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Perfect Palindromic Lac Operator DNA Sequence Exists As a Stable

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Perfect Palindromic Lac Operator DNA Sequence Exists As a Stable Proc. Natl Acad. Sci. USA Vol. 80, pp. 1797-1801, April 1983 Biochemistry Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo (psoralen/chromosome structure) RICHARD R. SINDEN, STEVEN S. BROYLES, AND DAVID E. PETTIJOHN Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262 Communicated by William B. Wood, December 17, 1982 ABSTRACT A perfect palindromic 66-base pair (bp) DNA se- MATERIALS AND METHODS quence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66- Bacterial Strains, Growth Conditions, and Plasmid Prep- bp linear form or as two 33-bp cruciform arms. The fraction of the aration. Escherichia coli K-12 strains pOCE12/HB101 and sequence in the cruciform depends on the superhelical density of pOCE13/HB101 containing a single and two tandem 66-bp the plasmid DNA. Relaxed DNA contains no cruciforms. The pal- palindromic lac operator DNA sequences, respectively, cloned indrome in the cruciform structure is cut by EcoRd endonuclease into pMB9 were used (23). Supercoiled plasmid molecules were at the base of the cruciform arms, releasing 33-bp fragments; when purified and molecules of varying superhelical density were in the linear form only 66-bp fragments are produced. The cru- prepared as described (24) by using a HeLa topoisomerase ex- ciform structure is fixedbytrimethylpsoralen crosslinks inthe cru- tract (25). Superhelical densities were measured as described ciform arms. This together with the EcoRP cutting provides an as- (26). say for the cruciform structures in the DNA of living cells. Using Photobinding of 4,5',8-Trimethylpsoralen. 4,5',8-Trime- this assaywe showthatthecruciform structure rarelyif ever exists thylpsoralen (Me3psoralen) and irradiation conditions have been in vivo, but after DNA isolation >90% of the sequence is in cru- described in detail (27). Samples were irradiated by using two ciforms. Results suggest that theplasmid DNA as organizedin vivo General Electric F15T8 BLB bulbs at an incident light in- either lacks sufficient torsional tension to form this cruciform or tensity of 1.6 kJ-m 2-min-'. The dose ranges used in vitro were the palindrome is restrained in the linear form by other bound <48 kJ m-2. For photobinding in cells, cells were perme- molecules. abilized by treatment with EDTA (28). Two-minute exposure to EDTA was terminated by the addition of MgCl2. Photo- A palindromic or inverted repeated DNA sequence has the binding immediately followed permeabilization (dose range, unique property that base pairing can potentially occur not <24 kJ m2). only between the two complementary strands of DNA but also Gel Electrophoresis. Restriction enzymes noted in the text within each single DNA strand. In the latter configuration bases (Bethesda Research Laboratories) were used according to the at the symmetric center of the palindrome are at the ends of manufacturer's directions. Plasmid DNAs and restriction frag- "hairpin" or cruciform arms with respect to flanking, non- ments were analyzed by electrophoresis on 0.8% agarose gels palindromic sequences (Fig. 1). Palindromes have been iden- as described (24). For determination of the superhelical den- tified in DNA from many organisms and are often found at sity of various DNAs, electrophoresis was as described by Keller operator and transcription termination regions in bacterial and (26). Thirty-three- and 66-bp fragments were analyzed on 14 bacteriophage DNA (1-3), as well as in DNA replication origins X 10 X 0.15 cm 5% polyacrylamide gels in 40 mM Tris borate of prokaryotes (4-6) and eukaryotes (7-11). Considering the buffer (pH 8.3) containing 10% glycerol. Current was kept