Proc. Natl. Acad. Sci. USAVol. 86, pp. 5281-5285, July 1989Biochemistry

Calf thymus histone H1 is a recombinase that catalyzesATP-independent DNA strand transfer

(homologous painng/mitotic recombination/homology)

ICHIRO KAWASAKI, SHOJI SUGANO, AND HIDEO IKEDA*Department of Molecular B3iology, The Institute of Medical Science, The University of Tokyo, P.O. Takanawa, Tokyo 108, Japan

Communicated by James D. Watson, April 17, 1989

ABSTRACT An activity that catalyzes the strand transferfrom linear double-stranded tetracycline-resistance gene (tetr)DNA to circular M13mp8-tetr viral DNA was detected in acrude extract from calf thymus. This activity was purified tonear, if not complete, homogeneity as judged by NaDodSO4/polyacrylamide gel electrophoresis. We have tentatively namedthis protein calf thymus strand-transfer protein 1 (CTST1).The apparent molecular mass of the protein was 35 kDa by gelelectrophoresis. Its sedimentation coefficient was approxi-mately 1.5 S in glycerol gradient centrifugation. These valuesled us to examine the possibility that CTST1 is histone H1.Western blot analysis of CTST1 with anti-rat liver histone H1antiserum showed that CTST1 crossreacts with the serum,indicating that CTST1 is histone H1. The mobility of CTST1was identical to one of the subtypes of calf thymus histone H1by NaDodSO4/polyacrylamide gel and acetic acid/urea/poly-acrylamide gel electrophoreses. We have also confirmed theabove conclusion by showing that calf thymus histone H1 hasa strand-transfer activity with a specific activity comparable tothat of CTST1. The reaction required homologous substrates,but neither Mg2+ nor ATP. The reaction also required stoi-chiometric amounts of protein. The purified CTST1 fractionlacked detectable exo- and endonuclease activities and alsolacked a DNA helicase activity.

Understanding of the molecular mechanism of genetic re-combination requires identification and characterization ofrecombination enzymes and their genes. In Escherichia coliand lower eukaryotes, a great deal of information has beenaccumulated about enzymes that catalyze DNA strand trans-fer, which is believed to be an initial step of DNA strandexchange. The E. coli recA gene (1) is known to code for arecombinase (2) that mediates strand transfer (3) and thenleads to the formation of the Holliday structure (4, 5). TheRecA protein first binds stoichiometrically to single-stranded(ss) DNA to form an intermediate in the formation of jointmolecules (6). The interaction with double-stranded (ds)DNA triggered by binding ofRecA protein to ssDNA resultsin partial unwinding of the duplex and finally leads to theformation of the joint molecule (7, 8). The bacteriophage T4UvsX protein, Ustilago maydis Recl protein, and Saccha-romyces cerevisiae strand-transfer protein a (STPa) havebeen purified and shown to catalyze strand-transfer reactionsin a fashion similar to the RecA protein (9-12).

Somatic recombination is known to occur in many mam-malian cells in vivo as well as in cultured cells. Moreover,homologous recombination between DNA introduced intocells and homologous chromosomal sequences, an eventcalled gene targeting, has also been shown (13, 14). Thus,somatic mammalian cells must possess the enzymatic ma-chinery for efficiently-promoting homologous recombination.

To date strand-transfer activities have been partially purifiedfrom mammalian cells (15-18) but have been neither identi-fied as a single protein nor characterized biochemically.

In this paper, an activity that catalyzes the strand transferfrom linear ds tetracycline-resistance gene (tet') DNA tocircular M13mp8-tetr viral DNA was detected in a crudeextract from calfthymus. This activity was purified to a singleband on a NaDodSO4/polyacrylamide gel and on an aceticacid/urea/polyacrylamide gel. Based on biophysical andimmunological properties, we have concluded that this ac-tivity is histone H1.

