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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 264, No. 22, Issue of August 5. pp. 12785-12790,1989 Printed in U. SA. Common Sites for Recombination and Cleavage Mediated by Bacteriophage T4 DNA Topoisomerase in Vitro* (Received for publication, January 30, 1989) Masatoshi ChibaS, Hatsushi Shimizug, Akiko Fujimotol, Hiroko Nashimoto, and Hideo Ikedall From the Department of Molecular Biology, The Institute of Medical Science, University of Tokyo, P. 0. Takannwa, Tokyo 108, Japan We have previously shown that purified T4 DNA topoisomerasepromotes illegitimate recombination be- tween two X DNA molecules, or between X and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acud. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous se- quences of X DNA. In this paper, we have examined the sequences of the recombinationjunctions produced by the recombination between two X DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of X recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites withthe topoisomerase cleavage sites, indicating that a capacity of the topoisomeraseto make a transient double-strandedbreak in DNA plays a role inthe ille- gitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAYJNNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4- base pair staggered fashion and produces 5“protrud- ing ends. DNA rearrangements are frequently found in the genomes of bacteriophages, bacteria, and higher organisms. One type of rearrangement is produced by illegitimate recombination events. These recombinations are distinguishable from ho- mologous recombination which requires extensive homology or from site-specific recombination which requires specific sites. Illegitimate recombination in bacteria has been explained mainly by three types of models. First, some DNA re- arrangements take place at short regions of sequence homol- ogy (1, 2). Based on these observations, Albertini et al. (2) suggest that deletion is formed by slippage during replication. * This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan (to H. I.) and from the Nissan Science Foundation. 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. $ Present address: Mitsubishi Kasei Corp. Kashima Plant Phar- maceuticals Development Dept., Sunayama 14, Hazaki-machi, Kash- ima-gun, Ibaragi 314-02, Japan. !i Present address: Japanese PatentOffice, 4th ExaminationDept., Kasumigaseki 1-3-1, Chiyoda-ku, Tokyo 100, Japan. 7 Present address: Dept. of Oncology, The Institute of Medical Science, University of Tokyo, P. 0. Takanawa, Tokyo 108, Japan. 11 To whom correspondence should be addressed. Tel.: 03-443-8111. Fax: 03-443-3893. Second, certain inverted repeats or palindromit sequences are unstable in bacteria and tend to form deletions. Glickman and Ripley (3) suggest that stem-loop or cruciform structure can serve as a substrate for excision or as a shortened template for DNA replication. Third, we have found that Escherichia coli DNA gyrase and calf thymus DNA topoisomerase I1 participate in illegitimate recombination in in vitro systems (4-6). Analyses of recombinants produced inoursystems reveal that homology is not required for the recombination (6, 7). E. coli DNA gyrase and calf thymus DNA topoisomerase I1 belong to the class of type I1 topoisomerases that participates in topological changes in the state of circular DNA such as the introduction or removal of supercoils, the formation of catenanes, and the knotting or unknotting of circular DNA (8-11). The abilities of the enzymes to bind to double- stranded DNA and make a double-stranded break might have a role in illegitimate recombination. In fact, we have found common sites for recombination and cleavage mediated by E. coli DNA gyrase (12). We have proposed that two DNA topoisomerase molecules bound to DNA can associate with each other and cause the exchange of DNA strands through the exchange of DNA topoisomerase subunits (5,6). We have recently shown that purified T4 DNA topoisom- erase promotes oxolinic acid-induced recombination between two phage X DNA molecules or between X and plasmid DNA in an in uitro system (13, 14). Since the recombinant DNA produced in the X-X recombination contain duplications or deletions, the cross-overs probably take place between non- homologous sequences of X DNA molecules. Since phage T4 DNA topoisomerase is also a type I1 topoisomerase, it is likely that it participates in illegitimate recombination in a mecha- nism similar to thatmediated by E. coli DNA gyrase and calf thymus DNA topoisomerase 11. The T4 topoisomerase-me- diated recombination system is particularly interesting, be- cause the purified enzyme alone can promote recombination and the frequency of recombination is remarkably high. It is also known that phage T4 DNA topoisomerase has an ability to promote oxolinic acid-induced DNA cleavage (15). This activity is, therefore, likely to be essential for the mechanism of recombination. These facts suggest to us thattheT4 topoisomerase-mediated recombination is a good system for studying the mechanism of illegitimate recombination. In the present communication, we report that cross-overs mostly take place between nonhomologous sequences of DNA and that cross-over sites are associated with T4 DNA topoi- somerase cleavage sites, suggesting that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in illegitimate recombination. MATERIALS AND METHODS Bacteria, Bacteriophage, and Plasmid Strains-The bacterial strains used were all derivatives of E. coli K12. Ymel supF was used 12785

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THE J O U R N A L OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 22, Issue of August 5. pp. 12785-12790,1989 Printed in U. S A .

