 21-nt single strands (Fig. lh). The gel shift pattern after exposure to high EBP-80 (Fig. 4) showed a single major band. However, the filter binding curve (not shown) was concave at low concentrations of EBP-80 and could not be fitted with the binding equation. Therefore, affinity was evaluated by com- petition for EBP-80 with the 64-bp duplex oligomer which is similar in overall length (Fig. 5A). The four-tailed construct competed as effectively as the 64-mer itself, indicating a comparable K d for binding (Fig. 1) and interaction with EBP- 80 at the same sites as the blunt-ended duplex.

Since EBP-80 has little affinity for single strands by them- selves, it seemed possible that the protein was binding at the

a b C d n ---

FIG. 4. Gel shift patterns by a, oligonucleotide probe h; b- d, oligonucleotides j , k, and i, respectively (see Fig. 1 for structures). The probes were end-labeled with 32P, and 0.1-ng sam- ples were incubated for 30 min at 20" C wih a, 0.25 and 0.5 pl of EBP- 80; b-d, 0, 0.1, and 0.25 pl of EBP-80. 1 pl of EBP-80 stock solution contained 0.5 pmol.

l o o 80 60

40

20

10

6

.2 .4 .6 .8 1.01.21.41.6 .2 .4 .6 .8 1.01.21.41.6 DILUTION FACTOR (LOG,,)

FIG. 5. Competition of atypical oligonucleotides against lin- ear blunt-ended duplex probes. A, structure h, with four single- stranded extensions flanking an 18-bp central duplex (Fig. 1) was competed against the 64-bp duplex End-labeled 64-mer (9 fmol per tube) was mixed with increasing levels of either unlabeled self or oligo-h up to 40-fold dilution, and 8 fmol of EBP-80 per tube was then added. The tubes were incubated at 20 "C for 35 min, and bound probe was collected on nitrocellulose filters. The level of uncompeted binding was also determined. The data are shown as log-log plots of the fraction of uncompeted counts versus the dilution factor: 0, 64- mer; A, h. The slopes are proportional to the log,, &. B, structures j , k , and 1, Fig. 1, were competed against the 32-bp probe as above. 0, 32-mer; 0,1; A, j , and A, k.

ends of the central duplex region, i.e. at the points where the strands diverged. To determine whether single-stranded seg- ments can participate in binding, we examined the constructs designated i, j , k, and 1 in Fig. 1. The smallest one, a hairpin with 8 bp of duplex stabilized by a 9-nt loop, was incapable of binding EBP-80 (Fig. 4). However, when either the 5 ' - or 3'-end was extended with single strands, binding was observed in both the gel shift (Fig. 4) and filter binding competition (Fig. 5B) assays. The results by the two methods were con- sistent in showing that either single strand greatly facilitated binding and that there was a moderate (%fold) preference for the 5"extension (Fig. 1). The construct with both ends ex- tended (Fig. 11) aggregated extensively in the gel shift system. However, it could be examined by filter binding. In the competition assay (Fig. 5B), this construct had an affinity for EBP-80 comparable to that of the 32-bp blunt-ended duplex probe. These observations show that single-stranded DNA can participate in the binding of EBP-80 when associated with a region of duplex DNA.

Lack of Requirement for DNA Strand Ends-To determine whether binding requires the presence of strand ends, we prepared a dumbbell-shaped oligonucleotide in which a 38-bp double-stranded segment was flanked by 4-nt hairpin turns (Fig. lm). This construct displayed a gel shift pattern (Fig. 2) identical to that of the blunt-ended 64-bp duplex. The same results were obtained when the closed oligomer was made to contain 22 bp of double-strand region flanked by 20-nt loops (Fig. In).

A competition experiment was carried out to test whether the blunt-ended 32-bp duplex and the dumbbell oligonucleo- tide interacted with EBP-80 via the same DNA binding sites (Fig. 6A). The dumbbell competed as effectively with the 32- bp oligomer for binding to EBP-80 as did the 32-mer itself. The affinities of constructs m and n were also measured from direct binding data (not shown), yielding Kd values of 25-30 k 5 PM. Thus, EBP-80 does not require free strand ends to interact with DNA. A dumbbell structure was tested for binding by Ku, with comparable results (25).