MATERIALS AND METHODSMaterials. Calf thymus was purchased from Seibutsu

Zairyo Center (Tokyo). M13mp8 viral and covalently closed,supercoiled replicative form (RFI) DNAs were prepared aspublished (19). 4X174 viral and RFI DNAs were purchasedfrom Bethesda Research Laboratories. M13mp8-tetr RFIDNA was constructed by ligation of EcoRI- and Sma I-digested M13mp8 RFI DNA with an EcoRI- and Bal I-digested pBR322 fragment [base pair (bp) 4361-bp 1444]. Oneof the pBR322 DNA tetr fragments (276 bp) was prepared bydigesting pBR322 RFI DNA with BamHI and Sal I. One ofthe cX174 DNA fragments (348 bp) was prepared by digest-ing 4X174 RFI DNA with Hap II. The 3' ends were labeledby filling recessed 3' ends of staggered restriction sites byusing the Klenow fragment of E. coli DNA polymerase I inthe presence of [a-32P]dCTP. Unreacted 32P-labeled nucleo-tides were removed by gel filtration. Rabbit anti-rat liverhistone H1 antiserum was kindly provided by Y. Ohba and H.Yasuda (Kanazawa University, Kanazawa, Japan). Calf thy-mus histone H1 was from Boehringer Mannheim. Restrictionenzymes, T4 DNA ligase, and the Klenow fragment ofDNApolymerase I were from Takara Shuzo (Kyoto, Japan).Proteinase K was from Merck. Phenylmethylsulfonyl fluo-ride, antipain, and pepstatin A were from Sigma. Biotin-conjugated goat anti-rabbit IgG was from Tago. Avidin-biotin complex and biotin-conjugated peroxidase were fromVector Laboratories.

Strand-Transfer Assay. The modified strand-transfer assaydeveloped by McCarthy et al. (20) was used. The reactionwas carried out in a 30-,l volume containing 25 mM Tris HCl(pH 7.5), 10 mM MgCl2, 2 mM ATP, 1 mM dithiothreitol,bovine serum albumin (100 ,&g/ml), 10 AM viral ssDNA ofM13mp8-tetr or 4X174, 0.67 ,M 32P-labeled dsDNA frag-ments of pBR322 tetr gene (276 bp) or 4X174 RFI DNA (348bp), and an aliquot of the enzyme fraction. The mixture wasincubated at 30°C for 10 min. The reaction was terminated byadding 3 ,ul of stop mixture containing 250 mM EDTA, 5%

Abbreviations: RFI DNA, covalently closed, supercoiled replicativeform DNA; ssDNA, single-stranded DNA; dsDNA, double-strandedDNA; CTST1, calf thymus strand-transfer protein 1; tetr, tetracy-cline-resistance gene.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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5282 Biochemistry: Kawasaki et al.

(wt/vol) NaDodSO4, and proteinase K to a final concentra-tion of 0.6 mg/ml. The mixture was incubated at 370C for 10min. After adding 3 1.L of a dye mixture containing 0.25%bromophenol blue and 60%o (vol/vol) glycerol, the mixturewas loaded on a 1% agarose gel containing ethidium bromide(0.5 ,ug/ml). After electrophoresis at 1 V/cm for more than 6hr, the gel was photographed under ultraviolet light, dried onDEAE-paper (Bio-Rad), and autoradiographed at -80TC.

Detection of ssDNA on a Strand-Separating Gel. After thestrand transfer, the mixture was brought to 100 mM potas-sium phosphate (pH 6.8) and then suspended in 50 1A ofhydroxylapatite (Bio-Gel HT; Bio-Rad) equilibrated with 100mM potassium phosphate. The suspension was incubated at37TC for 30 min to adsorb most of the unreacted 32P-labeleddsDNA and then centrifuged. The supernatant was collectedand subjected to strand-separating polyacrylamide gel elec-trophoresis according to the method of Maniatis et al. (21).Under the condition described above, approximately 30%o ofthe 32P-labeled ssDNA was recovered, and more than 95% ofthe unreacted 32P-labeled dsDNA was removed.