Common Sites for Recombination and Cleavage Mediated by Bacteriophage T4 DNA Topoisomerase in Vitro*

(Received for publication, January 30, 1989)

Masatoshi ChibaS, Hatsushi Shimizug, Akiko Fujimotol, Hiroko Nashimoto, and Hideo Ikedall From the Department of Molecular Biology, The Institute of Medical Science, University of Tokyo, P. 0. Takannwa, Tokyo 108, Japan

We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination be- tween two X DNA molecules, or between X and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acud. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous se- quences of X DNA. In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two X DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of X recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the ille- gitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAYJNNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4- base pair staggered fashion and produces 5“protrud- ing ends.

DNA rearrangements are frequently found in the genomes of bacteriophages, bacteria, and higher organisms. One type of rearrangement is produced by illegitimate recombination events. These recombinations are distinguishable from ho- mologous recombination which requires extensive homology or from site-specific recombination which requires specific sites.

Illegitimate recombination in bacteria has been explained mainly by three types of models. First, some DNA re- arrangements take place at short regions of sequence homol- ogy (1, 2). Based on these observations, Albertini et al. (2) suggest that deletion is formed by slippage during replication.

* This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan (to H. I.) and from the Nissan Science Foundation. 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.

’$ Present address: Mitsubishi Kasei Corp. Kashima Plant Phar- maceuticals Development Dept., Sunayama 14, Hazaki-machi, Kash- ima-gun, Ibaragi 314-02, Japan. !i Present address: Japanese Patent Office, 4th Examination Dept.,

Kasumigaseki 1-3-1, Chiyoda-ku, Tokyo 100, Japan. 7 Present address: Dept. of Oncology, The Institute of Medical

Science, University of Tokyo, P. 0. Takanawa, Tokyo 108, Japan. 11 To whom correspondence should be addressed. Tel.: 03-443-8111.

Fax: 03-443-3893.

Second, certain inverted repeats or palindromit sequences are unstable in bacteria and tend to form deletions. Glickman and Ripley (3) suggest that stem-loop or cruciform structure can serve as a substrate for excision or as a shortened template for DNA replication. Third, we have found that Escherichia coli DNA gyrase and calf thymus DNA topoisomerase I1 participate in illegitimate recombination in i n vitro systems (4-6). Analyses of recombinants produced in our systems reveal that homology is not required for the recombination (6, 7).

E. coli DNA gyrase and calf thymus DNA topoisomerase I1 belong to the class of type I1 topoisomerases that participates in topological changes in the state of circular DNA such as the introduction or removal of supercoils, the formation of catenanes, and the knotting or unknotting of circular DNA (8-11). The abilities of the enzymes to bind to double- stranded DNA and make a double-stranded break might have a role in illegitimate recombination. In fact, we have found common sites for recombination and cleavage mediated by E. coli DNA gyrase (12). We have proposed that two DNA topoisomerase molecules bound to DNA can associate with each other and cause the exchange of DNA strands through the exchange of DNA topoisomerase subunits (5,6).

We have recently shown that purified T4 DNA topoisom- erase promotes oxolinic acid-induced recombination between two phage X DNA molecules or between X and plasmid DNA in an in uitro system (13, 14). Since the recombinant DNA produced in the X-X recombination contain duplications or deletions, the cross-overs probably take place between non- homologous sequences of X DNA molecules. Since phage T4 DNA topoisomerase is also a type I1 topoisomerase, it is likely that it participates in illegitimate recombination in a mecha- nism similar to that mediated by E. coli DNA gyrase and calf thymus DNA topoisomerase 11. The T4 topoisomerase-me- diated recombination system is particularly interesting, be- cause the purified enzyme alone can promote recombination and the frequency of recombination is remarkably high. It is also known that phage T4 DNA topoisomerase has an ability to promote oxolinic acid-induced DNA cleavage (15). This activity is, therefore, likely to be essential for the mechanism of recombination. These facts suggest to us that the T4 topoisomerase-mediated recombination is a good system for studying the mechanism of illegitimate recombination.

In the present communication, we report that cross-overs mostly take place between nonhomologous sequences of DNA and that cross-over sites are associated with T4 DNA topoi- somerase cleavage sites, suggesting that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in illegitimate recombination.

