Double-strand Cleavage and Strand Joining by the Replication

Vol. 264, No. 21, Issue of July 25, pp. 12627-12632,1989 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Double-strand Cleavage and Strand Joining by the Replication Initiator Protein of Filamentous Phage f l * (Received for publication, January 27, 1989)

David Greenstein$ and Kensuke HoriuchiB From the Rockefeller University, New York, New York 10021

aL., 1979) because gpII requires asupercoiled substrate for nicking (Meyer and Geider, 1979b). gpII also functions a t a step beyond nicking: DNA molecules that have been nicked by gpII still require gpII for their unwinding and replication (Geider et al., 1982). Upon completion of a round of synthesis, gpII functions in the termination reaction by cleaving and circularizing the displaced single strand (Harth et al., 1981). gpII also has site-specific topoisomerase activity since, in the presence of M e , the RFI DNA is converted by gpII i n vitro to a mixture of RFII (nicked-circle) and RFIV (Meyer and Geider,1979b). Fig. 1 shows thetopography of thegpII substrate, the (+)-strandorigin of DNA replication. gpII binds to thecore origin in a two-step fashion (Greenstein and Horiuchi,1987; Greenstein, 1989).The first binding step involves interaction of two gpII molecules with an inverted repeat (P-7 shown in Fig. 1) at the center of the core origin to forma binding intermediate, complex I. The second binding step involves addition of two more protein molecules to complex I, resulting in formation of the functional structure, complex 11. Of these two protein molecules, one binds to and contacts repeat 6; the other gpII molecule protects the nicking site in a sequence-independent fashion. In addition, gpII bends the origin DNA upon binding; both complexes I and I1 are bent (Greenstein, 1989).’ Purified gpII nicks the replication origin at a specific site in the presence of M F (Meyer et al., 1979). When Mn2+is The gene I1 protein (gpI1)’ of filamentous phages (fl, M13, substituted for M%+, the enzyme produces a double-strand Geider, 1979b; Dotto et and fd) is a multifunctional protein that plays several key cleavage at the same site (Meyer and al., 1981). This activity of the enzyme has been a useful one roles in phage (+)-strand DNA replication. First, it binds to we have the (+)-strand origin (Geider et al., 1982; Horiuchi, 1986; to monitor during its purification. In this paper, of the Mn” determined the structure of the cleavage product Greenstein and Horiuchi,1987) and introducesa specific nick in the (+)-strand of supercoiled replicative form DNA (RFI) reaction. The cleavage product has an unusual structure in form a telomere(Meyer et al., 1979). The 3”hydroxyl end of the nick serves which the (+)- and (-)-strands are linked to like hairpin. Formation of this product proceeds in a two-step as the primer for (+)-strand rolling-circle-typereplication (GilbertandDressler, 1968). Theproducts of a round of reaction: nicking at the origin is followed by strand joining. rolling-circle synthesis are a single-stranded circle and a re- In contrast to nicking in the presence of M$’, the Mn2+laxed, covalently closed, double-stranded circle (RFIV). The dependent cleavage activity does not require a supercoiled RFIVDNA must be supercoiled by Escherichia coli DNA substrate andshows reduced DNA sequence requirements. gyrase for another round of replication to ensue (Horiuchiet

The replication initiator protein (gene I1 protein (gpII)) of bacteriophage f l is a multifunctional protein that plays central roles in initiation and termination of phage DNA replication. It introduces a nick at a specific site on the (+)-strandof supercoiled replicative form DNA. The 3”hydroxyl end of the nick serves as the primer for (+)-strandrolling-circlereplication. Upon completion of a round of synthesis, gpII cleaves and circularizesthe displaced singlestrand. When Mn2+ is included in the buffer instead of Mg2+, gpII cleaves both strands. In this paper, we investigate the mechanism of the Mn2+-dependent double-strand cleavage activity of gpII. This reaction, unlike nicking in thepresence of Mg2+,does not require superhelicity. The reaction proceeds in two kineticsteps: first nicking of the (+)-strand, and then cleavage of the (-)strand. The nucleotide sequence requirement for nicking is reduced compared to that in thepresence of Mg2+. The product of the double-strand cleavage has an unusual structure. The left end is a telomere-like hairpin since the (+)- and (-)-strands are joined, as demonstrated by base sequencing. The right end has a onebase 3’-overhang. This reaction probably reflects the cleavage-joiningactivity of gpII in the termination event.