Preparation of Extract from Calf Thymus Gland. A whole-cell extract was prepared from calf thymus gland. All pro-cedures were carried out at 4°C. Frozen calfthymus gland (20g) was thawed and homogenized in 40 ml of buffer Acontaining 0.1 M KCl [buffer A contains 20 mM potassiumphosphate (pH 7.5), 1 mM EDTA, 1% ethanol, 0.5 mMphenylmethylsulfonyl fluoride, antipain (2 ,ug/ml), pepstatinA (0.8 ,g/ml), and 10mM 2-mercaptoethanol]. Solid KCl wasadded with stirring to make 0.3 M KCL. The mixture wascentrifuged and the supernatant was discarded. The precip-itate was mixed with 40 ml of buffer A containing 2 M KCL.The suspension was then mixed with 4.5 g of polyethyleneglycol (Mr, 6000) to give 7.5% (wt/vol). The mixture wascentrifuged and the supernatant (48 ml) was collected.

Purification ofa Strand-Transfer Protein. The crude extractcontaining about 300 mg of protein was added to 192 ml ofKCl-free buffer A to make 0.4 M KCl. Hydroxylapatite(Bio-Gel HT, 75 ml) suspended in buffer A containing 0.4 MKCl and 20 mM potassium phosphate was mixed with the calfthymus extract. The suspension was filtered and the adsor-bent was successively washed with bufferA containing 0.4 MKCI followed by increasing concentrations of potassiumphosphate. The strand-transfer activity was eluted between150 mM and 500 mM potassium phosphate. The hydroxyl-apatite fraction containing the activity (69 mg ofprotein in 330ml) was dialyzed against buffer B containing 50 mM KCl[buffer B contains 20 mM potassium phosphate (pH 7.5), 0.1mM EDTA, 10%o glycerol, 1% ethanol, 0.5 mM phenylmeth-ylsulfonyl fluoride, antipain (2 I&g/ml), pepstatin A (0.8,ug/ml), and 10 mM 2-mercaptoethanol] and centrifuged. Thesupernatant was loaded onto a phosphocellulose column(P11, Whatman, 1.6 x 3 cm) and washed with buffer Bcontaining 50 mM KCl, and activity was eluted with a lineargradient of50mM to 1 M KCI in buffer B. The strand-transferactivity was detected at 0.8 M to 1 M KCL. The phosphocel-lulose fraction with activity (6.9 mg of protein in 15 ml) wasdialyzed against buffer B containing 50 mM KCl and loadedonto a ssDNA-cellulose column (10 mg of DNA in S g ofcellulose, 1.6 x 7 cm), and activity was eluted with a lineargradient of50mM to 2M KCl in buffer B. The strand-transferactivity was detected at about 0.6 M KCL. The ssDNA-cellulose fraction with activity (-5 mg ofprotein in 57 ml) wasdialyzed against buffer B containing 0.1 M KCl and loadedonto a hydroxylapatite HPLC column (Tonen TAPS, 2.1 x 10cm), and activity was eluted with a linear gradient of 20 mMto 400mM potassium phosphate in buffer B containing 0.1 MKCL. The strand-transfer activity was eluted at 350mM to 400mM potassium phosphate. The protein fraction with activitywas dialyzed against buffer B containing 0.1 M KCI and 20mM potassium phosphate, concentrated by loading onto a

small phosphocellulose column and by eluting activity with 2M KCl, and dialyzed again with buffer B containing 50 mMKCl and 10% glycerol. The hydroxylapatite HPLC fractionwas sedimented at 45,000 rpm for 60 hr in Beckman SW50.1rotor through a 15-35% glycerol density gradient in buffer Bcontaining 50 mM KCl. Fractions corresponding to 1.5 S, inwhich the strand-transfer activity was detected, were pooled.The glycerol density gradient fraction is nearly homoge-nous-more than 95% pure-as judged by a Coomassiebrilliant blue-stained NaDodSO4/polyacrylamide gel.Other Methods. ssDNA-cellulose was prepared by the

method of Alberts and Herrick (22). Acetic acid/urea/polyacrylamide gel electrophoresis was carried out accordingto the method of Smith (23). Protein concentration wasdetermined by the method of Bradford (24) in early purifi-cation steps and in late steps determined spectrophotomet-rically using an extinction coefficient of 1345 cm'-M-1 at 275nm (25) and a molecular mass for calf thymus histone H1 of21 kDa (26).