MATERIALS AND METHODS

Bacteria, Bacteriophage, and Plasmid Strains-The bacterial strains used were all derivatives of E. coli K12. Ymel supF was used

12785

12786 Recombination and Cleavage by Phage T4 DNA Topoisomerase

for the preparation of X phages and for the assay of total phage titers. HI96 Su- recAl (X Dam15 FZam96B Sam7 Ram5) (16) was used for the preparation of extracts. NS428 recA (X cZ857 b2 AamIl Sam7) and NS433 recA ( X cZ857 b2 Eam4 Sam7) (17) were used for the preparation of packaging extracts. NI103 (Alac-pro thi strA endA sbcBI5 hsdR supE/F' traD36 proA+ proB+ laciq lacZAMI5 was used for the assay of M13 phage and for the preparation of M13 ssDNA and M13 dsDNA. Phage X imm434cZ Dam15 FZam96B b538 red3 and X imm434cI Sam7 Ram5 int6 red3 were used as parental phage for recombination (16). They are called Xb538DF and XSR, respectively, in this paper. X cZ857 Sam7 was used for the preparation of X DNA. M13mp19 was used for cloning of DNA fragments of phage X and for

junctions of X DNA. sequence analysis. pUC9 was used for cloning of recombination

Media, Buffers, and Enzymes-Media and buffers used in this paper were described previously (16). T4 DNA topoisomerase was kindly provided by B. Alberts (University of California, San Fran- cisco) (18). Restriction enzymes used were as follows: AccI (Boehrin- per), AuaI (Takara), AuaII (Takara), AluI (Takara), BamHI (Takara), BglII (Takara), BstEII (Bethesda Research Laboratories), ClaI (Boeh- ringer), EcoRI (Takara), EcoRV (Toyobo), H i d 1 (Boehringer), HpaI (Takara), MluI (Takara), PuuII (Takara), ScaI (Takara), Sal1 (Boeh- ringer), and SstII (Bethesda Research Laboratories).

Cloning and Sequencing of Recombination Junctions-DNA of XMMl recombinant phage was digested with PuuII. A fragment of 1800 bp' containing a recombination junction was purified by agarose gel electrophoresis and cloned with pUC9 vector. The cloned fragment was further mapped by restriction enzymes and subcloned into M13mp19. The sequence of the recombination junction was deter- mined by the dideoxy method. Similarly, the following restriction fragments containing recombination junctions were cloned into pUC9 and used for sequencing: HpaI fragment of 5600 bp from XMM3; HindIII-ScaI fragment of 2000 bp from XMM.I; ScaI-SmaI fragment of 3400 bp from XMM7; BamHI-SmaI fragment of 3600 bp from XMM8; MluI-PuuII fragment of 1000 bp from XMM14; PstI fragment of 2600 bp from XMM18.

Preparation of Fragments Containing Recombination Sites of Pa- rental X DNA-To prepare the fragments containing recombination sites on parental X DNA, they were cloned from X cZ857 Sam7 DNA into M13mp19 vector. X cZ857 Sam7 DNA was digested by an appro- priate restriction enzyme(s) and electrophoresed on polyacrylamide gel. DNA fragments containing recombination sites were recovered from polyacrylamide gel and cloned into M13 vector. For the analysis of the XMM3 recombination junction, a PstI-EcoRV fragment of 302 bp (bp 8524-8826 in X coordinate) which contains the recombination site on parental X b538DF DNA was isolated from X cZ857 Sam7 and ligated at PstI-SmaI site of M13mp19. This clone was called M13 MM3DF (Fig. la). M13 MM3DF RFI DNA was prepared from this phage, and, after digestion with HindIII and BanI, a fragment of 252 bp was isolated. This fragment contained a X DNA of 240 bp (bp 8524-8764 in X coordinate) connected by a M13 sequence of 12 bp at bp 8524. For the analysis of XMM8 recombination junction, two kinds of fragments which contain the recombination sites from both parents were used. A SstII-PuuII fragment of 164 bp (bp 20553-20697) which contained a recombination site on the parental X b538DF DNA was isolated from X cZ857 Sam7 DNA and ligated at SmaI site of M13mp19. This clone was called M13 MM8DF (Fig. IC). A fragment (212 bp) which was prepared by digestion of M13 MM8DF RFI DNA with EcoRI and HindIII contained a X DF sequence of 164 bp (bp 20533-20697) flanked by M13 sequences of 16 and 32 bp at both ends. Another fragment of 1679 bp (bp 21273-22952) which contains the site from parental X SR DNA was isolated by digestion of X cI857 Sam7 DNA with EcoRV and ligated at SmaI site of M13mp19. This clone was called M13 MM8SR (Fig. lb). M13 MM8SR RFI DNA was prepared from this clone and, after digestion with HindIII and EcoRI, a fragment of 1729 bp was isolated. This fragment contained a X DNA of 1679 bp (bp 21273-22952) flanked by M13 sequences of 35 and 15 bp.

