substrate specificity of hela endonuclease r

10
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 265, No. 19, Issue of July 5, pp. 1084%10850,199O Printed in U.S.A. Substrate Specificity of HeLa Endonuclease R A G-SPECIFIC MAMMALIAN ENDONUCLEASE* (Received for publication, September 29, 1989) Jonathan Gottliebg and Nicholas Muzyczkag From the Department of Microbiology, State University of New York, Stony Brook Medical School, Stony Brook, New York 11794 We examined the substrate specificity of endonucle- ase R (endo R) a mammalian endonuclease that cleaves G-C-rich DNA sequences. The best substrates for dou- ble-stranded cleavage were homopolymeric stretches of poly(dG)*poly(dC). Plasmids which contain other G- rich sequences were also cleaved but at a reduced frequency. These included the telomeric sequences, d(G4T2) and d(GlmaA), which were cleaved at approxi- mately one-third the frequency of d(G),*d(C),. The alternating copolymer d(GA) and the terminal se- quences of adeno-associated virus d(GlmsT/A) were also cut. Poly(dA)*poly(dT) and the alternating copolymer d(GC), were not detectably cleaved. Although endo R has a nicking activity which converts supercoiled plas- mids to nicked circular DNA, the nicking activity is random with respect to plasmid sequences. Specific cleavage of G-rich sequences appears to occur by a concerted double-stranded mechanism. The cleavage pattern within the G-rich runs suggests that cleavage can occur anywhere within the G-rich region. Product ligation experiments indicate that a limited number of cleavage events (l-2) occur/molecule. Inasmuch as the best substrates for endo R are d(G),*d(C), and telo- merit sequences, we suggest that endo R may directly recognize and cleave DNA that contains G* G base pair- ing. Eukaryotic genomes contain a disproportionate number of sequences which are rich in G.C base pairs (Behe, 1987; Beasty and Behe, 1988). These exist either as single motifs (for review, see Wells et al., 1988) or as moderately repeated tandem arrays (Jeffreys et al., 1985; Nakamura et al., 1987; Hoffmann-Liebermann et al., 1986). They are suspected of being involved in several biological functions, including im- munoglobulin rearrangement (Nikaido et al., 1982), gene expression (Cooney et al., 1988; Hentschel, 1982; Larsen and Weintraub, 1982; Htun et al., 1984, 1985; Yu and Manley, 1986; Kilpatrick et al., 1986), DNA recombination (Mokarski and Roizman, 1981; Wohlrab et al., 1987; Weinrab et al., 1988; Konopka, 1988; Nakamura et al., 1987; Gottlieb and Muzy- czka, 1988), chromosome alignment or segregation (Sen and *This work was supported by Program Project Grant 5 PO1 CA2814607 from the National Cancer Institute and Grant ROlGM3572302 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported in part during the course of this work by National Institutes of Health Training Grant 5 T32 AI07110 to the Department of Microbiology at the University of Florida. § To whom correspondence should be addressed. Gilbert, 1988), and the maintenance of chromosomes ends (Blackburn and Szostak, 1984). At the moment the only clear evidence for the involvement of G-rich sequences in a cellular function is in their role as telomere sequences. In addition to their possible biological functions, G-rich sequences are unusual in that they are capable of adopting non-B, non-Z secondary structures either as linear homopol- ymers or when they are present in supercoiled plasmid DNA (Arnott et al., 1983; Henderson et al., 1987; Evans and Efstra- tiadis, 1986; Lyamichev et al., 1986; Sen and Gilbert, 1988; McCall and Brown, 1985; Pulleyblank et al., 1985; Rajagopol and Feigon, 1989; Sundquist and Klug, 1989; Williamson et al., 1989). Several laboratories have isolated G-specific mam- malian DNA endonucleases which specifically cleave runs of poly(dG) .poly(dC) (Ruiz-Carillo and Renaud, 1987; Low et al., 1987; Gottlieb and Muzyczka, 1988). In a companion report we described the purification of one of these endonu- cleases, endo R,’ to near homogeneity (Gottlieb and Muzy- czka, 1990). In this report we continue our characterization of endo R by describing its substrate specificity. We suggest that endo R recognizes and specifically cleaves DNA that contains G. G base pairing. EXPERIMENTAL PROCEDURES Cell Culture, Viruses, and Purification of Endo R-HeLa S3 cells were maintained at 37 “C in suspension culture in Eagle’s minimal essential medium supplemented with 5% calf serum, 1% glutamine, penicillin, and streptomycin. Wild-type adenovirus 2 was prepared from a freeze-thaw lysate of HeLa S3 cells as previously described (Samulski et al., 1983). Endo R was purified from adenovirus-infected HeLa spinner cells as described (Gottlieb and Muzyczka, 1990). Unless otherwise indicated, fraction V was used. Recombinant Clones and Enzyme Substrates-All recombinant clones were maintained in either the recA host HBlOl (Boyer, 1969) or the recBC, recF, sbcB host JC8111 (Boissy and Astell, 1985), as described (Gottlieb and Muzvczka, 1990). The AAV wild-t.ype plasmid pSM620 (Fig. 1) contains a full copy ofthe AAV genome-i&xted by G. C tailing into the PstI site of pBR322. The plasmid contains 19 bp of poly(dG) .poly(dC) [(dG .dC).] at the left pBR322/AAV junction and 29 bp of G. C at the right junction (Samulski et al., 1983; Gottlieb and Muzyczka, 1988). This includes one G. C bp which forms part of the PstI site and 2 G. C bp of the AAV-terminal sequence which flank the G. C tail. The plasmid pGM620D contains the right terminal PstI fragment of the wild-type AAV plasmid, pSM620, subcloned into the PstI site of pBR322 and contains the parental 29 bp of poly(dG)+ polv(dC) tail present at the right AAV/vector junction of pSM620 iG&tlieb and- Muzyczka, 1988). The plasmid bGMlOO8 was con- structed bv removing the PstI-BssHII frament (AAV nucleotides 4260-4658) from pGG620D and religating t<e parental molecule after producing blunt ends with the Klenow fragment of DNA polymerase I. The plasmid contains the 31-bp poly(dG) .poly(dC) tail plus the terminal 18 bp of the terminal AAV sequence, nucleotides 4659-4676. This plasmid had previously been reported to contain a 29 bp run of ’ The abbreviations used are: endo R, endonuclease R; AAV, adeno- associated virus; bp, base pair; kbp, kilobase pair. 10842 by guest on April 7, 2018 http://www.jbc.org/ Downloaded from

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Page 1: Substrate Specificity of HeLa Endonuclease R

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 19, Issue of July 5, pp. 1084%10850,199O Printed in U.S.A.