RESULTSDetection of a Strand-Transfer Activity in Crude Calf Thy-

mus Extracts. Crude calf thymus extracts containing strand-transfer activity were prepared with buffers containing var-ious concentrations of KCl. The activity was assayed bymeasuring strand transfer of 32P-labeled, ds tetr DNA toM13mp8-tetr viral DNA (Fig. 1). The activity was detectedwhen the extracts were made with buffers containing 1.0 M,1.5 M, and 2.0 M KCl but not with buffer containing 0.6 MKCl (data not shown). The extract prepared with 2.0 M KClbuffer had the strongest activity and was used for furtherpurification of the strand-transfer activity.

Purification of the Strand-Transfer Activity. The strand-transfer activity in the crude calfthymus extract was purifiedextensively. In the glycerol density gradient centrifugation,the strand-transfer activity was detected at a position corre-sponding to 1.5 S (Fig. 2B). NaDodSO4/polyacrylamide gelelectrophoresis of the glycerol gradient fractions showed that

S 32P-pBR322 lineards DNA276 bp

'. i~ ~ 14ff

Ml 3mp8-tetr> /< ~~~circular ss DNA

R I m\1r 8670 ntd6233 6238 \

FIG. 1. Linear dsDNA and circular ssDNA substrates. Therestriction sites in the tetr gene ofpBR322 DNA were used to prepare32P-labeled dsDNA substrates. A BamHI4Sal I fragment of 276 bpwas used most of the time. RI, EcoRI; B, BamHI; S, Sal I; Bl, BalI; Sm, Sma I. Numbers indicate map coordinates of pBR322 (thickline) or M13mp8 (thin line). Stars indicate positions of 32p label.

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A L f r7 9 11 13 14 16 18 20 21 23 M kDa

_ -97.4

N -66.2

_ -42.7

- _ _ _

_ - 31.0

-21.5

_ - 14.4

4 3 2 1 S

B fr8 9 10 11 12 13 14 15 16 17 18 19 20 21

aE "sS __U - -JM

H1 (the latter protein exhibited three major bands, Fig. 3A).A similar result was obtained when the proteins were sub-jected to acetic acid/urea/polyacrylamide gel electrophore-sis, except that both proteins formed a single band (Fig. 3B).Furthermore, a Western blot analysis of CTST1 with anti-histone H1 antiserum showed that CTST1 crossreacts withthe antibody (Fig. 3C). Finally, we have detected a strand-transfer activity in a calf thymus histone H1 preparation witha specific activity comparable to that of CTST1 (data notshown). We hence concluded that CTST1 is one of thesubtypes of histone H1. We have confirmed the aboveconclusion by peptide mapping analysis (I.K., S.S., H.Yasuda, Y. Ohba, and H.I., unpublished data).

Properties of the Reaction Catalyzed by CTST1. Whenincreasing amounts of the purified CTST1 were added to areaction mixture, the formation ofjoint molecules increasedto up to iO% of the total input 32P-labeled DNA and thenreached a plateau (data not shown). At a saturation level, theconcentration of CTST1 was 9 ,ug/ml of a reaction mixture,which corresponds to 20 nucleotides of ssDNA per oneCTST1 molecule. The reaction, therefore, requires stoichi-ometric amounts of the protein. DNA strand-transfer reac-tion catalyzed by CTST1 requires homologous ds- and ss-DNAs (Fig. 4B). To quantitate the requirement of homology,the bands corresponding to the joint molecules were excisedfrom the gel and 32p radioactivity was measured. The reactionwith M13mp8-tetr ssDNA and tetr dsDNA produced a prod-uct having about 20 times as much radioactivity as that with4X174 ssDNA and tetr dsDNA. As a positive control, thereaction with 4X174 ssDNA and 4X174 dsDNA formed a

4 3 2 1 S

FIG. 2. Detection ofa strand-transfer activity in a glycerol densitygradient. (A) The hydroxylapatite HPLC fraction with strand-transferactivity was sedimented through a 15-35% glycerol gradient. Twenty-three fractions were collected and a 60-1g aliquot of each 200-jAdfraction was subjected to NaDodSO4/4-20%o polyacrylamide gradientgel electrophoresis. The gel, which was stained with Coomassiebrilliant blue R-250, is shown. Sedimentation coefficient values (S)were calculated from positions of markers that were simultaneouslysedimented in other tubes and are indicated at the bottom ofA and B.Lanes: L, a hydroxylapatite HPLC fraction; fr. 7-23, glycerol gradientfractions; M, molecular mass markers (low range standards; Bio-Rad),molecular masses of which are shown on the right. (B) A 6-pl aliquotof each glycerol fraction as indicated was assayed for strand-transferactivity. ds, 32P-labeled linear dsDNA; JM, joint molecule.