Zn Vitro DNA Cleavage Reaction-Phage T4 DNA topoisomerase- mediated cleavage reaction was carried out in a mixture containing (in 50 pl) 1.2 pg of T4 DNA topoisomerase, 5 ng of 3ZP-labeled DNA fragment, 40 mM Tris.HC1 (pH 7.8), 60 mM KCl, 10 mM MgCl,, 0.5 mM dithiothreitol, 0.5 mM EDTA, 0.5 mM ATP, 30 pg/ml bovine serum albumin and 0.5 mg/ml oxolinic acid. Samples were incubated at 30 "C for 60 min. Sodium dodecyl sulfate (1 p1 of a 10% solution) was added, and the solution was heated at 70 "C for 5 min. Then

The abbreviation used is: bp, base pair(s).

HindIII Pat1 REC BanI EcoRV/SnaI 6233 6245/8524 8714 8764 8826/6268

~ 1 3 I I h

I REC HincII

(c) MMBDF A

6263 M13

I.-"-

4 Obp

FIG. 1. Structures of M13mp19 harboring segments of X DNA. a, M13 MM3DF contains the recombination site of the paren- tal Xb538DF DNA, which was found by the analysis of X MM3 recombinant DNA. M13 MM8SR (b) and M13 MM8DF ( e ) contain each of two recombination sites of the parental XSR and Xb538DF DNAs, respectively, which were found by the analysis of X MM8 recombinant DNA. Thick open block and thin open block indicate the sequences of X and M13 DNA, respectively. The nucleotides are numbered according to the X map coordinate defined by Sanger et al. (19) and the M13 map coordinate defined by Yanisch-Perron et al.

DNAs. (20). REC indicates recombination sites found in the parental X

proteinase K (2 pl of 1 mg/ml) and EDTA (1 pl of 0.1 mM) were added, and the solution was incubated at 37 "C for 1 h. The reaction was terminated by the addition of 50 pl of a stop solution which contains 0.6 M ammonium acetate, 0.1 M EDTA and E. coli tRNA (Sigma) at 100 pg/ml. The sample was treated with phenol and chloroform, precipitated by ethanol, and subjected to electrophoresis in an 8% polyacrylamide/urea gel. The samples of the Maxarn-Gilbert sequencing reactions for G+A and C+T were co-electrophoresed as size markers.

RESULTS

Cloning and Sequencing of Recombination Junctions Formed by T4 DNA Topoisomerase-mediated Recombination-Phage T4 DNA topoisomerase promotes recombination between two genetically marked X phage DNAs in vitro as shown previously (13). To examine the sequence specificity of the recombina- tion sites for the T4 DNA topoisomerase reaction, we have cloned the recombination junctions of recombinant phages formed by the T4 topoisomerase-dependent reaction and de- termined their nucleotide sequences. The restriction enzyme maps of the recombinant phages used for cloning of the junctions were shown in Fig. 2. The recombinant phage DNAs contain duplications or deletions at various sites, suggesting that the crossovers take place between nonhomologous se- quences of X DNA. Maps of a total of 16 recombinant strains, including 7 mapped previously (13), indicated that the recom- bination sites were distributed throughout the X phage chro- mosome.

To determine the sequences of recombination junctions of those recombinants, the recombinant DNAs were digested by restriction enzymes and cloned into pUC9. The cloned junc- tion fragments were further mapped by restriction enzymes and subcloned into M13mp19 (see "Materials and Methods"). The M13 clones thus obtained were sequenced.

Fig. 3 shows the sequences of junctions in the recombinants and the corresponding sequences of parental genomes. In XMMl DNA, a X sequence occurs up to bp 23815 and then another X sequence begins from bp 18694 (Fig. 3a). There is no homology between the parental X recombinants. Similarly, in XMM3 DNA and XMM4 DNA, there is no homology between the parental X recombination sites (Fig. 3, b and c ) . In XMM7 DNA, a X sequence occurs up to bp 27653 and then

Recombination and Cleavage by Phage T4 DNA Topoisomerase 12787 4 9 . 1

a"1

m 3 2 2 . 4

/ 1 7 . 9 ' I 3

m 4 , 50.1

3 2 . 1 Td ' a"5 -\

, 6 9 . 4

70.9'

a"6 , 7 4 . 5

7 75.6'

m 7 , 5 7 . 0

T 7

l S . 4 7 7

XHHS , 4 7 . 2

M I 4 , 3 3 . 0

ann1 s , 56.1 \

61.2'

I I I I I J 0 2 0 4 0 6 0 80 100

Z of X unit

FIG. 2. Structure of the am* recombinant phage DNAs. The recombinant phage DNAs were digested with various restriction enzymes. DNAs were electrophoresed in an agarose gel and structures of the recombinants were deduced. Cross-over points are shown by arrows with numbers that give the X map coordinate. Open rectangle, b538 deletion originally present on parental Xb538DF DNA.