Binding of EBP-80 to Circular Structures Containing Single to Double-Strand DNA Transitions-Ku is known to bind poorly to closed circular plasmid DNA (lo), and this has been confirmed for EBP-80 (see below). We examined the affinity of EBP-80 for small closed circular DNAs that contained a

) \ I 3 ' .; .b .6 .; 1!01:21f4116 ' .; .b .6 .;I 11O1121h1.6

DILUTION FACTOR (LOG,,)

FIG. 6. Competition of oligonucleotides lacking strand ends with the 32-bp blunt-ended probe. A , the dumbbell-shaped struc- ture, m (Fig. I), was competed against the 32-mer as described in legend of Fig. 5 . 0 , 32-mer; 0, m. B, the minicircle structures shown in Fig. 7 were competed for binding to the 32-bp probe. Competitors: 0, 32-mer (self); 0, minicircle with 30-bp bubble; A, minicircle with 30-nt single-strand gap; ., minicircle linearized with EcoR1; A, untreated minicircle. Several of the minicircle competitors were pres- ent in lower than nominal concentration in the binding mixtures; in these cases, the true slopes were established after the free EBP-80 had been saturated with competitor (breaks in the lines).

10550 EBP-80 Recognizes Single to Double-strand Transitions in DNA

single-stranded gap or a region of nonhomology (bubble). The minicircle forms shown in Fig. 7 were tested in a gel shift assay as competitors of the linear 64-bp oligonucleotide for binding of EBP-80. The constructs containing the 30-bp bubble of nonhomologous sequence and the 30-nt single- stranded gap both competed a t least as well as the linearized minicircle. On the other hand, the two completely double- stranded constructs, one the reconstructed and the other the native minicircle, both competed poorly. The same constructs were competed against the 32-bp duplex oligomer in the filter binding assay (Fig. 6B). The displacement of the competition lines for several of the minicircle derivatives was due to lower than nominal concentrations of these constructs in the assay mixtures. Once the free EBP-80 had been bound by the competing constructs (region of low slopes in the initial part of the binding curves), the lines describing competition by the bubble, gap, and linear forms were essentially parallel to the line of self-competition, while the complete minicircle com- peted poorly. The results are in accord with the gel shift data in showing that the gap- and bubble-containing constructs bind to EBP-80 with high affinity.

To test the possibility that these constructs were binding because they were being cut during incubation in the binding reaction, they and the 32-bp duplex oligomer were incubated for 35 min at 20 "C in the standard binding mixture with and without EBP-80 and then end-labeled with T4 kinase and [y- 32P]ATP at 20 "C for 15 min more. Acid-precipitable counts/ min for mixtures incubated with and without EBP-80 were as follows: 32-bp oligomer, 26,900 and 33,800, respectively; gap minicircle, 1530 and 5860; bubble minicircle, 615 and 485. There appears to have been no cutting of the minicircle constructs as evidenced by an increase in susceptibility to end labeling. Binding of EBP-80 inhibited end labeling of the gap minicircle, suggesting that it limited access of the kinase to the 5"strand end in this construct.

Preference of EBP-80 for A/T-rich Ends-Transitions be- tween single- and double-stranded DNA can occur at the ends of blunt-ended duplex DNA as a result of transient strand separation (melting or fraying). Melting is expected to be

a b C

more extensive a t A+T-rich than at G+C-rich ends. Accord- ingly, we tested the binding of EBP-80 to a 32-bp oligomer containing 7-bp tracts of A+T or G+C at the ends (Fig. 8). The binding curves indicated that the A+T-ended oligomer bound EBP-80 very tightly (Kd of 6 PM) with a 5-fold greater affinity than the G+C-ended form (Kd of 30 p ~ ) . Preferential binding of NFIV/Ku to A/T-enriched ends of DNA fragments was noted by de Vries et al. (16).

DISCUSSION

EBP-80 resembles the human nuclear autoantigen with respect to its nuclear location, the molecular weight of its subunits, its peptide sequence at two positions in each subunit, its reactivity with antibodies specific for Ku, and its tight binding to linear double-stranded DNA. EBP-80 may be identical to Ku, but this remains to be shown. Evidence has recently been presented for a family of genes encoding Ku p70-related polypeptides that differ by only a few nucleotide and amino acid substitutions (12). It was suggested that these structural variations might affect the functional properties of the individual gene products.