the distribution of a single polypeptide of apparent molecularmass of 35 kDa coincided with that of the activity (Fig. 2A).We, therefore, concluded that a polypeptide of apparentmolecular mass of 35 kDa catalyzes the DNA strand-transferreaction and we tentatively named it calf thymus strand-transfer protein 1 (CTST1). Based on the semi-quantitativemeasurement of the strand-transfer activity, the extent ofpurification in the final glycerol density gradient fraction isapproximately 700-fold.CTST1 Is Histone Hi. Ultraviolet absorbance of the puri-

fied protein solution exhibited a major absorption peak at 225nm and a minor absorption peak at 275 nm, which is largelyshifted from a standard absorption spectrum of proteins.Chromatographic and isoelectric properties indicate thatCTST1 is a basic protein. These properties led us to examinea possibility that CTST1 is histone H1. The molecular massof histone H1 is 21 kDa (26), but its apparent molecular massis known to be around 35 kDa as judged by NaDodSO4/polyacrylamide gel electrophoresis, suggesting a similarity ofthe molecular mass of CTST1 to that of histone H1. In fact,coelectrophoresis of CTST1 and calf thymus histone H1 ona NaDodSO4/polyacrylamide gel indicated that the mobilityof CTST1 coincides with that of the middle band of histone

A

kDa 1 2 3 4

B C1 2 1 2 3

97.4-66.2-

42.7-

31.0--

2 1.5-

FIG. 3. Identification ofCTST1 with histone H1. CTST1 and calfthymus histone H1 (Boehringer Mannheim) were subjected to twotypes of gel electrophoresis. (A) NaDodSO4/15% polyacrylamide gelelectrophoresis (pH 8.8). Lanes: 1, molecular mass markers asdescribed (Fig. 2A, lane M); 2, calf thymus histone H1 (2.0 ,ug); 3, amixture of a hydroxylapatite HPLC fraction of CTST1 (0.6 .g) andcalf thymus histone H1 (2.0 ,ug); 4, a hydroxylapatite HPLC fractionof CTST1 (0.6 ,ug). (B) Acetic acid/urea/20% polyacrylamide gelelectrophoresis (0.9 M acetic acid, pH 3/2.5 M urea). Lanes: 1, calfthymus histone H1 (1.2 jg); 2, a hydroxylapatite HPLC fraction ofCTST1 (1 jg). InA and B, gels were stained with Coomassie brilliantblue R-250. (C) Western blot analysis of CTST1 with rabbit anti-ratliver histone H1 antiserum. After NaDodSO4/polyacrylamide gelelectrophoresis, proteins were transferred to nitrocellulose. A stripcontaining molecular mass markers was stained with amido black10B. Strips containing calf thymus histone H1 (2.5 ,g) and ahydroxylapatite HPLC fraction of CTST1 (0.9 pZg) were incubatedwith a 1:500 dilution of rabbit anti-rat liver histone H1 antiserum andthen incubated with biotin-conjugated goat anti-rabbit IgG followedby incubation with avidin-biotin complex and biotin-conjugatedperoxidase. Immunospecific bands were visualized with a peroxi-dase substrate solution [4-chloro-1-naphthol (0.5 mg/ml) and 4 mMH202]. Lanes: 1, markers; 2, calf thymus histone H1; 3, a hydrox-ylapatite HPLC fraction of CTST1.

