Large palindromes in the X phage genome are preserved in a reck host by inhibiting X DNA replication ( recBC / sbcB / dnaBts / Escherichia cola )

A large palindrome carried by phage X has been shown to prevent growth of the phage on a rec' strain of Escherichia coli. The phage do form plaques on recBC sbcB strains, but the palindrome is not stable-deletions that either destroy the palindrome or diminish its size overgrow the original engineered palindrome-containing phage. We have prepared stocks of X carrying a palindrome that is 2 x 4200 base pairs long. These phage stocks are produced by induction of a lysogen in which the two halves of the palindrome are stored at opposite ends of the prophage and are of sufficient titer (109 phage per ml) to enable one-step growth experiments with replication-blocked phage. We find that the large palindrome as well as a lesser palindrome of 2 x 265 base pairs are recovered intact among particles carrying unreplicated chromosomes following such an infection of a rec' host. We propose that DNA replication drives the extrusion of palindromic sequences in vivo, forming secondary structures that are substrates for the recBC and sbcB gene products. The literature is sprinkled with reports of the inability to clone or maintain DNA sequences containing large regions of palindromic symmetry in Escherichia coli. In some cases the palindrome was lethal to the vector (1-3); in other cases, recA-independent deletions occurred that removed the symmetry of the palindrome (4-6). Leach and Stahl (7) reported that X carrying a 3200-base-pair (bp) palindrome could form plaques on a recBC sbcB strain of E. coli but not on a rec+ strain. The phage was viable, but the palindrome was initially unstable in the recBC sbcB host. The palindrome was stable, however, following symmetrical or near symmetrical shrinkage to a size of 600 ± 100 bp. Phage carrying this smaller palindrome remained inviable on the rec+ host. These results implicated the products of the recBC and sbcB genes, the recombination-related nucleases recBC gene protein and exonuclease I, in the destruction of palindrome-containing DNA. Leach and Stahl (7) proposed that palindromes in vivo might adopt a cruciform structure, which is topologically equivalent to a recombination intermediate, the Holliday junction, and that this structure might be cleaved by recBC gene protein and exonuclease I. It has been argued that cruciforms do not form in vivo from palindrome sequences due to kinetic barriers (8) and attempts to detect cruciforms in vivo using short artificial palindromes cloned in plasmids have been unsuccessful (8, 9). These considerations suggest that in vivo cruciform formation is not an inherent property of palindromes. However, the kinetic arguments, based on studies carried out in vitro with purified DNA, may not be applicable in vivo, and cruciforms might be detected under conditions other than those tested thus far. We describe here the construction of a X phage carrying a palindrome of 8400 ± 100 bp and show that the inviability and instability associated with both this palindrome and a 530-bp palindrome are dependent on replication of the phage DNA. The results of our studies imply that the activity of the DNA replication machinery facilitates the formation of nucleasesensitive secondary structures from palindrome sequences. MATERIALS AND METHODS Phage and Bacterial Strains. X phage used are described in Fig. 1. The E. coli K12 strains used are listed in Table 1. Construction of FS1596. JC7623 (A. J. Clark, University of California, Berkeley), which is recB21 recC22 sbcB15 lacyl str3l, was transduced to hflAJ with the aid of Tn5 in a linked pur gene (in strain SB41 from D. Hagen, University of Oregon, Eugene). The resulting strain was transduced to pur+, to eliminate TnS, with P1 grown on the hflA pur+ strain SB38 (D. Hagen). The resulting strain (FS1537) was P1transduced to hflB with the aid of a linked TnJO (12). JC7623 gives clear plaques with wild-type X; the hflA derivative (FS1537) gives turbid plaques; the hflA hflB derivative (FS1552) fails to allow wild-type X growth. FS1552 was transduced to lac+. The resulting Fstrain (FS1578) was mated to HfrC strain KL752 (K. B. Low, Yale University, New Haven, CN), which is lacZ813 lacI3 str'. A LacStrR colony was designated FS1596. FS1596 does not allow Xc' growth, confirming hflA hflB. It does allow XcI carrying a large palindrome to grow, confirming recB and/or recC plus sbcB. It is likely that the strain is both recB and recC and also lacI3 and is so designated in Table 1. FS1596 is a derivative of AB1157 (13). Density Gradient Experiments. X phage were density labeled by UV induction of lysogens grown in minimal medium containing [13C]glucose and 15NH4Cl. X DNA synthesis was blocked, but transcription, recombination, and packaging were permitted, by infecting a dnaBts strain (FA77) at 420C. FA77 was grown in broth at 260C to a titer of 1.5 x 108 cells per ml, then shifted to 420C for 10 min prior to infection with an equal volume of prewarmed phage at a titer of 1.5 x 109 plaque-forming units/ml. The cultures were supplemented with each of the four deoxynucleosides at 0.05 ,ug/ml and with glucose at 10 mg/ml to dilute out unincorporated isotope in the labeled stocks. The infected cells were aerated for 65 min at 420C, then sedimented and resuspended to eliminate unadsorbed phage. Finally, the cells were lysed with CHCl3 and eggwhite lysozyme. Experiments in which DNA synthesis was allowed were carried out as above except that a dnaB+ strain (594) was used, and the temperature was maintained at 370C throughout. The lysates obtained were brought up in cesium formate solution to a refractive index of 1.3775. Fractions were collected through the bottom of the tube after centrif*To whom reprint requests should be addressed. 1624 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. D ow nl oa de d by g ue st o n Ju ly 9 , 2 02 0 Proc. Natl. Acad. Sci. USA 84 (1987) 1625 A > , A IB Itt 11.6F40 4D E F