Proc. Natl. Acad. Sci. USA Vol. 74, No. 10, pp. 4219-4222, October 1977 Biochemistry

Nucleotide sequences near the origin of replication of bacteriophage fl (restriction mapping/complementary strand/DNA hairpin structure)

JEFFREY V. RAVETCH, KENSUKE HORIUCHI, AND NORTON D. ZINDER The Rockefeller University, New York, New York 10021

Contributed by Norton D. Zinder, July 15, 1977

ABSTRACT The nucleotide sequence of a region related to the initiation of the reaction in which single-stranded DNA gives rise to the replicative form (complementary strand synthesis) in bacteriophage fl has been determined. The sequence can be drawn in an extensively base-paired structure, i.e., a single hairpin-helix 55 bases long. DNA replication in the filamentous, single-stranded DNA bacteriophage (fl, fd, M13) is postulated to occur in three steps: (i) conversion of the parental single-stranded viral DNA (SS) into double-stranded replicative form (parental RF); (ii) replication of parental RF to form a pool of progeny RF; and (iii) asymmetric replication of single-stranded viral DNA by the replacement of old viral strands on the RF molecules by newly synthesized viral strands (1). Recently, Horiuchi and Zinder (2) identified the origin and direction of synthesis in vivo of the viral and complementary strands of fI DNA. Their results indicate that the origins of replication of viral and complementary strands are close to one another, the former within restriction fragment Hae III-G and the latter within Hae III-G or the proximal portion of Hae III-F (our unpublished data). Previously, Tabak et al. (3) demonstrated that M13 viral DNA was converted to a duplex (RFII) molecule by an Escherichia coli enzyme preparation. The discontinuity that they found in the complementary strand of the RFII synthesized in vitro was specifically located within the restriction fragment Hpa TI-H (see Fig. 1), implying that the origin of the complementary strand was located within this restriction fragment. Furthermore, Schaller et al. (4) isolated, in vitro, a specific DNA fragment from fd viral DNA that had been bound to RNA polymerase in the presence of E. coli unwinding protein. This DNA fragment maps to restriction fragment Hpa II-H. Finally, Vicuna et al. (5, 6) have described an in vitro system capable of selectively converting fd viral DNA and not OX 174 DNA to duplex (RFII) DNA. This RFII contains a unique gap in the complementary strand, which has been mapped to the HpaII-H fragment of the fd genome. Fig. 1 presents the physical and genetic map of bacteriophage f 1. Fragment Hpa II-H includes fragment Hae TII-G and the adjacent fragment Hae III-Fa (Hae III-F-Hae II-A; see Fig. 2) and should therefore contain both origins of replication. We determined the nucleotide sequence of this fragment by use of the Maxam-Gilbert method (14). The present paper reports the nucleotide sequence of restriction fragment Hae III-Fa and shows that this sequence is related to the origin of SS - RF replication, i.e., the origin of complementary strand synthesis. The costs of publication of -this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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FIG. 1. Physical and genetic map of bacteriophage fl. The outer six circles, representing the physical map, show the cleavage sites of the restriction enzymes R.Hha I, R.Hpa II, R-Hae III, R-Hae II, R-Eco RII, and R-Dpn II (refs. 7-11 and our unpublished data). The single R-HindII cleavage site is indicated by the arrow. The seventh circle represents the genetic map (12). Genes are indicated by Roman numerals. IG refers to the intergenic space between genes IT and IV (9, 13). The + and - indicate the sites of the origin of replication of the viral strand (+) and the complementary strand (-) (2). The location of some mutant sites within the genes are shown by the numbers on the outside of the genetic map. The inner circle is divided into 10 equal physical map units with the R-HindII cleavage site as the reference point. The polarity of the viral strand is such that the 5' - 3' direction corresponds to counterclockwise movement on the map.

The sequence has the potential to form an extensively basepaired structure. MATERIALS AND METHODS fl DNA. Preparation of nonradioactive, RF DNA has been described (15). Nucleases. Restriction endonucleases R-Eco RII, R-Hae II, R-Hae III and R-HindII were prepared as described (7, 9). Abbreviations: SS, single-stranded DNA; RF, replicative form DNA; RFI, covalently closed, circular, superhelical DNA; RFII, circular DNA with a break in one strand. The nomenclature proposed by Smith and Nathans (22) for restriction enzymes has been used; i.e., endo refers to endonuclease, R to restriction, Hin to Haemophilus influenzae, Hae to H. aegyptius, Hpa to H. parainfluenzae, Hha to H. haemolyticus, Eco to Escherichia coli, Dpn to Diplococcus pneumoniae, and Alu to Arthrobacter luteus.

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Biochemistry: Ravetch et al.

Proc. Natl. Acad. Sci. USA 74 (1977)

iipa 11-H Hpa II

G A T+C C G A T+C C G A lT+C C

Hinf Hinf Hae III

Hae III

!-

Hae III-G

Alu I Hpa II Hae II Hae III-Fa

FIG. 2. Detailed physical map of the Hpa IH-H fragment. The total length of Hpa IH-H is approximately 400 nucleotides; Hae III-G is 140 nucleotides and Hae III-Fa is 152 nucleotides.