Substrate Specificity of HeLa Endonuclease R A G-SPECIFIC MAMMALIAN ENDONUCLEASE*

(Received for publication, September 29, 1989)

Jonathan Gottliebg and Nicholas Muzyczkag From the Department of Microbiology, State University of New York, Stony Brook Medical School, Stony Brook, New York 11794

We examined the substrate specificity of endonucle- ase R (endo R) a mammalian endonuclease that cleaves G-C-rich DNA sequences. The best substrates for dou- ble-stranded cleavage were homopolymeric stretches of poly(dG)*poly(dC). Plasmids which contain other G- rich sequences were also cleaved but at a reduced frequency. These included the telomeric sequences, d(G4T2) and d(GlmaA), which were cleaved at approxi- mately one-third the frequency of d(G),*d(C),. The alternating copolymer d(GA) and the terminal se- quences of adeno-associated virus d(GlmsT/A) were also cut. Poly(dA)*poly(dT) and the alternating copolymer d(GC), were not detectably cleaved. Although endo R has a nicking activity which converts supercoiled plas- mids to nicked circular DNA, the nicking activity is random with respect to plasmid sequences. Specific cleavage of G-rich sequences appears to occur by a concerted double-stranded mechanism. The cleavage pattern within the G-rich runs suggests that cleavage can occur anywhere within the G-rich region. Product ligation experiments indicate that a limited number of cleavage events (l-2) occur/molecule. Inasmuch as the best substrates for endo R are d(G),*d(C), and telo- merit sequences, we suggest that endo R may directly recognize and cleave DNA that contains G* G base pair- ing.

Eukaryotic genomes contain a disproportionate number of sequences which are rich in G.C base pairs (Behe, 1987; Beasty and Behe, 1988). These exist either as single motifs (for review, see Wells et al., 1988) or as moderately repeated tandem arrays (Jeffreys et al., 1985; Nakamura et al., 1987; Hoffmann-Liebermann et al., 1986). They are suspected of being involved in several biological functions, including im- munoglobulin rearrangement (Nikaido et al., 1982), gene expression (Cooney et al., 1988; Hentschel, 1982; Larsen and Weintraub, 1982; Htun et al., 1984, 1985; Yu and Manley, 1986; Kilpatrick et al., 1986), DNA recombination (Mokarski and Roizman, 1981; Wohlrab et al., 1987; Weinrab et al., 1988; Konopka, 1988; Nakamura et al., 1987; Gottlieb and Muzy- czka, 1988), chromosome alignment or segregation (Sen and

*This work was supported by Program Project Grant 5 PO1 CA2814607 from the National Cancer Institute and Grant ROlGM3572302 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported in part during the course of this work by National Institutes of Health Training Grant 5 T32 AI07110 to the Department of Microbiology at the University of Florida.

§ To whom correspondence should be addressed.

Gilbert, 1988), and the maintenance of chromosomes ends (Blackburn and Szostak, 1984). At the moment the only clear evidence for the involvement of G-rich sequences in a cellular function is in their role as telomere sequences.

In addition to their possible biological functions, G-rich sequences are unusual in that they are capable of adopting non-B, non-Z secondary structures either as linear homopol- ymers or when they are present in supercoiled plasmid DNA (Arnott et al., 1983; Henderson et al., 1987; Evans and Efstra- tiadis, 1986; Lyamichev et al., 1986; Sen and Gilbert, 1988; McCall and Brown, 1985; Pulleyblank et al., 1985; Rajagopol and Feigon, 1989; Sundquist and Klug, 1989; Williamson et al., 1989). Several laboratories have isolated G-specific mam- malian DNA endonucleases which specifically cleave runs of poly(dG) .poly(dC) (Ruiz-Carillo and Renaud, 1987; Low et al., 1987; Gottlieb and Muzyczka, 1988). In a companion report we described the purification of one of these endonu- cleases, endo R,’ to near homogeneity (Gottlieb and Muzy- czka, 1990). In this report we continue our characterization of endo R by describing its substrate specificity. We suggest that endo R recognizes and specifically cleaves DNA that contains G. G base pairing.

EXPERIMENTAL PROCEDURES

Cell Culture, Viruses, and Purification of Endo R-HeLa S3 cells were maintained at 37 “C in suspension culture in Eagle’s minimal essential medium supplemented with 5% calf serum, 1% glutamine, penicillin, and streptomycin. Wild-type adenovirus 2 was prepared from a freeze-thaw lysate of HeLa S3 cells as previously described (Samulski et al., 1983). Endo R was purified from adenovirus-infected HeLa spinner cells as described (Gottlieb and Muzyczka, 1990). Unless otherwise indicated, fraction V was used.

Recombinant Clones and Enzyme Substrates-All recombinant clones were maintained in either the recA host HBlOl (Boyer, 1969) or the recBC, recF, sbcB host JC8111 (Boissy and Astell, 1985), as described (Gottlieb and Muzvczka, 1990). The AAV wild-t.ype plasmid pSM620 (Fig. 1) contains a full copy ofthe AAV genome-i&xted by G. C tailing into the PstI site of pBR322. The plasmid contains 19 bp of poly(dG) .poly(dC) [(dG .dC).] at the left pBR322/AAV junction and 29 bp of G. C at the right junction (Samulski et al., 1983; Gottlieb and Muzyczka, 1988). This includes one G. C bp which forms part of the PstI site and 2 G. C bp of the AAV-terminal sequence which flank the G. C tail. The plasmid pGM620D contains the right terminal PstI fragment of the wild-type AAV plasmid, pSM620, subcloned into the PstI site of pBR322 and contains the parental 29 bp of poly(dG)+ polv(dC) tail present at the right AAV/vector junction of pSM620 iG&tlieb and- Muzyczka, 1988). The plasmid bGMlOO8 was con- structed bv removing the PstI-BssHII frament (AAV nucleotides 4260-4658) from pGG620D and religating t<e parental molecule after producing blunt ends with the Klenow fragment of DNA polymerase I. The plasmid contains the 31-bp poly(dG) .poly(dC) tail plus the terminal 18 bp of the terminal AAV sequence, nucleotides 4659-4676. This plasmid had previously been reported to contain a 29 bp run of

’ The abbreviations used are: endo R, endonuclease R; AAV, adeno- associated virus; bp, base pair; kbp, kilobase pair.

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G-Specific Mammalian Endonuclease 10843

P R, TABLE I

P-

3 1 s 13

l I I .I. 6

Frequency of cleavage of G-rich sequences

Clone Inserted sequence” Frequency

pGM1008’ (C)31 100 pGMGC13* (C)l, 30 pGMGCS* (09 20

4 R pGA38 (CAhs 10 pGA14 (Wu 3 PGMC4A2 (G&h 30 pGMC4T (Cs.sT)s 30

GGCCACTCCCTCTCTGCC ; 0.8

pGMAAVter pGMAAVter2’ (TGGCCACTCCCTCTCTGCCh k pBR322 *vd (Gh 0 pGMaltGC (GCh 0 pEV136 Wss 0

’ The construction of the clones is described in detail under “Ex- perimental Procedures.” The sequences are listed as they appear in the clone on the clockwise strand in a 5’ to 3’ direction. The frequency of cleavage is defined as the percent of starting substrate cleaved in a standard assay with 1 unit of endo R. The amount of cleavage for pGM1008 was arbitrarily set to 100%. The complete sequences of the inserts are listed in Fig. 6.