another X sequence begins from bp 20684 (Fig. 3d). There is an overlap of 1 bp (A/T) at the junction. Hence the cross- over could take place at either side of the base pair. Similarly, in hMM8, there is an overlap of 1 bp (T/A) at the junction (Fig. 3e). In XMM14 DNA, a X sequence occurs up to bp 16040 and then another X sequence begins from bp 7476 (Fig. 3f). There is an overlap of 2 bp (AT/TA) at the junction. Hence, the cross-over could take place at three possible points around these 2 base pairs. In XMM18 DNA, a X sequence begins from bp 29695 (Fig. 3g). There is an overlap of 5 bp (GTATC/ CATAG) at the junction. Hence the cross-over could take place at six possible points around these 5 base pairs. In summary, among seven sets of recombination sites analyzed, there is no homology in three cases and homologies from 1 to 5 bp in four cases. Therefore, we concluded that homology is not essential in the recombination mediated by phage T4 DNA topoisomerase.

T4 DNA Topoisomerase Cleavage Sites-It is known that the T4 DNA topoisomerase cleaves double-stranded DNA and that oxolinic acid stimulates the cleavage (15). The ability of T4 DNA topoisomerase to cleave double-stranded DNA might play a role in illegitimate recombination. To examine the relationship between cleavage and recombination, we have determined the cleavage sites by measuring the lengths of cleavage products having a 32P-labeled 5' end and a free 3' end.

At first, we examined cleavage sites by T4 topoisomerase around the recombination site where the cross-over occurred in the recombinant XMM3 phage, located between bp 8713 and 8714 on X DF parental phage. A HindIII-Ban1 fragment of 252 bp was isolated from M13 MM3DF RFI DNA (Fig. la), labeled with 32P at both ends, and digested by PstI enzyme

23798- SR-23815 18694-DF-18711 \ I

( a ) AMMl 5'-CGGTTTGGAGGAATTGATAACGGTGCGGTGATTTAT-3'

ASR 5'-CGGTTn;GAGGMTTGATAAATTC!AAGCGAAATA-3'

ADF 5'-TATCTGAAAGTACTGATGAACGGTGCGGTGATTTAT-3'

1081 2- SR-10829 871 4 -DF- 8731 \ f

( b ) XMM3 5'-CTGATCCTfXlYX!AACAGAGCGATACCTGGCAGGCG-3'

ASH 5'-CIT;ATCCn;CIY;CAACAGGGGGGGCAGGn;AAGGAC-3'

ADF 5'-ACGGGCGAAGAGCTGGACAGCGATACCTGGCAGGCG-3'

24312- SR-24329 15610-DF-15627 \ I

(c) AMM4 5'-CGCCACTGTCCCTAGGACGAAGGTCCGGTGGATGGC-3'

ASR 5"CGCCACTGTCCCTAGGACCTATGTGCCGGAGCGG-3'

ADF 5'-ATCAGCGAAGGGCCGATTGAAGGTCCGGTGGATGGC-3'

27636- SR ~ 27653 20684 - DF - 20701

( d ) hMM7 5"GTTGTGTlTTACAGTA ATATAGCTTCAGCTGT-3'

ASR 5 ' -GTTG=ACAGTA TAGTCTGTTTTTTAT- 3 '

ADF 5"ATTCGGCAAAACGTGCA + ATATAGCTTCAGCTGT-3'

22882- SR ~ 22?00 2061 1- DF - 20628

(e) AMM8 5"TCTATAATTGGCATTGT TCAGCATCGCAGAGCA-3'

ASR 5"TCTATMTTGGCATTGT TATTGGTTTATXGAG-3'

XDF 5"AGGCTGCGGGAAGTGCG B TCAGCATCGCAGAGCA-3' 16021- SR 16040 73K DF- 7391

(f) AMM14 5"CCGCCGCGCCCGTTTAA TGCTGACCGGACATG-3'

ASR 5"CCGCCGCGCCCGTTTAA CGGATGCGCAGGATG-3'

ADF 5"CCCCTCTCAGCCGGGAA TGCTGACCGGACATG-3'

27201- SR 2 9 6 9 5 - t D F - 2971 4

27221

( g ) AMMl8 5"TATTAATGCATATAT TAT GCAATGTTTATGTA-3'

ASR 5"TATTMTGCATATAT TAT CCGMCGATTAGCT-3'

AD'? 5"TCGTTTCAGCTAAACG D TAT GCAATGTTTATGTA-3'

FIG. 3. Comparison between sequences around the X-X junc- tions and sequences of the parental XSR and hb538DF DNAs. Numbers represent the map coordinates of the X DNA sequence. Rectangle on the sequences represents homology at the sites of recom- bination. Vertical arrows indicate possible points of recombination on the parental XSR and Xb538DF DNAs. a, sequences around the X-X junction of XMM1; b, XMM3; c, XMM4; d, XMM7; e, XMMB; f, XMM14; g, XMM18. Open rectangles represent regions of homologies found in sequences of parental recombination sites.