Previous work from other laboratories demonstrated that Ku binds to the termini of linear double-stranded DNA (5). In competition experiments, closed circular pBR322 DNA competed poorly compared to the linearized form, and there was a direct relationship between binding and the number of free DNA ends generated by cutting the plasmid DNA with different restriction enzymes (10). The interaction of Ku with linear fragments derived from adenovirus DNA has been visualized by electron microscopy (16): rapid binding to the ends was followed by a slower energy-independent transloca- tion of the protein which progressed until the leading molecule encountered a more tightly bound protein or, absent such an event, until the DNA was fully occupied by Ku heterodimers a t a spacing of about 40 bp. DNase I footprinting of Ku- saturated DNA indicated a spacing of 30-35 bp, of which about 20 bp were strongly protected.

In the present study, binding to short duplex oligonucleo- tides was a function of sequence length. We observed little or

d e f

r

81 L

F

FIG. 7. Gel shift analysis of EBP-80 binding to minicircular constructs containing single- to double-strand DNA transitions. The following constructs were used as competitors against 0.1 ng (5.5 fmol) of 32P-labeled blunt-ended 64-bp oligonucleotide probe: lane a, uncompeted 32-mer; b-f, binding reactions carried out in the presence of the indicated competitor (structure shown below each panel). In b- d, the arrows point to the oligomer inserted into the minicircle: b, a 30-nt region of single-stranded DNA; c, 30 bp of nonhomologous sequence; d, 30 bp of homologous sequence. In e, the parent 350-bp minicircle, and in f , the EcoRI-linearized minicircle were used as competitors. In b- f, from left lane to right, competitors were present in 25-, 50-, and 100-fold molar excess. 55 fmol of EBP-80 were added to each tube. F, free probe; B, the region representing protein-DNA interactions.

EBP-80 Recognizes Single to Double-strand Transitions in DNA 10551

I

.22 .44 .66 .88 EBP-80 (nM)

FIG. 8. Binding of EBP-80 to 32-bp oligomers of random sequence with 7-bp stretches of A+T or G+C at the ends. The sequences were ATAAATA and GCGGGCG. The curves were calcu- lated from data obtained by the filter binding assay. 0 , A+T ends; 0, G+C ends. K d values used to calculate the curves were 6 pM and 30 p~ for the A+T- and G+C-ended constructs, respectively.

no reaction between EBP-80 and oligomers of 12 bp or less, and only weak binding to a 14-mer. Affinity increased ab- ruptly (more than 10-fold) upon addition of only 2 bp, and 2- %fold over further increments in length up to 32 bp. For double-stranded oligomers, a length of 16-18 bp appears to be necessary and sufficient for strong binding of EBP-80.

On the other hand, the hairpin structure containing a 9-nt loop and only 8 bp of duplex DNA (Fig. l i) , although itself unable to bind, was complemented by single-strand extensions to give a construct (Fig. lj) with high affinity for EBP-80. Thus, the binding needs of EBP-80 can be satisfied by single strands when they are associated with a short stretch of duplex DNA. As shown in Fig. 3, single strands alone have a very low affinity for EBP-80.

Paillard and Strauss (25) showed that DNA strand ends are not required for binding of Ku. This is confirmed for EBP-80 by the binding of the bubble-containing minicircle and the two dumbbell constructs (Fig. 1, m and n).

The observations that (a ) strand ends are not required for binding, ( b ) single strands can complement short duplex segments for binding, and ( c ) a minicircle containing a bubble of nonhomology binds EBP-80 much more strongly than completely double-stranded circles, all support the supposi- tion that EBP-80 recognizes and binds to DNA containing transitions between single- and double-stranded DNA.

Under this concept, binding to blunt-ended duplex mole- cules is due to the interaction of the protein with transiently melted (frayed) ends. This view is supported by the difference in binding affinity of EBP-80 for the A+T- and G+C-ended oligomers. A detailed study of the reaction kinetics, using tailed and blunt-ended oligomers containing appropriate se- quence variations, is needed to clarify the mechanism of recognition and binding. Chemical cross-linking of strand termini (26) may provide a definitive resolution of this ques- tion.

Paillard and Strauss (25) have described an experiment in which 320-bp linear DNA fragments were allowed to bind three molecules of Ku and then circularized by enzymatic ligation. Ku bound to these circularized fragments was found to resist displacement by sodium chloride concentrations as high as 2 M, whereas the protein was dissociated at 0.35 M salt after the fragment had been linearized. Thus, Ku, once bound and interiorized, is resistant to displacement unless a free DNA end is made available.