6.1 , u 30 I

*Frequencies of these clones were reported previously for a less pure preparation of endo R (Gottlieb and Muzyczka, 1988). They did not change when we used fraction V endo R and they are included here for comparison.

‘The sequences for pGMAAVter2 are repeated in an inverted orientation.

-1;: 4.4 30, ’ Natural poly(dG) stretches of 6 bp occur in pBR322 at nucleotides

2550 and 2197.

FIG. 1. Predicted endo R cleavage fragments of clones con- taining G-rich inserts. Top right, SphI digestion of endo R-cleaved substrate should produce 1.3- and 3.1-kbp fragments from pGM clones containing oligomer inserts in the PstI site of pBR322 (all clones except PGMC4A2). EcoRI digestion should produce 3.6- and 0.8-kbp fragments. Top left, PstI digestion of PGMC4A2, which contains an insert in the EagI site of pBR322 should produce 1.7- and 2.7-kilobase fragments. S = SphI site, P = PstI site, E = EcoRI,R = endo R site, filled square = oligonucleotide insert. See “Experimen- tal Procedures” for further descriptions of the clones and Table I for the sequence and frequency of cleavage of each. Bottom, digestion of pSM620 with BstEII (B) and endo R at one or both of the endo R sites will produce the indicated fragments.

plasmids were confirmed by DNA sequencing. The plasmid pGA38, a gift from Todd Evans (Columbia Univer-

sity), contains an insert of the alternating co-polymer d(GAh8, cloned into the EcoRI site of pUC9 (Evans and Efstratiadis, 1986). pGA14 is a subclone of pGA38 and contains an insert of d(GA),, cloned into the EcoRI site of pUC9. The plasmid pEV136, a gift from Eckhard Wimmer, is an infectious polio virus plasmid. It contains the complete polio virus cDNA sequence inserted at an EcoRI site. One of the two polio/vector junctions contains 18 bp G C; the other contains an 84 bp stretch of poly(dA):poly(dT) (Semler et al., 1984).

G.C (Gottlieb and Muzyczka, 1988), but the run of G.C in the plasmid DNA used in this study and in the companion study (Gottlieb and Muzyczka, 1990), was found to be 31 bp long. This was presum- ably due to an expansion of the G. C sequence during propagation in HBlOl (Hauswirth et al., 1984).

Endo R Cleavage Assays and Nuclease Assays-The standard endo R cleavage reaction was as described (Gottlieb and Muzyczka, 1990). Reactions (25 ~1) containing 0.2 pmol of plasmid DNA (0.5 pg of pGM1008) and 1-5 units of fraction V endo R were incubated at 37 “C for 1 h.

The plasmids pGMGC13, pGMGC9, pGMC4T, pGMaltGC, pGMAAVter, and pGMAAVter2 were constructed by inserting chem- ically synthesized oligonucleotides (Systec) into the PstI site of pBR322 (Fig. 1, pGM PstI series, Table I). The 5’ ends of the single- stranded oligomers were phosphorylated prior to ligation only when multiple tandem copies of the insert were desired. Positive clones were selected on the basis of their tetracycline resistance and ampi- cillin sensitivity. The plasmids pGMGC13 and pGMGC9 were pre- viously described (Gottlieb and Muzyczka, 1988) and contain 13 and 9 base pairs of poly(G) .poly(C) homopolymer, respectively. pGMaltGC contains 30 bp of alternating copolymer (GC), and pGMC4T contains the Dictyostelium telomeric repeat sequence (G2. 6Ah (Shampay et al., 1984). Two plasmids contain a portion of the terminal 19 bp of AAV (TTGGCCACTCCCTCTCTGC), in either a monomer form (pGMAAVter) or as an inverted dimer (pGMAAter2). The exact sequences of the inserts are indicated in Table I and Fig. 6. In each case, both strands of the oligonucleotides were synthesized so that the PstI sites flanking the inserts were maintained after the two strands of each oligonucleotide were annealed and ligated into pBR322. The AAV sequence has the general form (G1.3A/r).

The plasmid pGMC4A2 contains three copies of the Z’etrahymena telomeric sequence, (G,T,), (Blackburn and Szostak, 1984), which were chemically synthesized and inserted into the EagI site of pBR322. As in the case of the pGM PstI series, a duplex oligonucle- otide fragment was inserted so as to maintain the flanking EagI sites. The inserts and pBR322 flanking sequences of all of the constructed

DNA Labeling, Sequencing, and Primer Extension-For primer extension of endo R products, 2 pg of plasmid DNA were incubated with 4 units of endo R in a standard reaction mixture of 50 ~1 at 37 “C for 1 h. Following electrophoresis of the reaction mixtures, the form III product was isolated from a 1.2% low melting agarose gel and dissolved in 10 ~1 of water and 4 ~1 of 5 x reaction buffer (0.3 M Tris-HCl, pH 8.3, 0.375 M NaCl, 37.5 mM MgCl,, and 2.5 mM dithiothreitol). The reaction mixtures were divided into two 7-~1 portions, and 1 ~1 of either upper or lower strand pBR322/PstI primers (2 pmol/pl) was added to each portion. The solutions were heated to 100 “C for 5 min under paraffin oil and immediately frozen in a dry ice/ethanol bath. After thawing on ice, each reaction received 1 ~1 of a stock solution containing 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM TTP, 0.02 mM dATP, 1~1 of [a-32P]dATP (3000 Ci/mM, 10 &i/ rl), and 1 ~1 of avian myeloblastosis virus reverse transcriptase (10 units/J, IBI). After incubation at 42 “C for 10 min, 2 ~1 of chase solution (0.25 mM each of dGTP, dATP, dCTP, and TTP) were added, and incubation was continued at 42 “C for an additional 10 min. The reaction was terminated with the addition of 7 ~1 of stop buffer containing 1.6 pl of 0.25 M EDTA, 2 pl of 3 M sodium acetate, 0.2 ~1 of 2 mg/ml tRNA, and 3.2 ~1 of water. After ethanol precipi- tation with 3 volumes of 95% ethanol, the primer extension products were redissolved in 5 ~1 of sequence gel running buffer (Maxam and Gilbert, 1977) and heated to 100 “C for 3 min prior to gel electropbo- resis. The sequence at the site of endo R cleavage was determined by comparing the mobility of the primer extension products with dideoxy sequencing ladders produced using the same single-stranded primers with plasmid DNA that had not been cut with endo R. The primer which was used to sequence in the counter-clockwise direction on the plasmid was GGAGCTGAATGAAGCC. (In the case of pGMC4A2

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10844 G-Specific Mammalian Endonuclease

the primer was CATCCAGCCTCGCGTCG.) Because of the orien- tation of the inserts, this primer generally extended the G-rich strand and provided information about the positions of the cuts on the complementary C-rich strand of the plasmid. (See Table I for the orientation of each insert). The primer used for the clockwise direc- tion which was used to determine the cut sites on the G-rich strand was GCTAGAGTAAGTAGTT (or AGCCTTCGTCACTGGTCC in the case of pGMC4A2).