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B1 2 3 4 5 6 7 8

BA1 2 3 4 5 6 7

C1 2 1 2 3 4

.0 +

1 2 3 4

-JM1 --

JM2 -

ds2-

9I - d s 1 '

FIG. 4. Conditions for DNA strand-transfer reaction catalyzedby CTST1. The reaction mixture contained 6.6 ng of 32p_3'-end-labeled tetr DNA fragment (BamHI-Sal I fragment of276 bp),100 ng of M13mp8-tetr viral DNA, and 68 ng ofCTST1. (A) Reactionunder various conditions. Lanes: 1, 32P-labeled tetr DNA andM13mp8-tetr DNA only (no enzyme); 2, complete reaction mixture;3, ATP was omitted; 4, same as lane 3 except that 2 mM adenosine5'-[ythio]triphosphate was added; 5, same as lane 3 except that 2mM adenosine 5'-[p,yimido]triphosphate was added; 6, Mg2+ was

replaced by 10mM EDTA; 7, CTST1 was pretreated with proteinaseK (1 mg/ml) at 37°C for 30 min; 8, after reaction, the mixture was

treated with 0.15 M NaOH at 37°C for 15 min. (B) Homologydependence of strand-transfer reaction. Lanes: 1, no enzyme, sameas lane 1 in A; 2, viral ssDNA was omitted; 3, complete reaction,same as lane 2 in A (32P-labeled tetr dsDNA and M13mp8-tetr viralssDNA was used); 4, 32P-labeled tetr dsDNA was replaced by32P-labeled #X174 dsDNA (Hap II fragment of348 bp); 5, 32P-labeledtetr dsDNA and M13mp8-tetr viral ssDNA were replaced by 32P_labeled gX174 dsDNA and 4X174 viral ssDNA, respectively; 6,M13mp8-tetr viral ssDNA was replaced by qbX174 viral ssDNA; 7,M13mp8-tetr viral ssDNA was replaced by pBR322 RFI DNA. dsland ds2, 32P-labeled tetr dsDNA and 32P-labeled 4iX174 dsDNA,respectively: JM1 and JM2, joint molecules that comigrate withM13mp8-tetr viral ssDNA and 4X174 viral ssDNA, respectively.

product having radioactivity comparable to that withM13mp8-tetr ssDNA and tetr dsDNA (Fig. 4B, lanes 3 and 5).The structure of the product of the strand-transfer reaction

was examined. The product was eluted from the gel and waselectrophoresed again before or after treatment with 0.15 MNaOH. In the sample without NaOH treatment, joint mole-cules were found in addition to the ss- and dsDNA fragments,whereas only ssDNA fragments were detected in the samplesubjected to the alkali treatment (Fig. SA), indicating that thejoint molecules were formed by hydrogen bonding. Therelease of ss- and dsDNA fragments from the product withoutthe alkali treatment suggests that at least part of the jointmolecules observed in this reaction consisted of dsDNAfragments partially transferred to circular ssDNAs.To examine whether a completely transferred molecule is

formed in the strand-transfer reaction, either the plus or theminus strand of the dsDNA fragment was end-labeled sepa-rately and used for the strand-transfer assay. If completestrand transfer occurs, then a circular ssDNA and a minusstrand of the dsDNA fragment would be converted into aduplex joint molecule while the displaced plus strand of thedsDNA fragment would be released. The reaction withdsDNA labeled on the minus strand produced a producthaving twice as much radioactivity as that with dsDNAlabeled on the plus strand (Fig. SB). The partially asymmetrictransfer of the labeled strands suggests that the products area mixture of partially transferred joint molecules and com-pletely transferred joint molecules. We have, therefore,tested strand specificity ofssDNA fragments displaced by thestrand-transfer reaction. ssDNA fragments were found in thereaction with dsDNA labeled on the plus strand, but not in thereaction with dsDNA labeled on the minus strand (Fig. SC).

-JM--JM

S s(+-a

SS=M~~S"-ds 4= --ds-

FIG. 5. Structure of the joint molecule. (A) Re-electrophoresis ofthe joint molecule before or after alkaline treatment. The joint mole-cule, prepared in the complete reaction mixture and electrophoresedon an agarose gel, was extracted and re-electrophoresed on a 5%