(see "Materials and Methods"); the resulting fragment of 240 bp (bp 8524-8764) was purified and then subjected to oxolinic acid-promoted T4 DNA topoisomerase cleavage followed by proteinase K treatment as described (8). The lengths of the 32P-labeled strands were determined by electrophoresis in a denaturing gel alongside the Maxam-Gilbert sequencing prod- ucts obtained for each labeled strand of the original cleaved restriction fragment (21). Since the Maxam-Gilbert method

12788 Recombination and Cleavage by Phage T4 DNA Topoisomerase

generates DNA fragments that terminate in a 3"phosphate and from which the nucleotide of interest is removed, topoi- somerase-cleaved fragments run about 1.5 nucleotide posi- tions slower than the sequencing product cleaved at the same nucleotide (22). The result showed that strong cleavages were observed at two sites between bp 8717 and 8718 and between bp 8746 and 8747 (Fig. 4; see also Fig. 5a). Since T4 DNA topoisomerase cleaves DNA with a 4-bp stagger and a pro- truding 5' end, one cleavage event must occur between bp 8713 and 8714 on one strand and between bp 8717 and 8718 on the other strand. This cleavage site is in agreement with the location of the recombination event in XMM3.

We have also examined sites of cleavage by T4 topoisom-

1 2 3 4

G T c- A T - G

\G \A L

FIG. 4. DNA cleavage induced by T4 DNA topoisomerase in the presence of oxolinic acid in a X fragment of 240 bp. A PstI- Ban1 fragment was prepared from M13 MM3DF and end-labeled. The T4 topoisomerase-mediated cleavage reaction was carried out as described under "Materials and Methods." Lanes 1 and 2 are the Maxam-Gilbert sequencing reactions for G+A and C+T. Lane 3 is the cleavage reaction which contains T4 DNA topoisomerase. Lane 4 is the same as lane 3, except that the mixture did not contain the topoisomerase. Arrow indicates cleavage site.

22880 22890 22900 2291 0 22920 (b) XMM8 TTC ATCTATAATT GTATTGGTTT ATTGGAGTAG ATGCTTG

(SRI AAG TAGATATTAA CATAACCAAA TAACCTCATC TACGAAC

20590 20600 20620 ~ 6 3 0 ( c ) auMs AC AGAGGCTGCG GGAAIGTFCGG TATCAGCATC GCAGAGCAAA AGTGCGGC

( D F ) TG TCTCCGACGC C C T T C A C ~ T A G T C G T A G CGTCTCGTTT TCACGCCG

FIG. 5. Comparison of sites of recombination and cleavage on X DNA. The sites of recombination where the cross-overs were found in the indicated recombinant phages are shown by open triun- gles. Where multiple triangles are shown for one recombinant site, the cross-over could have occurred at any one of those positions. Locations and strands of cleavage sites determined by this paper are shown by large solid triangles (strong cleavage) and small solid triun- gles (weak cleavage). Cleavage sites identical to the recombination sites are also shown by staggered lines between double strands. The orientation of DNA is 5' to 3' from left to right in the upper strand.

AA

20600 2061 0 1 2 0 6 2 0 2 0 6 3 0 5'-CTGCG GGAAGTGCGG TATCAGCATC GCAGAGCAAA AGTGC-3' 3"GACGC CCTTCACGCC ATAGTCG.TAG CGTCTCGTTT TCACG-5'

t FIG. 6. Mode of DNA cleavage induced by T4 DNA topoi-

somerase in the presence of oxolinic acid in a LM13 recom- binant fragment. A HindIII-HincII fragment of 174 bp was pre- pared from M13 MMSDF and end-labeled. The labeled fragment was cleaved by T4 DNA topoisomerase in the presence of oxolinic acid. A strong cleavage site detected in this experiment was compared for the cleavage site found in Fig. 5c.

erase near the recombination sites where the cross-over oc- curred in the recombination XMM8 phage, located between bp 20610 and 20612 on X b538DF parental phage and between bp 22899 and 22901 on X SR parental phage. For the latter recombination site, a HindIII-EcoRI fragment of 1729 bp was isolated from M13 MM8SR (Fig. lb), end-labeled, and di- gested with PstI (see "Materials and Methods"). The resulting fragment of 542 bp contained a X sequence of 527 bp (bp 21273-22952) that is connected with a M13 sequence of 15 bp and was cleaved by T4 topoisomerase. A strong cleavage was observed between bp 22900 and 22901 and the cleavage site agreed with one of the putative recombination sites (Fig. 5b).