Gel Electrophoresis-Denaturing polyacrylamide-urea sequencing gels were as described (Maxam and Gilbert, 1977). All sequencing gels were run at 55-60 “C to prevent compression of palindromic sequences (Lusby et al., 1980). For denaturing agarose gel electropho- resis, 1.5% horizontal agarose gels were poured in 50 mM NaCl and allowed to equilibrate in excess running buffer (30 mM NaOH, 1 mM EDTA) for at least 1 h. Samples were incubated for 5 min in gel running buffer containing 0.2 N NaOH prior to electrophoresis at 30 volts for 24 h.

RESULTS

Substrate Specificity for Double-stranded Endo R Cleav- age-In previous work we demonstrated that endo R would produce double-stranded cuts in plasmids that contained runs of poly(dG) +poly(dC). The best substrate (Gottlieb and Mu- zyczka, 1988), which was used for the purification of endo R (Gottlieb and Muzyczka, 1990), was pGM1008. This plasmid contains a 31 bp stretch of G.C inserted into the PstI site of pBR322. When we tested plasmids which contained shorter stretches of G. C with endo R fraction V, we found that they displayed a lower frequency of specific cleavage which was essentially proportional to the decrease in length of the G. C sequence. The substrate pGMGC13 (13 bp of G.C) was cleaved at 30% and pGMGC9 (9 bp of G+C) was cleaved at 20% of the level observed with pGM1008 (Fig. 2, Table I). The relative frequencies of cleavage for these three plasmids is the same as that reported previously for less pure prepara- tions of endo R (Gottlieb and Muzyczka, 1988). As before the length of the G.C sequence in pGMGC9 appears to be the limit of enzyme recognition. We saw no detectable cleavage of the naturally occurring stretches of poly(dG)e in pBR322 (Fig. 1, Table I) and (dG)7 in X-bacteriophage DNA (not

1.7 - b J

1.3 - : b4 I

FIG. 2. Endo R cleavage of pGM clones containing G-rich sequences. Standard reaction mixtures of 25 ~1, containing 0.4 pmol of the indicated pGM clones, were digested with 1 unit of endo R at 37 “C for 1 h. The reactions were terminated with the addition of proteinase K stop solution (see “Experimental Procedures”), phenol extracted, ethanol precipitated and digested with either SphI (all clones except pGMC4A2) or PstI (pGMC4A2). After electrophoresis on a neutral 1.4% agarose gel, the products were stained with ethidium bromide. See the legend to Fig. 1 for the expected fragment sizes and Table I for the endo R target sequence in each clone. The marker lane (M) contains a Pat1 digest of pSM620 which produces fragments of 4.4,2.4, 1.5,0.51, and 0.45 kbp in length; the two smaller fragments were run off the sel.

shown) when we used endo R fraction V. These results suggest that a minimum of 8 or 9 G residues are necessary for site- specific double-stranded cleavage by endo R and that the frequency of cleavage increases in direct proportion to the length of the homopolymer chain.

We also reported previously that some sequences which were not homogeneous runs of G. C could be cleaved by endo R (Gottlieb and Muzyczka, 1988). To determine systemati- cally what types of substitutions in the poly(dG) chain were tolerated, we obtained or constructed plasmid substrates con- taining a variety of repeating polymeric sequences and tested them for their ability to be cleaved by endo R. The design of these substrates is shown in Figs. 1 and 3 and the sequence of the inserts and cleavage frequencies are listed in Table I. The first of these was the plasmid pEV136, an infectious polio clone which contains 18 bp of dG .dC at the 5’ end and an 84-bp stretch of dA. dT at the 3’ end of the polio virus cDNA sequences inserted in the plasmid (Semler et al., 1984). When incubated with endo R and BglII, pEV136 produced two 5.7- kbp fragments of identical size which were the result of cleavage exclusively at the poly(dG) site; no detectable cleav- age was observed at the dA. dT site (Fig. 3). We also tested plasmids that contained an insert of the alternating co-poly- mer d(GA)38 or d(GA)l., (pGA38 and pGA14, respectively; Evans and Efstratiadis, 1986). These were cleaved by endo R at approximately one-tenth the frequency of homogeneous G. C runs of comparable size (Table I and Fig. 3). Thus, homo-

pGA38

R RS M

-2.7 -

Fml- w bn* -1 8

--.94

18

pEVl36

M R RB

m -Fm2

-Fm3 -57

2.4- -

1.5- -

- ((3,

FIG. 3. Endo R cleavage of pGA38 and pEV136. Top, 1 ,ug of either pGA38 or pEV136 was incubated with 1 unit of fraction V endo R under standard reaction conditions in a volume of 25 pl. After phenol extraction and ethanol precipitation, half of each reaction was further digested with either ScaI (pGA38) or BglII @EV136) and both the undigested (R) and the restriction enzyme digested, endo R- treated products (RS and RB) were fractionated on 1.4% agarose gels and stained with ethidium bromide. Bottom, restriction maps of pGA38 and pEV136. Filled boxes represent the poly(GA) insert for pGA38 (GA), and the poly(dG) and poly(dA) inserts for pEV136 t(G) n and (A),, respectively). Numbers indicate the size of the prod- ucts (kbp) that would be expected from a restriction enzyme digest of endo R products cleaved at (GA), in pGA38, and at (G), plus (A), in pEV136. The size markers (M) were the same as those described for Fig. 2.

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G-Specific Mammalian Endonuclease

polymers of (dA) were not recognized as substrates for cleav- age, but substitutions of (dA) in stretches of poly(dG) were tolerated and resulted in a decrease in cleavage activity. This was not the case with alternating polymers of GC. A plasmid which contained an insert of poly d(GC)SO, pGMaltGC, was not detectably cleaved by endo R (Fig. 2, pGMaltG-C, Table I).