MATERIALSANDMETHODS

-

* This work was supported in part by grants from the National Science Foundation and the National Institutesof Health. The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be hereby marked “aduertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by Training Grant AI 07233 from the National Institutes of Health. Present address: Dept. of Molecular Biology, Massachusetts General Hospital, Boston,MA 02114. I To whom correspondence should be addressed: Rockefeller University, 1230 York Ave., New York, NY 10021-6399. ’ The abbreviationsused are: gpII, gene I1 protein; RFI,supercoiled replicative form DNA; RFIV, relaxed,covalentlyclosed,doublestrandedcircular DNA; RFIII,full-lengthlineardouble-stranded DNA; RFII, nicked circular DNA; bp, base pair(s).

Enzymes, DNA Manipulations, and Origin Plasmids-Restriction enzymes were from New England Biolabs. T4 polynucleotide kinase was from Pharmacia LKB Biotechnology Inc.DNA polymerase I Klenow fragment was from Bethesda Research Laboratories. Calf intestinalalkalinephosphatase wasfromBoehringer Mannheim. Gene I1 protein was purified to homogeneity according to the method of Greenstein and Horiuchi (1987). Identical results were obtained with gpII isolated according to the method of Meyer and Geider (1979a). Plasmid and phage R F DNAs were prepared according to Maniatis et al. (1982) or Zinder and Boeke(1982). Procedures for restriction enzyme digestion and endlabeling of restriction fragments were described by Maniatis et al. (1982). The origin-containing plasmid pDG117, which contains the wild-type core origin and part of

12627

D. Greenstein and K. Horiuchi, manuscript in preparation.

Strand J o i n i n g by the fl Replication Initiator Protein

12628 Nick 0

-30 I

40 I

1

GPI

- B"_ 0

100 I

I

I

I

IHF

IHF

I 8

+ 200

+ 150

"

2'

Z',

Initiation Termination I

I

L

Core origin

Replicolion enhancer

FIG. 1. The (+)-Strand replication origin of bacteriophage f l . The origin region is shown as a horizontal line. The vertical arrow marks the site of nicking by gpII (0)(Meyer et al., 1979). DNA replication begins at the nick and proceeds rightward. The core origin sequence and the replication enhancer sequence are indicated (Dotto et al., 1984; Johnston and Ray, 1984). DNA sequences protected by gpII or the E. coli integration host factor (ZHF) are shown by bars above ((+)-strand) and below ((-)-strand)the line. The leftward hatched bar(site 1) shows the region stronglyprotected by the integration host factor, and the rightward hatched bar (site 2) shows the region weakly protected by it (Greenstein etal., 1988). The region protected by gpII is shown by the open bar. Repeatedsequences within the core origin are indicated by arrows CY and 0 (5"TGGAC3') or y and d (5'-TGGAAC-3'). Arrows I , 2', and 2" are sequences within the replication enhancer that nearly match the consensus sequence for integration host factor binding. The (+)-strand origin contains signals for both the initiation and termination of DNA synthesis (Horiuchi, 1980; Dotto and Horiuchi, 1981; Dotto et al., 1982a). Sequences required for initiation and termination of DNA replication, respectively, are shown.

by Maxam-Gilbert sequencingfollowing their isolation on preparative DNA sequencing gels. The radioactive DNA fragments were eluted from the preparative DNA sequencing gel by suspension of the gel slice in 400 pl of N buffer (0.1 M Tris-CI (pH 7.9), 0.6 M NaOAc, and 2.5 mM EDTA) a t 37 "C overnight, followed by precipitation with ethanol. Sequencing of the Cleavage Product--RFIII-Mn*+ was sequenced by the method of Maxam and Gilbert(1977) using the BamHI site of pDG117 as the site of labeling. The BamHI end-labeled fragment that extended to the site of cleavage by gene I1 protein was isolated on a formamide (70%)-polyacrylamide (8%) gel and purified by electroelution. Following the base-specific cleavage reactions,the sample was loaded onto a formamide (70%)-polyacrylamide (8%) DNA sequencing gel. RESULTS