For the other recombination site of the XMM8 recombinant, located between bp 20610 and 20612 on Xb538DF, a HindIII- EcoRI fragment of 212 bp was isolated from M13 MM8DF (Fig. IC), end-labeled, and digested by BarnHI (see "Materials and Methods"). The resulting fragment of 185 bp contained a X sequence of 164 bp (bp 20533-20697) that is flanked by M13 sequences of 16 and 5 bp and was cleaved as described before. Strong cleavages were observed between bp 20613 and 20614 and between bp 20626 and 20627; and these cleavage sites agreed with none of the putative recombination sites (Fig. 5c). However, when we searched for weak cleavage sites, two weak cleavage sites between bp 20606 and 20608 agreed with the putative recombination sites (Fig. 5c).

To examine cleavage sites on the other strand of the frag- ment examined in Fig. 5c, a HindIII-EcoRI fragment of 212 bp from M13 MM8DF was end-labeled and digested by HincII. The resulting fragment of 174 bp contained a X sequence of 128 bp (bp 20569-20697) which is connected with a M13 sequence of 27 bp. A strong cleavage was observed between bp 20617 and 20618 (Fig. 6). Since strand cleavage

Recombination and Cleavage by Phage T4 DNA Topoisomerase 12789

was observed between bp 20613 and 20614 in the other strand, it is concluded that the cleavage took place in the 4-bp staggered fashion and produced 5"protruding ends.

DISCUSSION

In this paper, we first investigated the requirement for homology of recombination sites in the recombination me- diated by T4 DNA topoisomerase. The present results indicate that there is either no homology or 1-5-base pair homologies between the parental DNAs in seven combinations of X re- combination sites. Based on the facts that no homology was detected in three combinations of the X recombination sites among a total of seven recombinants, we concluded that homology is not essential for the recombination mediated by phage T4 DNA topoisomerase.

The next question is whether there are common sites for recombination and cleavage mediated by T4 DNA topoisom- erase. The result of T4 topoisomerase-mediated cleavages in the regions of interest on X DNA are summarized in Fig. 5. All three recombination sites tested coincide with sites of cleavage mediated by the topoisomerase, but one of these is a weak cleavage site. In order to assess the statistical signifi- cance of the results, we have estimated the probability of obtaining by chance two successful matches in three frag- ments (the match with a weak cleavage site in XMMS(DF) was disregarded for this purpose), taking into account that each fragment contains several cleavage sites of comparable strength to the one at the recombination site. In the fragment corresponding to XMM3(DF), there is one match among four cleavage sites in 80 bp. Around the XMM8(SR) site, there is one match in one cleavage site in 100 bp. Near the other one site without strong matching cleavage, there are five cleavages in 110 bp around the XMM8(DF) site. Considering that each topoisomerase cleavage produces two ends, either of which could match a recombination site, the probability is:

Therefore, it is likely that the association of recombination sites with topoisomerase cleavage sites did not occur by chance.

As we previously suggested in the case of DNA gyrase cleavage (12), one can speculate about the mechanism of formation of the recombination junction as follows; in the XMM3 recombinant phage, one 5"protruding end in XDF is formed at bp 8714; the corresponding end in XSR may be at bp 10829. Only two bases would form correct base pairs, with mismatches at the other two positions (Fig. 7). In another recombinant phage, there would also be mismatched base pairs in the putative junctions. These mismatched base pairs might be repaired during in vitro packaging or after infection into indicator bacteria, because a single burst experiment with

1 0 8 2 0 1 0 8 2 9 8 7 1 4 8 7 2 7

l a l I M M 3 5 : G C T G C A A C A G A L C G A T A C C T G G C A 3 : I \ I I

3 C G A C G T T G T C ' & ] T A T G G A C C G T S

2 2 0 8 7

( b ) X M M 8 S ' A A T T G G C A T T C G G T A T C A G C A T C G 3 ' 3 ' T T A A C c G T A 1 ' & ] T A G T C G T A G C S '

I 2 2 8 9 6 2 0 6 0 6 2061 9

I

FIG. 7. Possible nucleotide sequences of recombination in- termediates at the junctions. Based on the nucleotide sequences for the recombination sites and those for the cleavage sites in X DNA, we suggested the nucleotide sequences of recombination junctions. The map coordinates represent h DNA sequences.

bp 8 7 1 8 GCCAGGTAT CGCT GTCCAGCT bp 8 7 3 9 GAGCTGCAT ATCG AAGTTTTC

bp 2 0 6 1 3 GTGCGGTAT CAGC ATCGCAGA bp 8 7 4 7 CAGGAAAAC TTCG ATATGCAG

bp 2 0 6 3 1 TGCCGCACT TTTG CTCTGCGA bp 2 0 6 9 0 GCTGAAGCT ATAT CTTCTGCA bp 2 0 6 9 2 CAGCTGAAG CTAT ATCTTCTT bp 2 2 8 9 6 AACCAATAC ATAC AATGCCAA