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R-Hpa II was purified as described by Sharp et al. (16). R.Hha I was purchased from New England Biolabs. R-HinfI was prepared according to the procedure of R. J. Roberts (personal communication). Haemophilus influenzae serotype f, Dingles was grown at 370 in brain heart infusion supplemented with hemin at 10 ig/ml and NAD at 2 Aug/ml. Cells were harvested in stationary phase, and 10 g of frozen cells were suspended in 20 ml of 10 mM Tris-HCl, pH 7.9/10 mM 2-mercaptoethanol. Cells were disrupted by sonication and debris was removed by centrifugation at 100,000 X g for 90 min. Supernatant was made 1.0 M in NaCI and applied to Bio-Gel A-0.5m (2.5 X 100 cm) and eluted with 1.0 M NaCl/10 mM Tris-HCl, pH 7.9/10 mM 2-mercaptoethanol. Peak fractions were combined and dialyzed against 10% (vol/vol) glycerol/10 mM KPO4, pH 7.4/10 mM 2-mercaptoethanol/0.1 mM EDTA (PC buffer). The material was then applied to a phosphocellulose column (1.2 X 25 cm) previously equilibrated with PC buffer and eluted with a linear gradient from 0 to 1.0 M KC1 in PC buffer. Peak fractions were combined and dialyzed against PC buffer. The material was then applied to a DEAE-cellulose column (0.9 X 25 cm) previously equilibrated with PC buffer and eluted with a linear gradient from 0 to 0.3 M KC1 in PC buffer. The peak

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B FIG. 3. Isolation of 5'-32P-labeled Hae III-Fa. A portion (8.25 X 105 cpm) of a mixture of 5'-32P-labeled restriction fragments Hae III-D, E, and F was digested with 40 units ofHae II (Biolabs) in a 50-,ul reaction mixture containing 5.6 mM Tris-HCl, pH 7.4/5.6 mM MgCl2 for 3 hr at 37°. The cleavage products were fractionated on a 10% polyacrylamide gel containing 40 mM Tris/20 mM sodium acetate/2 mM EDTA, pH 7.2 at 80 V. B indicates the position of the bromphenol blue dye marker.

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FIG. 4. Radioautograph of the cleavage products of Hae III-Fa. Reaction conditions are those described by Maxam and Gilbert (14). G and A were methylated with dimethylsulfate for 20 and 25 min, respectively, at 20°. Bases modified at G were cleaved in 1 M piperidine at 900 for 30 min. Methylated adenosines were released by treatment in 0.1 M HCO at 0° for 120 min and the polynucleotides were cleaved by heating at 900 for 30 min in 0.1 M NaOH. T and C in H20 and 2.5 M NaCl, respectively, were treated with 18 M hydrazine for 20 and 25 min, respectively, at 200. Polynucleotides were cleaved by heating in 1.0 M piperidine at 900 for 1 hr. The cleavage pWoducts of the four reactions were fractionated on a 30 X 40 X 0.15 cm slab gel of 20% polyacrylamide/7 M urea. Three sets of samples were run at 25 V/cm for 12, 24, and 36 hr (right to left).

fractions were combined and dialyzed against PC buffer. The final purification was an AH-sepharose 4B (Pharmacia) column (0.9 X 25 cm) previously equilibrated with PC buffer and eluted with a linear gradient from 0 to 0.6 M KCl in PC buffer. Peak fractions were pooled, dialyzed against PC buffer containing 50% glycerol, and stored at -20°. Bacteriophage T4 polynucleotide kinase (ATP:5'-hydroxylpolynucleotide 5'-phosphotransferase, EC 2.7.1.X) was a generous gift of C. Yehle. Alkaline phosphatase (E. coli) [orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] was purchased from Worthington. 5'-Terminal Labeling. [y-32P]ATP (about 1000 Ci/mmol) was prepared by the method of Glynn and Chappell (17). DNA restriction fragments were 5'-terminally labeled by the method of Subramanian et al. (18) in which 1 ug of DNA was first treated with 0.2 jig of E. coli alkaline phosphatase in a 100,l-M reaction containing 5 mM Tris-HCl, pH 7.9/5 mM MgCl2 at 370 for 1 hr. The reaction mixture was then extracted with phenol, the organic and aqueous phases were separated, and

Proc. Nati. Acad. Sci. USA 74 (1977)

Biochemistry: Ravetch et -al.

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Table 1. Comparison of pyrimidine tracts of ori-DNA and Fa hairpin Fa hairpin ori-DNA Relative molar Pyrimidine Pyrimidine Relative molar Comments tract yield yield* tract* Absent 0.8 T5C

Nucleotide sequences near the origin of replication of bacteriophage f1.

Proc. Natl. Acad. Sci. USA Vol. 74, No. 10, pp. 4219-4222, October 1977 Biochemistry Nucleotide sequences near the origin of replication of bacteriop...
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