An explanation for this phenomenon is suggested by con-

sidering the possible mode of interaction between EBP-80 (Ku) and duplex DNA ends. Although affinity is influenced to some extent by base composition (Fig. 81, binding appears to be essentially sequence-independent and therefore related to general properties of DNA. We speculate that transient strand opening at the ends of linear duplex DNA provides an opportunity for EBP-80 to interact cooperatively with multi- ple unpaired residues in the single strands. Reversal of bind- ing, which entails breaking these protein-DNA interactions and re-establishment of base pairing, requires a degree of freedom available only at the ends of the duplex DNA. Even there, the energy requirements for simultaneous opening of the several protein-DNA bonds may be met only rarely. We have found that in 0.1 M KCl, release of 30- and 32-bp duplex oligomers and the four-tailed construct h (Fig. 1) was ex- tremely slow, with 50% exchange times of 14-15 h in the presence of 100-fold excess unbound oligomer and immeas- urably slower release with no displacer added.3

The octamer-binding variant Ku-2 was reported to bind to circular plasmid that had been lightly digested with DNase I, indicating that the protein could bind to single-stranded nicks as well as double-stranded ends (15). A similar observation has been reported for Ku (5). Under our suggested mode of interaction, nicks provide points of transient strand separa- tion which permit binding.

Because of its binding to free DNA ends, Ku antigen has been proposed to play a role in DNA repair and/or transpo- sition (9). In addition, several studies have implicated Ku or Ku-like proteins in the regulation of transcription (5). We have observed that EBP-80 enhances transcription in vitro much more effectively from closed circular than from linear templates (2, 3). It has been difficult to imagine how this functional effect was related to the end-binding properties of

The fact that EBP-80 can bind to interior strand openings and single-strand gaps, while consistent with a role in DNA repair and/or recombination, also suggests new possibilities for participation of this and other Ku-related factors in tran- scriptional regulation. Relevant to the specific case of EBP- 80 binding to the IAP LTR is the fact that its target site contains an 11-bp A+T-rich segment (AAACAATAAT): strand separation can occur in such regions in supercoiled DNA under certain conditions of salt and temperature, the free energy of the helix-coil transition being derived from the relaxation of superhelical stress attending reduction of twist in the unwound portion (27). The presence of a C/G residue (conserved in all IAP LTRs) would greatly shorten the life- time of neighboring A/T pairs (28) and facilitate strand opening. Binding of the factor may be complemented by sequence-specific interactions at the enhancer core motif. Similar motifs are not found in the Ku-binding oligomers derived from the transferrin and U l sRNA genes (13, 14) or containing the eukaryotic octamer sequence (15). Neverthe- less, A+T-rich tracts are not uncommon in upstream regions of eukaryotic genes, and some of these could provide points of entrance for EBP-80 (Ku) from which it could translocate to a functional position. The LTR binding site for EBP-80 has an interesting parallel in the case of bacteriophage T4, where a transcriptional enhancer element was found to be a break in the nontranscribed DNA strand rather than a specific sequence (29). The possibility that EBP-80 can bind directly to the strand opening associated with transcription complexes also needs to be explored.

The capacity to bind at points of strand separation is reminiscent of topoisomerase enzymes, e.g. SV40 large T

EBP-80.

M. Falzon, J. W. Fewell, and E. L. Kuff, unpublished data.

10552 EBP-80 Recognizes Single to Double-strand Transitions in DNA antigen which has helicase activity and binds at the DNA 4. Mimori, T., Akizuki, M., Yamagata, H., Inada, s., Yoshida, s., and Homma,

replication fork in an energy-independent manner (30). How- 5. Reeves, W. H. (1992) Rheum. Dis. Clin. North Am. 18, 391-414 ever, we have been unsuccessful thus far in demonstrating 6. Chan, J. Y. c., Lerman, M. I., Prabhakar, B. S., Isozaki, O., Santisteban,

activity of this type with isolated EBP-80. The function of P., Kuppers, R. C., Oates, E. L., Notkins, A. L., and Kohn, L. D. (1989)

this and other Ku-related proteins remains to be demon- 7. Reeves, w. H., and Sthoeger, z. M. (1989) J. Biol. Chem. 264,5047-5052 J. Biol. Chem. 264,3651-3654

strated, perhaps best through the use of more complex sys- 8. Yaneva, M., Wen, J., Ayala, A., and Cook, R. (1989) J. Biol. Chem. 2 6 4 ,

13407-13411 terns. 9. Mimori, T., Ohosone, Y., Hama, N., Suwa, A,, Akizuki, M., Homma, M.,

Griffith, A. J., and Hardin, J. A. (1990) Proc. Natl. Acad. Sci. U. S. A . 87, 1777-1781

M. (1981) J. Clin. Inuest. 6 8 , 611-620

AckWwledgments-We thank C. Klee for microsequence analysis 10. Mimori, T., and Hardin, J. A. (1986) J. Biol. Chem. 261,10375-10379 of peptides, s. G. widen for help with the chromatography, H. E. 11. Griffith, A. J., Blier, P. R., Mimori, T., and Hardin, J. A. (1992) J. Biol.