polyacrylamide gel before or after alkaline treatment with 0.15 MNaOH at 3rC for 15 min. Lanes: 1, 32P-labeled tetr DNA alone wastreated with alkali; 2, thejoint molecule was treated with alkali; 3, thejoint molecule was electrophoresed without alkaline treatment; 4,32P-labeled tetr DNA alone was electrophoresed without alkalinetreatment. (B) Strand specificity of transferred DNA. Strand-transferreaction was carried out with 32P-labeled tetr DNA labeled on eitherthe plus or the minus strand. Reaction products were electrophoresedin agarose gel as in Fig. 2B. Lanes: 1, reaction with tetr DNA labeledon the plus strand; 2, reaction with tetr DNA labeled on the minusstrand. Equal amounts ofradioactivities were added to both reactions.(C) Strand specificity of ssDNA displaced by the strand-transferreaction. Strand-transfer reaction was carried out as in B. Reactionproducts were electrophoresed on strand-separating polyacrylamidegel after hydroxylapatite batch treatment to remove most of theunreacted 32P-labeled tetr dsDNA. Lanes: 1, heat-denatured 32p_labeled tetr DNA labeled on the plus strand; 2, native 32P-labeled tetrDNA; 3, reaction with tetrDNA labeled on the plus strand; 4, reactionwith tetr DNA labeled on the minus strand. ds, native 32P-labeled tetrDNA; ss, a mixture ofplus and minus strands of32P-labeled tetr DNA;ss(+), plus strand of 32P-labeled tetr DNA; JM, joint molecule.

Radioactivity in the former ssDNA band was comparable tothat in the band of joint molecule. These results led us toconclude that the minus strand that originated from the nativeDNA fragment was partially or fully transferred, resulting inthe formation of a joint molecule carrying either a dsDNAfragment or a minus ssDNA fragment.

Neither ATP nor other nucleotides were required for theactivity. Adenosine 5'-[-thio]triphosphate and adenosine5'-[fi,y-imido]triphosphate also did not affect the activity(Fig. 4A, lanes 3-5). Unexpectedly, Mg2+ was not requiredfor the reaction (Fig. 4A, lane 6). The reaction was ratherinsensitive to salt: 200 mM NaCl did not inhibit the reaction,but 400 mM NaCl did (data not shown). The optimal tem-perature was 37°C. A reaction lag period was not detected.The strand-transfer activity was totally inactivated by treat-ment with proteinase K (Fig. 4A, lane 7).

In the most purified fraction (glycerol gradient fraction),we could detect neither 5' to 3' nor 3' to 5' exonucleolyticactivities using a 5'- or 3'-end-labeled, native or heat-denatured DNA fragment as a substrate (data not shown). Wecould not detect endonucleolytic activities using M13mp8viral or RFI DNA as a substrate (data not shown). Weexamined whether CTST1 has a DNA helicase activity butfailed to detect such an activity, since 32P-labeled tetr DNAcould not be changed into denatured form upon incubationwith CTST1 (data not shown). In addition, neither DNA-dependent nor DNA-independent ATPase activities havebeen detected (data not shown).

DISCUSSIONIn the present study, we have purified the strand-transferactivity from calf thymus and shown that the purified protein

A

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formed a single band on NaDodSO4/polyacrylamide gelelectrophoresis. We tentatively named the protein calf thy-mus strand-transfer protein 1 (CTST1). Mobilities deter-mined by two types ofgel electrophoresis and crossreactivityagainst anti-rat liver histone H1 antiserum led us to concludethat CTST1 is one of the subtypes of histone H1.Mammalian histone H1 is classified into several subtypes.

On NaDodSO4/polyacrylamide gel, calf thymus histone H1forms three major bands, and CTST1 corresponds to themiddle band. Histone H1 is further separated into five toseven subtypes by various column chromatographic methods(27). We do not know which subtype CTST1 corresponds to.Some of the subtypes are expressed only in a specific celldivision cycle or in a specific tissue. It is particularly note-worthy that histone Hit is expressed only in testis (28). Thisprotein might have high strand-transfer activity, becausemeiotic cells are known to undergo recombination at anefficiency two or three orders of magnitude higher than thatin somatic cells.The assay system used in this paper is the modified transfer

reaction developed by McCarthy et al. (20). This reactionincludes strand transfer of 32P-labeled, ds tetr DNA toM13mp8-tetr viral DNA. Joint molecules produced by thestrand-transfer reaction were unstable at alkaline pH, indi-cating that the transferred strand was connected with viralDNA by hydrogen bonding as expected. When the strand-transfer assay was carried out with increasing concentrationsof CTST1, the extent of the formation ofjoint molecules wasmaximally about iO% of the total linear dsDNA molecules.Since one strand in the labeled dsDNA molecule could betransferred to the ssDNA, only 20% of the total transferableDNA was transferred. The reaction mixture might lackanother component such as a ssDNA binding protein, whichis required for yeast strand-transfer protein isolated by Su-gino et al. (29). The ability of CTST1 to detect DNA homol-ogy and promote strand transfer is, therefore, reminiscent ofa recombinase for homologous recombination.