More complex sequences with less symmetrical substitu- tions of the poly(dG) sequence were also substrates for endo R. This was demonstrated by two clones, pGMC4A2 and pGMC4T, that contain generic forms of the Tetrahymena and Dictyostelium telomeric sequences, respectively (Blackburn and Szostak, 1984; Shampay et al., 1984). Both the Tetrahy- menu sequence d(G4T,). d(&A,) and the Dictyostelium se- quence d(G2.6A). d(C&T) were cleaved at approximately one- third the frequency of homogeneous G. C stretches of similar length, e.g. pGM1008 (Fig. 2, Table I). We also constructed two clones (pGMAAVter and pGMAAVter2) which contained portions of the terminal 19-bp sequence of AAV. This se- quence has’the general form d(Gl.3A/r) and had appeared to be a minor substrate for endo R (Gottlieb and Muzyczka, 1988). The plasmid pGMAAVter contains a single copy of this sequence and pGMAAVter2 contains two inverted copies that have been inserted into the PstI site of pBR322. Both of these constructs were cleaved by endo R at relatively low frequencies (Fig. 2). As expected, the frequency of cleavage observed with pGMAAVter was approximately 1% of that observed with pGM1008 (Gottlieb and Muzyczka, 1988). The clone which contained an inverted dimer of the AAV recog- nition sequence was cleaved at a 5-fold higher frequency (Fig. 2 and Table I).

Endo R Rapidly Converts Supercoiled Plasmids to Nicked Circular DNA-Analysis of the endo R products at several time points during the digestion of pSM620 (Fig. 4) revealed that supercoiled input plasmid DNA was quickly converted to the nicked circular plasmid form. Virtually all of the input supercoiled DNA (Fig. 4, A and C, Fml) disappeared within 8 min, well before significant amounts of linear molecules (Fm.3) had accumulated. Linear plasmid molecules produced by one double-stranded cut were observed as early as 3 min into the reaction, while the production of AAV and pBR322 linears (Fig. 4A, AP), which requires double-stranded breaks at both of the AAV/pBR junctions within the plasmid, were detected only after incubation for 15 min. When the products of the endo R reaction were digested with BstEII (Fig. 4B) it appeared that the accumulation of linear plasmid DNA (as well as free AAV and pBR322 DNA) were due to specific cleavage at the AAV/pBR322 junctions and correlated well with the decrease in form II molecules (Fig. 4C). This sug- gested that double-stranded cleavage might proceed through a nicked circular intermediate as an obligatory step and that form II molecules are produced by site-specific nicking of the input plasmid. This would mean that endo R acted by a mechanism similar to that of other G-specific endonucleases, including endonuclease Sl (Schon et al., 1983; Hanvey et al., 1988a), the mitochondrial endonuclease (Low et al., 1987; Cummings et al., 1987), and endo G (Cote et al., 1988). In support of this, endo R also has significant activity on single- stranded DNA. As reported earlier (Gottlieb and Muzyczka, 1988), we were unable to demonstrate the production of acid- soluble products when endo R fraction V was incubated with single-stranded circular Ml3 DNA. However, under standard reaction conditions single-stranded Ml3 DNA was digested to oligonucleotides of 200-400 base pairs in length (not shown). This occurred in the time required to specifically cleave 50% of an equivalent amount of duplex substrate.

C

100, 4 3K - l - s 75. 1’ l L\ .

0s is Fml 0 30 60 90 120 150

Minutes FIG. 4. Time course of cleavage reaction. Two pmol of super-

coiled pSM620 plasmid DNA (Fml) was incubated with 20 units of fraction V enzyme at 37 “C in a reaction volume of 0.5 ml. Aliquots of the reaction (50 ~1) were removed at the indicated times and the reactions were terminated with the addition of an equal volume of stop solution (see “Experimental Procedures”), phenol extracted, and ethanol precipitated. Half of each time point was digested with BstEII and both the uncut (panel A) and BstEII digested (panel B) samples were fractionated on a 1.4% agarose gel. Fm.3 = linear plasmid, A and P are linear monomer AAV and pBR322 molecules, respectively. See Fig. 1 for the origin of the B&E11 and endo R digestion fragments. Panel C, the conversion of the undigested plasmid forms (Fm.2 and Fm2 in panel A) to linear endo R products (Fm3) are plotted as a function of time. The appearance of the 3.0-kilobase (3K) fragment from BstEII digestion of the endo R products was used as a measure of the level of specific cleavage of the supercoiled substrate. Values are plotted as a percent of the total starting DNA which was deter- mined by densitometer measurements of the photographic negative. In the case of the 3.0-kbp fragment, the level of cleavage at 150 min was arbitrarily assigned a value of 100%.

Several observations indicated that the nicking activity on double- and single-stranded substrates and the G-specific double-stranded cleavage activity were due to the same en- zyme. First, the nicking and G-specific cleavage activities copurified during the last two purification steps (gel filtration and poly G-agarose), and the ratio of the activities in the enzyme obtained from these two steps (fractions IV and V) was the same. Second, during gel filtration and poly G-agarose chromatography, assays across the peaks of double-stranded nicking activity and G-specific endonuclease activity indi- cated that the peaks were coincident (not shown). Finally, attempts to separate the nicking activities from the site- specific cleavage activity by further fractionation of fraction V enzyme were unsuccessful and resulted in the loss of all nucleolytic activity. Thus, it appeared that endo R had at least three activities. It nicked double-stranded substrates, cut single-stranded DNA, and specifically cleaved sequences rich in G .C base pairs within duplex DNA. We estimated that endo R can generate 10 random double-stranded nicks or single-stranded cuts/l specific G. C cleavage event.

Nicking of Duplex DNA Is Random; Only the Double-

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stranded Cleavage Activity Is Specific for G-rich Sequemes- Acting on the assumption that the nicks produced early in the endo R reaction were specifically made within runs of G. C, we isolated form II circular molecules produced within the first 3 min of the reaction and tried to sequence the position of the nicks. During this time, the bulk of the supercoiled plasmid substrate had been converted to form II DNA (Fig. 4, A and C). This strategy for determining endo R sequence specificity is similar to the one first used by Schon et al. (1983) for mapping the cuts made by Sl endonuclease within G-rich sequences. However, in spite of repeated attempts, we could not demonstrate that any specific nicks had been made within the poly(dG) .poly(dC) sequence of form II pGM1008 DNA within the first 3 min (not shown). When we repeated this experiment as a time course with unfractionated endo R products (i.e. a mixture of fm1, fmI1, and fmII1 products), we did not observe G-specific cuts by primer extension until 1.5 min into the reaction, approximately the time (see Fig. 4, A and C) when the first endo R cleaved linear molecules ap- peared (not shown). We were forced to conclude that the nicking activity of endo R on duplex substrates was random and that only the double-stranded cleavage reaction was spe- cific for runs of G.C. This was confirmed by the results of the experiment shown in Fig. 5. The plasmid pGM1008 was incubated with endo R, and the products of the reaction were analyzed at several time points by fractionation on both neutral and denaturing agarose gels (Fig. 5, A and B, respec- tively). Digestion of a specifically cleaved form II intermediate