C o n s e n s u s RNAY NNNN RTNY

1

FIG. 8. Nucleotide sequences of eight strong cleavage sites cleaved by T4 DNA topoisomerase. Six strong cleavage sites shown in Fig. 5 and two other strong cleavage sites detected in a X DNA region (bp 20662-20711) were compiled. From these sequences, a consensus sec 3' from left to sites.

lence was deduced. The orientation of DNA is 5' to ght. Arrow indicates the T4 topoisomerase cleavage

I hMMl

A M M 3 A M M l

A M M 3 XMM4

( ( ( ( (

XMM4 ( D F ) A M M 7 ( SR )

A M M 8 ( S R ) A M M 7 ( D F )

X M M 8 ( D F ) hMMl 4 ( S R ) A M M 1 4 ( D F ) AMMI8 ( S R ) AMMI8 ( D F )

C o n s e n s u s RNAY NNNN RNNY

FIG. 9. Nucleotide sequences of 14 recombination sites. Fourteen recombination sites shown in Fig. 3 were compiled. From these sequences, a consensus sequence was deduced. The orientation of DNA is 5' to 3' from left to right. Arrow indicates putative T4 topoisomerase cleavage sites.

S R ) GGAATTGAT'TCAA ATTCAAGC D F ) GTACTGATG AACG GTGCGGTG SR ) TGCCAACAG GGGG GGCAGGTG D F ) GAGCTGGAC AGCG ATACCTGG SR ) CCCTAGGAC TGCT ATGTGCCG

GGGCCGATT GAAG

CGTGCAGAA GATA ACAGTATTA TGTA

GCATTGTAT GTAT AAGTGCGGT ATCA GTTTAATAT CCGG CGGGAAAAT GTGC ATATAGTAT CGCC AAACGGTAT CAGC

GTCCGGTG GTCTGTTT TAGCTTCA

GCATCGCA TGGTTTAT

ATGCGCAG TGACCGGA GAACGAAC AATGTTTA

the recombinant phage produced in vitro in the presence of T4 DNA topoisomerase has not yet revealed any evidence of mismatched bases in the recombinant phage particles.2 The 5'-protruding ends might be joined covalently to the 3' end by the action of T4 DNA topoisomerase, even if the mis- matched base pairs are not repaired before joining.

In order to deduce a consensus sequence for T4 topoisom- erase cleavage sites, we have used six strong cleavage sites shown in Fig. 5 and two other strong cleavage sites detected in a X DNA region (bp 20662-20711). After the compilation of the sequences, we have deduced a consensus sequence, 5'- RNAYJNNNNRTNY-3' (Fig. 8). The extent of matching was 75% among the eight cleavage sites used. The value indicates that the consensus is not complete as was the cases of E. coli DNA gyrase (23) and vertebrate DNA topoisomerase I1 (24). It is noteworthy that this consensus sequence exhib- ited diad symmetry. T4 DNA topoisomerase consists of three subunit proteins of 57,48, and 18 kDa, known to be coded by genes 39,52, and 60, respectively (25,26). The native form of T4 topoisomerase seems to be a dimer (18,27). It is therefore conceivable that T4 topoisomerase binds DNA as a dimeric subunit structure and recognizes DNA at least at two sites. This notion is also consistent with the subunit exchange model which is originally proposed for the interpretation of mechanism of DNA gyrase-mediated illegitimate recombina- tion (5).

M. Kumagai and H. Ikeda, unpublished results.

12790 Recombination and Cleavage by Phage T4 DNA Topoisomerase

It should be noted that some of the recombination sites where recombination occurred to generate the i n vitro X recombinant closely resemble known topoisomerase sites. We have collected seven combinations of the recombination sites from the data of Fig. 3. Sequences closely, if not completely, resemble the consensus sequence for T4 topoisomerase-me- diated cleavage (Fig. 9). The extent of matching of the recom- bination site sequences to the topoisomerase cleavage consen- sus sequence was 74%) which is considerably higher than the value expected from a random collection of sequences.

I t is noteworthy that all of the type I1 topoisomerases examined so far, E. coli DNA gyrase, phage T4 DNA topoi- somerase, and calf thymus DNA topoisomerase 11, have the ability to promote illegitimate recombination. These enzymes might be involved in i n vivo recombination in bacteria, bac- teriophage, and eukaryotic systems. We have already shown that E. coli DNA gyrase participates in deletion formation in E. coli (28).3 We have also obtained evidence for participation of topoisomerase in illegitimate recombination i n uivo of T4 DNA.4

Acknowledgments- We thank Dr. B. Alberts for providing T4 DNA topoisomerase and Dr. D. A. Shub for critical reading of the manuscript.

1.

2.

3.

4.

5.

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