Anomalous Migration in Alkaline Agarose Gels-The first indication that RFIII(full-length linear) formed from RFI by gpII in the presence of Mn2+ (RFIII-MnZ+) had an abnormal structure was its anomalous migration in alkaline agarose gels (Fig. 2). RFIII-Mn" (indicated by X L in Fig. 2) migrated slowly on alkalineagarose gels (Fig. 2, lane 3), whereas RFIII produced by the restrictionenzyme BamHI co-migrated with the unit-length linearsingle strands of the phage(Fig. 2, lane 5). RFIII-Mn2+ exhibited anomalous migration whether or not itwas extracted with phenol or treated with Pronase prior to electrophoresis (data not shown). Thus, the anomalous migration is a property of the DNA and is not due to the formation of covalent complex with gpII. The migration of RFIII-Mn2+ in neutral agarose gels was the same as that of the replication enhancer as a 151-base pair (bp) fragment flankedby RFIII molecules produced by the restriction enzyme BamHI. restriction enzyme recognition sequences for EcoRI and BamHI, was After denaturationwithalkali followed by neutralization, described (Greenstein andHoriuchi, 1987). The defective origins A83 (a deletion from nucleotides -14 to +3), pD30 (a 13-bp insertion at RFIII-Mn" migrated at the position of RFIII in a neutral co-migrated nucleotide +8), pD29 (a deletion from the left end to nucleotide +5), agarose gel, whereas RFIII prepared with BamHI and A+29 (a deletion from nucleotide +29 to the right end) were mainly with linear single strands under the sameconditions. previously described by Dotto etal. (1982a, 1984). Derivatives of these This indicates that RFIII-Mn2+ renaturesmore rapidly than defective origin plasmids that contain convenient restriction sites RFIII produced by digestion with BamHI. (described by Greenstein and Horiuchi (1987))were used to localize (+)- and (-)-Strands Are Joined-In order to characterize the siteof nicking in the presenceof Mn". the cleavage product, we mapped the cleavage end points and Cleavage Reaction-Cleavage reactions were carriedout as deof both terminiby chemical sequencscribed by Meyer and Geider (1979b). Purified gene I1 protein (5-20 established the structure ng) was incubated with fl origin-containing DNA (0.1 ng to20 pg) in ing. As shown in Fig. 3, the cleavage site inpDG117 is flanked 20 pl containing 20 mM Tris-CI (pH 8.0), 80 mM KCI, 5 mM dithio- by a BamHI site and anEcoRI site. These sites were used to threitol, and 5 mM MnCI2 for 30 min a t 30 "C. The reactions were end-label fragments in order to determine the structure of the terminated by addition of 1pl of stop mixture (0.2 M EDTA (pH8.0), cleavage product. Labeling was carried out by 3'-end filling 20% sucrose, 1%sodium dodecyl sulfate, and0.01% bromphenol blue) and analyzed on a 0.6% agarose gel, an 8% polyacrylamide gel, or an with the Klenow fragment of DNA polymerase I (lanes 3-6) alkaline agarose gel (in which case the stop mixture was not added) or 5'-end labeling with T 4 polynucleotide kinase following treatment with calf intestinal alkaline phosphatase (lanes1, as described (Maniatis et al., 1982). The nature of the products or their distribution was not affected by the method of stopping the reaction. Mapping of Cleavage Sites-The origin-containing plasmid pDG117 (20 p g ) was cleaved with gene I1 protein in the presence of Mn2+(described above), and the cleavage product (RFIII-Mn'+) was isolated on a 0.6% agarose gel. Theorigin-containingplasmid of Mn2+ (pDG117) was also nicked by gene I1 protein in the presence after a brief incubation (1 min), and thenicked product (RFII-Mn") was isolated on a 0.6% agarose gel. The gel-purified DNA samples were eluted electrophoretically, precipitated with ethanol, and further purified by extraction with phenol and precipitation with ethanol. Purified RFII-Mn" (10 pg) and RFIII-Mn" (10 pg) were each divided into two portions. One portionwas digested withBamHI (set B), and the other portionwas digested with EcoRI (set E). Sets B and E were each divided in half. Half of each set was end-labeled by treatment with T4 polynucleotide kinase and [y-"PIATP following treatment with calf intestinal alkaline phosphatase. The other half of each set was end-labeled by treatment with the Klenow fragment of DNA polymerase I and [w3'P]dATP. Following end-labeling, set B was digested with EcoRI, and set E was digested with BamHI. The endlabeled restrictionfragments were analyzed on apolyacrylamide (F)F)-urea (50%)DNA sequencing gel or polyacrylamide (8%)-formamide (70%) sequencing gel. Maxam-Gilbert (1977) sequencing reactions performed on the 151-bp BamHI-EcoRI fragment served as markers. The identityof the individual bands inFig. 3 was confirmed