A

Fml-

B 0 5 153060M

4.4 - II) I, - 3.6 M

m- 0.8

FIG. 5. Specificity of endo R nicking activity. A reaction mixture of 500 ~1 containing 1.0 pmol of pGM1008 in standard reaction buffer was incubated with 20 units of endo R (fraction V) at 37 “C. At the indicated times, 50-~1 aliquots were removed and treated as described for Fig. 4. Half of each time point was digested with EcoRI, denatured with the addition of 0.1 volume of 2 N NaOH, and fractionated on a 1.5% denaturing agarose gel (panel B), as described under “Experimental Procedures.” Time points not digested with EcoRI were fractionated on a 1.0% non-denaturing agarose gel (panel A). The lane marked M in panel B contains pGM1008 that has been digested with PstI and EcoRI to produce the 0.8- and 3.6-kbp marker fragments (see Fig. 1).

with a one-cut restriction enzyme would yield nicked form III molecules, which would be indistinguishable from unreacted substrate on native agarose gels, but would produce a discrete band (0.8 kbp) on a denaturing agarose gel. Nicked circular plasmid was the predominant product early in the reaction (Fig. 5A) and should result in the early accumulation of single- stranded fragments that were the result of a cut within the poly(dG) sequence. These could be distinguished from prod- ucts formed from double-stranded cuts which occurred rela- tively late in the reaction. The results show that the produc- tion of the 0.8-kbp fragment produced from the EcoRI diges- tion of the endo R products (Fig. 5B) accumulated slowly and corresponded closely to the accumulation of linear plasmid DNA by endo R in the absence of EcoRI digestion (Fig. 5A). (The accumulation of the reciprocal 3.6-kbp endo R/EcoRI fragment was obscured by a fragment present in the starting substrate.) This indicated that the formation of nicked cir- cular DNA very early in the reaction (5 min) was the result of random nicking of the substrate which was not specific for the poly(dG) sequence. Further, the nicking activity did not appear to be specific for any other sequence in pGM1008. Aside from the apparently specific nicks already present in the starting substrate and the 0.8-kilobase fragment generated by double-stranded cleavage at the poly(G) sequence, no other unique fragments were seen (Fig. 5B).

Sequence at the Site of Double-stranded Cleavage-The sequence at the site of endo R cleavage was determined for several of the substrates listed in Table I containing various lengths of poly(dG) .poly(dC), alternating copolymers of GA, telomeric sequences and the AAV recognition sequences (Figs. 6-8). In these studies, linear endo R-cleaved product DNA was isolated by electrophoresis on agarose gels and then annealed to primers which hybridized to sequences flanking the endo R cleavage site. The mobility of the primer extension products were compared with the sequence of the insert using the same single-stranded primer. Separate primers were used for the G-rich and C-rich strands. (Figs. 6 and 7, “Experimen- tal Procedures”). A summary of the cleavage patterns for most of the clones is presented in Fig. 8.

Cleavage of homopolymers of poly(G) .poly(C) occurred throughout the G. C insert (Fig. 6, pGMGC13 and pGM1008; Fig. 8). There was an apparently higher frequency of cleavage at the 3’ end of both strands of the insert in all of the homopolymeric clones. This effect was especially pronounced at the 3’ end of the poly(dG) strand in pGM1008 at the junction of the AAV and poly(G) sequences. Cutting in the C-strand in this clone also demonstrated a slight preference for the 3’ end. This pattern of cleavage was confirmed when the similar AAV/poly(G) .poly(C) junction of pGMPstD was sequenced by the Maxam-Gilbert (1977) method (not shown). The site of cleavage in clones that contained only poly(dG) . poly(dC) inserts and no AAV sequences (pGMGC13, Figs. 6 and 8; pGMGC9, Fig. 8) was also slightly skewed toward the 3’ end of both strands and cleavage was confined almost entirely to the G 1 C insert.

We then examined the cleavage pattern in substituted G- rich polymers. In substrates that contained the alternating copolymer (GA),, and (GA)38 (Fig. 8), cutting occurred throughout the polymer insert between the G and A residues on the lower strand and between the C and T residues on the upper strand. In these clones, cleavage was evenly distributed within the CT strand but more pronounced at the 5’ and 3’ insert/vector junctions in the GA strand (Fig. 8).

The cleavage pattern observed with plasmids containing telomeric sequences exhibited more periodicity. In the plasmid pGMC4T (Fig. 7), which contains the sequence d(G2.6A)5,

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G GCATR

C RGCAT

Fro. 6. Sequence at the site of endo R cleavage within poly d(G),. The sites of endo R cleavage on each strand were determined by primer extension from primers flanking the G-rich inserts of the substrates pGMGC13 (GCl3) and pGM1008 (1008)). Linear endo R- cleaved molecules were isolated from a 1.4% agarose gel. One pg of linear product DNA was annealed to one of two primers (G or C primer) which flanked the G-rich insert, and the primers were ex- tended with reverse transcriptase to the position of cleavage (R lanes). In the lanes marked G, C, A, or T of each panel, the same primer was used with uncut circular DNA to determine the sequence of the substrate in the vicinity of cleavage. In the lanes marked G, the primer that was used extended the strand that contained the C-rich residues. This indicated the position of cleavage on the G-rich strand. Lanes that are marked C, on the other hand, involved the use of a primer t.hat extended the G-rich strand of the substrate (see “Experimental Procedures”).

cleavage of the CT strand occurred throughout the insert with a particularly strong cut occurring at regular intervals imme- diately after the first C residue of every repeat. A preference for cleavage at the 3’ end of the CT strand was observed, corresponding to the region containing 6 continuous G . C base pairs (Figs. 7 and 8). Curiously, a strong preference for cutting was seen at the 3’ end of the GA strand, even though the longest stretches of G.C base pairs are at the 5’ end of this strand. In contrast, the pattern of cutting observed with the plasmid pGMC4A2, which contains the sequence d(G4TJ9, exhibited little preference for either end of the insert (Figs. 7 and 8). The cleavage pattern for this clone was repetitive and symmetrical throughout the oligonucleotide insert. The most prominent cleavages appeared to occur in directly opposite positions on each strand at the boundary of the G. C and T. A sequences within each repeat.

Cleavage of the plasmids pGMAAVter and pGMAAVter2, which contain single and inverted double copies of the AAV- terminal sequences, respectively, was contained within the C- rich region of the AAV recognition sequence and showed a preference for the 3’ end of each strand (Fig. 8). This is especially noticeable in pGMAAVter2, where the cut sites are

C4T G C

GCATR GCATR

10847

CM G C

GCATR RGCAT

4 i

FIN. 7. Sequence at the site of endo R cleavage within te- lomeric. See legend to Fig. 6 and “Experimental Procedures” for details. C4T and C4A2 indicate the plasmid substrates pGMC4T and pGMAAVter2, respectively (see Table I).