Typical strand-transfer proteins, E. coli RecA, bacterio-phage T4 UvsX, and U. maydis Recl, require ATP for strandexchange (3, 11, 30). Studies with RecA protein indicate that100-300 bp of duplex DNA are easily unwound upon forma-tion of a paranemic joint, resulting in the interwinding of theminus strand of the linear duplex DNA with the plus ssDNA.This step does not require ATP hydrolysis (8). It is, therefore,conceivable that CTST1 does not require ATP or othernucleotide cofactors. Many strand-transfer proteins havebeen purified or partially purified from eukaryotic cells andhave also been shown to be ATP-independent (17, 20, 29).CTST1 is, however, distinguishable from other eukaryoticrecombinases with respect to the requirement of Mg2+.

Histone H1 is known to have a role for stabilizing nucle-osome structure. Upon treatment of a chromatin with 0.6 MKCl, the histone H1 is released and the chromatin changes toa loose structure. It is, therefore, thought that histone H1plays a role in keeping the chromatin in a compact structure.The primary structure of histone H1 is divided into threedomains: (i) an amino-terminal domain of 40 amino acids, (ii)a highly folded central domain of about 75 amino acids, and(iii) a carboxyl-terminal domain ofabout 95 amino acids (31).The terminal domains have a role in binding to DNA. HistoneH1 molecules are also known to form a homopolymer struc-ture (32), possibly bearing multiple DNA binding domains.

Such a histone H1 homopolymer might promote the forma-tion of a presynaptic linkage between two DNA molecules.Which domain of histone Hi is responsible for the strand-transfer activity is an important subject for further analysis.

We thank Drs. Y. Ohba and H. Yasuda for gifts of histone H1 andanti-histone H1 rabbit antiserum and Drs. J. Inselburg and D. A.Shub for critical readings of the manuscript. This research wassupported by grants from the Ministry of Education of Japan andfrom the Nissan Science Foundation.

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C. M. (1979) Proc. Natl. Acad. Sci. USA 76, 1638-1642.4. Ikeda, H. & Kobayashi, I. (1979) Cold Spring Harbor Symp.

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Biochemistry: Kawasaki et al.

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Proc. Natl. Acad. Sci. USA 87 (1990)

Biochemistry. In the article "Calf thymus histone H1 is arecombinase that catalyzes ATP-independent DNA strandtransfer" by Ichiro Kawasaki, Shoji Sugano, and HideoIkeda, which appeared in number 14, July 1989, of Proc.Natl. Acad. Sci. USA (86, 5281-5285), the authors requestthat the following retraction be noted.We have learned from J. Svaren and R. Chalkley that short

double-stranded DNA denatures upon drying after ethanolprecipitation (33). Svaren and Chalkley have also suggestedthe possibility that we observed renaturation of single-stranded DNAs mediated by histone H1 rather than strandtransfer. Accordingly, we tested for possible contaminationby single-stranded DNA in the labeled linear double-strandedDNA preparation and found a small amount of single-stranded DNA. Next we asked whether a double-strandedDNA preparation that is free of single-stranded DNA couldbe a substrate for the strand-transfer reaction and observedthat only a trace of joint molecules was formed in thecomplete reaction mixture containing the single-stranded-DNA-free preparation. We, therefore, retract our formerconclusion that histone H1 mediates a strand-transfer reac-tion. Rather, our results confirm that histone H1 mediatesrenaturation of complementary single-stranded DNAs, asdescribed by Cox and Lehman (34).

33. Svaren, J. & Chalkley, R. (1987) Nucleic Acids Res. 15,8739-8754.

34. Cox, M. M. & Lehman, I. R. (1981) Nucleic Acids Res. 9,389-400.

1628 Retraction