XL"

:g F+ R

FIG. 2. Migration of RFIII-Mn2' on alkaline agarose gels. Purified gene I1 protein (approximately 20 ng) was incubated with f l RYI (approximately 200 ng) in 20 pl containing 20 mM Tris-CI (pH 8.0), 80 mM KCI, 5 mM dithiothreitol, and either 5 mMMgC12 (lane 2) or 5 mM MnCI2 (lane 3 ) for, 30min a t 30 "C. Following the incubation, the samples were loaded on a 0.6% alkaline agarose gel (Maniatis et al., 1982). After electrophoresis, the DNA was stained with ethidium bromide. Lane 1 contained no gene I1 protein. Lane 4 contained f l single-stranded DNA, which contained both the circular (SS,) and linear (SS,) forms. Lane 5 contained f l RFI cleaved with the restriction enzyme BamHI, which cleaves fl RFI once. XL (for cross-linked) designates the position of the anomalously migrating species thatresults from incubation of f l RFI with gpII inthe presence of Mn2+. In alkaline gels, RFI (lane 1 ) and RFIV (lane 2) co-migrate at theposition marked RF.

Strand Joiningby the fl Rc?plication ProteinInitiator

12629 b

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m

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RFll L RFlll

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+92 FIG. 3. Mapping of cleavage sites. The cleavage sites were mapped as depicted at the bottom. RFII-Mn” (lanes 1, 3,5, and 7) and RFIII-Mn’+ (lanes 2, 4, 6, and 8 ) were treated with BamHI (lanes 1-4) or EcoRI (lanes 5-8) and end-labeled by reactionwith the Klenow fragment of DNA polymerase I (lanes 3-6) or T 4 polynucleotide kinase(lanes 1,2, 7, and 8). The labeled DNA wasthen digested withEcoRI (lanes 1-41 or BamHI (lanes 5-8) and analyzed by electrophoresis on urea (8 M)-polyacrylamide (8%)sequencing gel. Maxam-Gilbert (1977) reactions performed ontheBamHI-EcoRI fragment from pDG117 (5”end-labeled at theBamHI site) served as markers. The sizes of the various fragments in nucleotides are indicated at the side of the gel. Each fragment is given a symbol that indicates the origin of the fragment as shown in the picture at the bottom.

-63

2, 7, and 8). After end labeling, the DNA was digested with a restriction enzyme (EcoRI for DNA labeled at the BarnHI site, and BarnHI forDNAlabeled at the EcoRI site) and subjected to electrophoresis on a urea (8 M)-polyacrylamide (8%) sequencing gel. The two fragments, labeled atthe BarnHI site, that extend to the cleavage site migrate anomalously on the standard sequencing gel (104- and 108-base fragments indicatedby filled squares inFig. 3); the fragments migrate as triplets in thisgel. The two fragments with labels on the(+)- and (-)-strands, respectively, differ in size because the fragment labeled on the (-)-strand was end-filled, which adds four nucleotides. Initial attempts to sequence these fragments chemically using urea (8 M)-polyacrylamide (8%)sequencing gels were unsuccessful. The sequence could be read up to the nicking site; then the fragments began to migrate anomalously, and resolution of the bandswas lost. In contrast, on a formamide (70%)-polyacrylamide (8%)sequencing gel, the fragments migrated assingle bands of 121 and 125bases, respectively (Fig. 4a and data not shown). These fragments (121- and 125-base fragments) were purified and sequenced chemically using polyacrylamide gels containing 70% formamide. In Fig. 4b, we see the results obtained by sequencing these fragments surrounding theleft side of the gpII cleavage site. In particular, note that nucleotides which derive from the (+)-strand and those which derive from the (-)-strand are linked at the cleavage site. The same resultwas obtained whether the (+)-strand was labeled with T4 polynucleotide kinase (left panel in Fig. 4b) or the (-)-strand was labeled with the Klenow fragment of DNA polymerase I (right panel in Fig. 46). The right side of the cleavage product has the structureof a one-base 3‘-overhang, although approximately 5% of the