,,,,,,,,,,, 11~,,1.1,1.11111111,111 IllI... ~GH1008 ATTGCCGCGCAGAGAGGGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTGCAGGCAT

(G)u TRACGGCGCGTCTCTCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGACGTCCGTA L ,,,,,, .I ,,,‘““““*‘*“““““““‘”

.,.Y,,,lliil.~ pGMGC13 ATTGCTGCAGCCCCCCCCCCCCCTGCAGGCAT

(G)13 TAACGACGTCGGGGGGGGGGGGGTCGTCCGTA

‘III”‘*’

,,.iIII... pGMGC9 ATTGCTGCAGCCCCCCCCCTGCAGGCAT

(6) g TAACGACGTCGGGGGGGGGACGTCCGTA ^‘lf”^^’

91 ,,,~..,l,.,.l....,~l~l,~~. PGHC~T ATTGCTGCACCTCCCTCCCCTCCCCCTCCCCCCTGCAGGCAT (G2.6A) 5 TAACGACGTGGAGGGAGGGGAGGGGGAGGGGGGACGTCCGTA IL., ‘,“‘,,‘A”““““’ ,,

TII I II II pGMC4A2 (G4 Tz)g ttt ttt ttt ttt 3

II I I Ii 4 I II I I 1 III 1. II pGAl4 GGCCAGTGAATTCGGCiC;CTCTCTCTCTCTCTCTCTCTCTCTCCCG~TTCCGCG (GA) 14 CCGGTCq:““Al”l”“~~G~G~G~~AG~G~G~G~G~G~G~~AGAG~~~~T~GGCGC

!I VIII I II PGHAAVTER ATTGCTGCAGGCCACTCCCTCTCTGCGCGCTGCAGGCAT (GI-33j4 TAACGACGTCCGGTGAGGGAGAGACGCGCGACG/CCGTA

11 tt ttt TI.F.I7II

PGMAAVTERZ ATTGCTGCAGAGCGCGCAGAGAGGGAGTGGCCATGGCCACTCCCTCTCTGCGCGCTCTGCAGCAAT (GI-3 fj8 TAACGACGTC:CGCGCGT~?~~~~~TCACCGGTACCGGTGAGGGAGAGACGCGCGAGACGTCGTTA

FK. 8. Summary of the cleavage sites. The sequence data obtained with G-rich substrates are summarized. Only the sequence of the oligomer insert and the flanking vector sequences are shown. Long arrows represent major cleavage sites, and short arrows repre- sent minor sites (approximately 25-50% of t.he intensity of the major bands). The relative intensities apply only within the same strand, and no attempt was made to compare the frequency of cleavage between complementary strands or between separate clones.

localized exclusively within the C-rich region of each strand and appeared to produce sizable 3’ overhangs.

Religation of Endo R Products-To determine whether one

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TABLE II Sequences of cloned endo R products

Five rg of pCM1008 or pGMC4A2 were cleaved with 10 units of fraction V endo R in a standard cleavage reaction mixture of 125 ~1. After phenol extraction, the linear products were isolated from a 1.2% agarose gel and religated with T, fiNA ligase in a reaction volume of 20 ~1 for 24 h at 15 “C!. The ligation mixture was adiusted to a DNA concentration of 20 pg/ml, and the entire ligation mixture for each clone was used to transform JC8111 cells. Five subclones of each type were sequenced as described under “Exnerimental Procedures.”

ClOllS Length of No. of Length of endo starting insert clones R-treated insert

pGM1008 (G)SI 3 (G)31 1 (Gh9

1 (G)Iz pGMC4A2 (G,T& 5 (GdT&

or more than one cleavage occurred within each stretch of G- rich sequence, we cloned the linear DNA products of an endo R digestion and sequenced several clones. This was done by isolating the linear plasmid product from agarose gels, ligating the linear DNA with Tq DNA ligase to form monomer circles, and transfecting JC8111 cells with the ligation mixture. A total of 10 clones were isolated and sequenced, five each from a pGMlOO8 and a pGMC4A2 digestion (Table II). All of the pGMC4A2 clones and three of the pGM1008 clones contained the original parental sequence. Two of the pGM1008 clones had suffered deletions within the poly(dG) &poly(dC) sequence of 12 or 19 bp. We concluded that generally only a limited number of cleavage events (l-2) had occurred/plasmid mole- cule under the conditions of the standard endo R assay used for DNA sequencing.

DISCUSSION

In general, the level of cutting by endo R depends on the length of the recognition sequence and the amount of substi- tutions in the G (or C) strand for a given length. Plasmids that contain homopolymeric stretches of poly(dG) *poly(dC) were the best substrates for endo R. The difference in cleavage frequency observed in clones that contained stretches of poiy(dG) of varying length indicated that a minimum of 8 or 9 bases of G residues were required for cleavage and that the frequency of cleavage increased in direct proportion to the length of the homopolymeric chain, to a maximum of approx- imately 40 base pairs (compare pGM1008, pGMGC13, and pGMGC9, Table I). Substitutions or variations in the G sequence reduced the level of cleavage, where the overall frequency depended on the length of the recognition sequence and the percentage of G. C base pairs in this sequence (Table I, compare pGM1008 and pGA38, pGA38 and pGA11). That the presence of G sequences was essential was confirmed by the observation that the homopolymer, poly(dA)s3 4 poly(dT)a, was not cleaved by endo R (Table I, pEV136).

The type and the extent of substitution in the G . C polymer were also important. Insertion of A or T residues into the poly(G) chain were tolerated, but resulted in significantly lower cleavage frequencies. Substrates that contained the 25 bp Dictyostelium telomeric sequence, d(G2-&, which is a polypyrimidine/polypurine chain (80% G), or the Z’etruhy- mena telomeric sequence, d(G4T&, in which purines are mixed with pyrimidines (67% G), were cleaved at approxi- mately the same frequency, corresponding to about 30% of the level observed with the poly(dG)sl clone. Therefore, mixed purine and pyrimidine residues in the recognition sequence do not significantly affect the cleavage frequency. However, cleavage could not be detected in the alternating copolymer poly d(GC)20 (another mixed purine:pyrimidine) whereas the

copolymer d(GA)se was cleaved at a frequency of 10%. Appar- ently the substitution of C residues within the G stretch or possibly the fact that d(GC), can form Z DNA inhibited endo R. The lower frequency for d(GA)38 may reflect the level of G substitution (50%). Similarly, the low frequency of the AAV recognition sequence d(Gim3T/A) may reflect its short length (13 bp) or to the lack of mirror symmetry (Mirkin et al., 1987). In fact when the AAV sequence was duplicated, the frequency of cleavage increased &fold.