Choy and s. AdhYa for the Plasmid pSA508, and E. M. Tan for 12. Chou, C.-H., Wang, J., Knuth, M. W., and Reeves, W. H. (1992) J. Exp. providing antiserum against human Ku. We also thank C. Klee and Med. 176,1677-1684 K. K. Lueders for critical reading of the manuscript. 13. Roberts, M. R., Miskimins, W. K., and Ruddle, F. H. (1989) Cell Regulation

14. Knuth, M. W., Gunderson, S. I., Thompson, N. E., Strasheim, L. A., and

Chem. 267,331-338

1,151-164

Note Added in Proof-Recently, Ku has been shown to mediate Burgess, R. R. (1990) J. Biol. Chem. 266,17911-17920 the binding and activation of a 3 5 0 - k ~ ~ DNA-dependent protein 15. May, G., SUtton, c., and Gould, H. (1991) J. Bid . Chem. 26633052-3059 kinase (PK) (Davir, A., Peterson, S . R., Knuth, M. W., Lu, H., and

16. de Vries, E., van Driel, W., Bergsma, W. G., Arnberg, A. C., and van der

Dynan, W. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11920-11924; 17. Stuiver, M. H., Coenjaerts, F. E. J., and van der Vliet, P. C. (1990) J. Exp. Vliet, P. C. (1989) J. Mol. Biol. 2 0 8 , 65-78

Gottlieb, T. M., and Jackson, S. P. (1993) Cell 72, 131-142). PK is known to phosphorylate sites in spl, p53, H ~ P g O , s~~~ antigen, 18. Griffith, A. J., Craft, J., Evans, J., Mimori, T., and Hardin, J. A. (1992) Med. 1 7 2 , 1049-1054

and the C-terminal domain of RNA polymerase 11 (reviewed by 19. Allaway, G. P., Vivino, A. A,, Kohn, L. D., Notkins, A. L., and Prabhakar, Mol. Biol. Reports 1 6 , 91-97

Anderson, C. W., and Lees-Miller, S . P. (1992) Crit. Reu. Eukaryotic B. S. (1990) Biochem. Biophys. Res. Commun. 168,747-755 G~~~ E ~ ~ ~ ~ ~ ~ , 2, 283-314). the work cited above, binding and 20. Graham, F. L., Smiley, J., Russell, W. C., and Nairn. R. (1977) J . Gen. activation was affected by DNA fragments Or oligonucleotides, k by 21. Stone, K. L., LoPresto, M. B., Crawford, J. M., DeAngelis, R., andWilliams, interaction of Ku with DNA ends. The present results suggest a K. R. (1989) in A Practical Guide to Protein and Peptide Purification for means by which KuPK could enter upstream regulatory sites con- Microsequencing (Matsudaira, P. T., ed) pp. 31-47, Academic Press, New

taining regions of strand separation or stem-loop structures. As 22, ~ ~ l ~ ~ ~ , M,, and Kuff, E, L. (1988) J , viral. 6 2 , 4070-4077 suggested by others, Ku could then provide a sliding tether which 23. Reeves, W. H. (1985) J. Exp. Med. 161,18-39 permits the activated PK to approach its target proteins. Conceivably, 24. Francoeur, A,"., Peebles, c. L., Gompper, p. T., and Tan, E. M. (1986) KuPK could bind to and track with advancing ''transcription hub- 25, paillard, s,, and Strauss, F, (1991) ~ ~ k i ~ Acids R ~ ~ . 19,6619-5624 bles," maintaining the C-terminal domain of Pol 11 in phosphorylated 26. Cowart, M., and Benkovic, S. J. (1991) Biochemistry 30,788-796 form. 27. Murchie, A. I., H., Bowater, R., Aboul-ela, F., and Lilley, D. M. J. (1992)

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