.. I

,

FIG. 4. Structure of the cleavage product. a, denaturing gel analysis of the cleavage product. The sample from Fig. 3 (lane 2) (RFIII-Mn” 5”end-labeled at the RamHI site) was analyzed on a formamide (70%)-polyacrylamide (8%)gel (shown in the rightmost lane). The Maxam-Gilbert (1977) sequencing reactions that served as markers aredescribed in the legend to Fig. 3. b, sequencing of the left side of the cleavage product. The 121- and 125-base fragments, 5’- and 3’-end-labeled at theBamHI site, respectively, were purified on a formamide(70%)-polyacrylamide (8%)gel and subjected to chemical sequencing. The left and right panels show the sequencing respectively. reactions for the 5’- and 3”end-labeledfragments, Analysis was performed on a formamide (70%)-polyacrylamide (8%) gel. The nucleotide sequenceis diagramed betweenthe two sequencing gel panels. The arrowhead shows the T residue a t position -1. c, mechanism of the Mn2+-dependent double-strandcleavage activity. The DNA sequence of the nicking site region is shown, with the (+)strand being the top strand. The two steps of the reaction are shown: 1) nicking at theorigin and 2) strand joining, as described in the text.

right-end productshave blunt ends. The terminal base on the (+)-strand is the A residue a t position +1 (Fig. 3, lanes 2, 6, and 8), and the terminal base on the (-)-strand is the A residue a t position -1 (Fig. 3, lane 8).This is shown both by sizing of the fragments in Fig. 3 and by chemical sequencing to verify their identities (data not shown). The sequencing information indicates that the T residue a t position -1 on the (+)-strand is linked to the A residue a t position -2 on the (-)-strand, as shown in Fig. 4c. Kinetics of Double-strand Cleauage-The kinetics of doublestrand cleavage is shown in Fig. 5a. The origin-containing plasmid DNA is nicked quickly (within 1 min of incubation; Fig. 5a, third lane) and converted to the linear form more slowly (complete conversion after 1 h; Fig. 5a, seventh lane). These nicked molecules are true intermediates since they are convertedtothelinear formwhen theyare purified and subsequently re-incubated with gpII (data not shown). Mapping of the nick (Fig. 3, lanes I, 3, 5, and 7) shows it tobe a t aunique site on the (+)-strand, the nicking site, between nucleotides -1 and +l. Only plasmids containing fl origins are substrates. pBR322 is not a substrate for either nicking ordouble-strand cleavage (datanotshown).The enzyme cannot use a random nick. When fl origin-containingplasmid DNA is nicked randomly with DNase I, it can be converted to the linear form upon incubation with gpII; however, the double-strand cleavage occurs at the origin, and the random nick remains as a single-strand break (data not shown). Cleavage in the Presence of Mn2+Does Not Require Superhelicity-Since molecules nicked at sites other than thegpII nicking site can serve as substrates for Mn2+-dependentdou-