Taken together our studies of the substrate specificity of endo R cleavage suggest that the consensus sequence for enzyme recognition is the general sequence d(G,T/A,),,n 2 andp 2 9. Sequences of this sort are exactly those which have been shown to adopt a number of non-B, non-Z structures when they are present in supercoiled plasmids or as free polymers (Pulleyblank et al., 1986; Evans and Efstratiadis, 1986; Cristophe et al., 1985; Lyamichev et al., 1985, 1986; Lee et al., 1979, 1984; Cantor and Efstratiadis, 1984; Henderson et al., 1987; Sen and Gilbert, 1988; Lyamichev et al., 1989; Sundquist and Klug, 1989; Williamson et al., 1989). The best characterized of these is H DNA (Lyamichev et al., 1985, 1986; Cristophe et al., 1985; Lee et al., 1979), a triple-stranded structure in which the 3’-half of the C strand is protonated and pairs with the 3’-half of the G strand and the 5’-half of the C strand to form the triplex C.G.C (Lyamichev et al., 1988; Htun and Dahlberg, 1988, 1989; Mirkin et al., 1987; Hanvey et al., 1988a, 1988b; Rajagopol and Feigon, 1989; Johnston, 1988; Voloshin et al., 1988; Kohwi and Kohwi- Shigematsu, 1988). The remaining 5’ end of the G strand is unpaired in such structures and presumably this accounts for the unusual asymmetric sensitivity of these sequences to the single-stranded endonuclease Sl (Larsen and Weintraub, 1982; Schon et al., 1983; Hentschel, 1983; Htun et al, 1984; Mirkin et al., 1989). Whether the triple-stranded structure forms and which half of which strand becomes the donor (or extra strand) in the triplex depends on a number of parame- ters. These include the pH (Rajagopol and Feigon, 1989; Mirkin et ai., 1987; Lyamichev et aZ., 1988; Htun and Dahl- berg, 1988), the level of plasmid superhelicity (Htun and Dahlberg, 1989; Mirkin et al., 1987), the sequence of the G- rich region (Hanvey et al. 1988a; Budarf and Blackburn, 1987; Htun and Dahlberg, 1989), salt concentration (Hanvey et al., 1988a, 1988b; Htun et al., 1984; Evans and Efstratiadis, 1986), and the presence of Me (Kohwi and Kohwi-Shigematsu, 1988).

The two best substrates for endo R are d(G),.d(C), and telomeric sequences. Although it is reasonably certain that some G-rich sequences (e.g. d(GA),) form the C. G’ C triplex described above, it is not precisely clear what type of strut- tures are formed by d(G), . d(C), or telomeric sequences. Fur- ther, there is relatively little information about the structure of these two types of sequences in relaxed circular plasmids, in the presence of M$+, and at pH 7.5 (i.e. the endo R reaction conditions). Since the structures for the substrates are not well defined, we can only speculate about the elements re- quired for enzyme recognition.

The most likely structure for d(G),.d(C), is a triple- stranded molecule of the type G. G. C, which was suggested by Kohwi and Kohwi-Shigematsu (1988) for d(G),.d(C),. This structure can form in the presence of Mg2’ and is relatively insensitive to pH over the range 6.0-7.5 (Kohwi and Kohwi-Shigematsu, 1988). In addition it is capable of forming under low salt conditions (Hanvey et al., 1988a). Judging by its Sl sensitivity, the telomeric sequence d(GdT& also may be able to form G.G.C triplexes (Budarf and Blackburn, 1986). Finally, oligomers containing telomere or telomere-like

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G-Specific Mammalian Endonuclease 10849

sequences have been reported to interact by G. G base pairing at neutral or slightly alkaline pH (Henderson et al., 1987; Sen and Gilbert, 1988). Therefore, we suggest that endo R may recognize polymers that contain G. G base pairs or G.G.C base triplexes.

Behe, M. J. (1987) Biochemistry 26,7870-7875 Boissv. R.. and Astell. C. R. (1985) Gene (Amst.) 36. 179-185

Several observations support this possibility. First, neither the nicking activity nor the single-stranded nuclease activity of endo R appeared to be involved in the G-specific cleavage reaction as is the case for other G-specific endonucleases. We could detect no G-specific nicks in form II products, and the accumulation of products that contained cuts within the G- rich sequences corresponded to the accumulation of linear product DNA and not form II molecules. Further, deletions would have been expected within the G-rich region if the mechanism of cleavage involved randomly placed sequential nicks on opposite strands. Yet, cleavage by endo R did not result in many deletions. Taken together, these observations suggest that G-specific cleavage does not occur by sequential nicking of the two strands of the substrate but rather by a mechanism in which both strands are recognized and cut at the same time. Second, the most potent competitive inhibitor of endo R was the oligonucleotide d(G)12 (Gottlieb and Mu- zyczka, 1990). Presumably, at least a portion of this existed as a G.G base-paired hairpin under the conditions of the endo R reaction (Henderson et al., 1987). This would explain the fact that d(C)iz, single-stranded DNA, and double- stranded DNA were essentially equivalent in their ability to inhibit endo R cleavage while d(Ghz was a lo-loo-fold more potent inhibitor (Gottlieb and Muzyczka, 1990).

One difficulty with this proposal is that the cleavage pattern is essentially uniform for the most efficiently cleaved sub- strates. If the endo R substrate is an inherently asymmetric triplex and on average there were only l-2 cleavage events/ molecule, then we should have seen an asymmetric cleavage pattern with respect to the two ends of the G-rich region. A possible explanation is that both the 5’ and the 3’ isomeric forms of the triplex can form under the conditions of the reaction in the absence of superhelicity (for discussion, see Htun and Dahlberg, 1989). Another difficulty is the regular pattern of cleavage seen with d(G4T&. This might be ex- plained by arguing that within a d(G4T& triplex the A and T residues are unable to form T. T . A base pairs. Nevertheless, other difficulties remain. For example, we have no good ex- planation for the asymmetrical cleavage that was seen with pGMAAVter2 or within the G strand of d( GA),. Clearly other substrate structures (Kohwi and Kohwi-Shigematsu, 1988; Henderson et al., 1987; Sen and Gilbert, 1988; Lyamichev et al., 1987, 1989; Sundquist and Klug, 1989; Williamson et al., 1989) and enzyme mechanisms are possible. Presumably, as either the substrates or the enzyme are better defined the correct explanation will become clear. Finally, it is worth noting that the striking cleavage activity of endo R on telom- eric sequences suggests that endo R may have a role in the resolution of telomeres during cellular DNA replication or chromosome segregation. In addition, there is a substantial amount of variation in the size of telomere sequences in somatic cells (de Lange et al., 1990) and endo R may be involved in controlling the length of these sequences.

Acknowledgments-We thank T. Evans, A. Efstratiadis, and W. Holloman for substrates.

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