Strand J o i n i n g by the f l Replication Initiator Protein

12630

ble-strand cleavage (see above), the reaction does not require were not cleaved (data notshown). This means that the initial superhelicity. This was confirmed further by digestion of a nicking reactiondoes not requiresuperhelicity inthe presence restriction fragment containing the fl origin (Fig. 5b). The of Mn2+. 151-bp origin fragment from pDG117 was cleaved to an 88Mn2+ Reduces the DNA Sequence Requirement for Nickbp anda 63-bp fragment.Fragments not containingfl origins ing-The DNAsequence requirements for nickinginthe presence of Mg2+ have been found to extendfrom 4base pairs a b before the nicking site to28 base pairs downstream (Dottoet al., 1982a, 1982b, 1984). This sequence contains repeatsj3 and y and most of repeat 6 (see Fig. 1). The DNAsequence requirements for the Mn2+-dependent double-strand cleavage reaction have not been determined. Therefore, we analyzed the Mn2+-dependentcleavage reaction using mutant origins as substrates. Surprisingly,some mutants that lack the nick+nicked ~ 8 bp 8 ing site were found to be substrates for gpII in the presence c linear "circular of Mn2+, although the product of the reaction was RFII instead ~ 6bp3 of RFIII (Fig. 6). Themutant pD30was constructed by insertion of a 10-bp linkerat thefilled-in HinfI site a t position +8 (Dotto et al., 1984). As a consequence of the insertion, the FIG. 5. Mn2+-dependent double-strand cleavage activity. a, from time course of the cleavage reaction. Purified gene I1 protein (12.5 wild-type nicking sequenceis positioned 13 bp upstream ng) was incubated with fl RFI (250 ng) in 20 pl containing 20 mM its normal position. DNase I footprinting experiments indiTris-CI (pH 8.0), 80 mM KCI, 5 mM MnCIZ,and 5 mM dithiothreitol. cate that the pD30 origin binds gpII and yields the 40-bp The reactions were incubated for various lengths of time a t 30 "C as protection patternlike the wild-type origin: the inserted linker follows for the first through seuenth lanes, respectively: no incubation, sequence is protected,whereas the displaced nicking sequence 30 s, 1 min, 5 min, 10 min,30 min, and 1 h. Following the incubation, is not (Greenstein and Horiuchi, 1987). This binding is not the reactions were stopped by addition of 1 pl of stop mixture, and the products were analyzed by electrophoresis on a 0.6% agarose gel functional because the pD30 origin cannot be nicked by gpII, containing ethidium bromide (0.5 pg/ml). b, cleavage of a restriction and the origin is defective for initiation (Dotto et al., 1984). fragment.The 151-bp BamHI-EcoRI origin-containingrestriction As shown in Fig. 6 (upper),the pD30 origin is nicked by gpII fragment from pDG117 (approximately 10 fmol) was incubated with in the presence of Mn2+. Since pD30 contains a wild-type gene I1 protein (approximately25 ng) under Mn2+cleavage conditions nicking sequence, which is nevertheless displaced from its (see above) for 1 h. The restriction fragment hadbeen 5"end-labeled we deterby treatment with (yR2P]ATP and T4 polynucleotide kinase follow- normal position with respect to the binding repeats, ing treatment withcalf intestinal alkaline phosphatase. The cleavage mined the position of the nickproduced by gpIIin the products were analyzed onan 8% polyacrylamide gel and detectedby presence of Mn2+. The nick was mapped in a similar way as autoradiography. depicted in Fig. 3. The results (summarized in Fig. 6 (lower)) indicate thatpD30 is nicked at a single site that corresponds to the normal position of the nick relative to the binding pD30 p029 repeats: the displaced nicking sequence is not nicked. In the case of two deletions that extend from the left past the nicking site (pD29 and A83), gpII nicked the defective origins in the presence of Mn2+ (Fig. 6 (upper) and data not shown). For pD29, the RFII molecules were a mixture, containing one of four closely spaced nicks on the (+)-strand in the region where the nicking would occur in the wild-type origin (data summarized inFig. 6 (lower)).The major site of nicking corresponded to theposition of the wild-type nick in t+l "0 relation to the binding sequence. The nick for the mutant -10 "0 *I? Q? A83 was not located. ~ ' ~ ~ ~ M T ~ c K u c mC C A T C T U X TT C T A T 7 - ~1 ' wt ~ " ~ c ~ ~ ~ ~ ~ ~ ~ u c ~ ~ The three binding repeats p, y, and 6 are needed for the $'-CgtTCtttu-