at prevalence of palindromes at such important regulatory sites, 6-8 mA to avoid heating. it has been tempting to speculate about possible biological sig- nificance of cruciform structures. The thermodynamics of cruciform formation and their sta- RESULTS bility have been discussed in detail (12-14). Recent studies EcoRI Cuts Arms of Cruciform Structures from Super- with single-strand nucleases and electron microscopy have coiled DNA. The base sequence of the 66-bp palindrome in- provided evidence that cruciforms do exist in supercoiled DNA serted into the EcoRI site of pMB9 and the possible cruciform (14-18), as first suggested by Lebowitz and co-workers (19, configuration of this sequence are shown in Fig. 1. Digestion 20). The probability of cruciform existence depends on the of the linear form by EcoRI restriction endonuclease produces superhelical density of DNA (18). This evidence seems con- a linear 66-bp fragment. Digestion of the cruciform structure vincing that palindromic DNA sequences under sufficient tor- produces two 33-bp hairpins or cruciform arms. DNA frag- sional tension can exist as cruciforms; however, we wished to ments of this size released by EcoRI have been observed (23). develop a procedure for assaying cruciforms that can be used This test can be applied to distinguish whether the palin- to detect these structures in vivo. dromic sequence is in a linear or cruciform structure. Psoralen crosslinks in double-helical DNA (21) should fix When supercoiled pOCE12 DNA was digested with EcoRI cruciforms and thus provide a basis for their detection in vivo the cloned 66-bp palindromic fragment was produced, as ex- (22). Here we describe the application of this approach to pected. In addition, a 33-bp fragment expected for the cru- studies in vitro and in vivo of a perfect 66-base pair (bp) pal- ciform arm was consistently observed (Fig. 2). A 33-bp frag- indromic lac operator sequence cloned into pMB9 (23). ment was not observed in pOCE12 DNA, linearized with HindIII prior to treatment with EcoRI. The observance of the The publication costs of this article were defrayed in part by page charge 33-bp fragment suggested that EcoRI was recognizing and cut- payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: bp, base pair(s); Me3psoralen, 4,5',8-trimethylpsoralen. 1797 Downloaded by guest on September 28, 2021 1798 Biochemistry: Sinden et aL Proc. Natl. Acad. Sci. USA 80 (1983) EcoRI A. EcoRk A-.1 ABCDEFGH I JKLMNOPQ -10 I 1 1 21 314 41 51 61 71 81 TATAGGAGCATAGAATTCCACAAATTGTTATCCGCTCACATTCCACAT6TG6ATT6TGAGCGGATACAATM6TGGAATTCTMTTTTCGCT ....ItTI I-ri I~ "-IIIIfrr -^a ATATCCTCGTATCTfAAGGTGMAACAATAGGCGAGTGTTAACGTGTACACCTTAACA CTCGCCTATTGTTAAACACCAGuATTAMAAA s 66-bp palindromic operator I I' 76-bp palindromic sequence pOCE12 A-T B. C G A T C G 31-C G T A-4111 C. NATIVE DUPLEX CRUCIFORM 35 5 21 38 66 AT A 'I il AATT C _ GG C 66 l1*! I.-i*61 TTAA A T |denaturation C G 66 C G 33 T A ~~T T AATT--6' A AI T A A EcoRI A T renoturation I A.1 1 A T ----TTAA G C-71 A T FIG. 2. Electrophoretic analysis of EcoRI digests of supercoiled and -20 -l0 T A 81 91 cA AT relaxed pOCE12 DNA. Direction of electrophoresis is top to bottom. CTTATCAATATAAGGA TTTTCGCTTAAGAACTT GAATAGTTATATCCTCG AAAAAGCGAACTCTTGM Lanes A-E contain digests of 10 jig of supercoiled pOCE12 DNA at TA TT D. CROSS-LINKED CROSS-LINKED constant DNA concentrations (100 and 5, 20, 50, 100, and 200 T A NATIVE DUPLEX CRUCIFORM Ag/ml) C G units, respectively, of EcoRI per ml. Lanes G-K contain digests with T A EcoR! T A A T constant EcoRI/DNA ratios (1 unit/pg of DNA) but increasing con- A T Cl A T 3 66 _ centrations of both enzyme and DNA; the former were 10, 20, 40, 100, G C AATT r- G C - - TTAA 4 and 1,000 units, respectively, per ml. In lane G the amount of applied T A DNA was greater than in lane which enhances the 33-bp band. Re- I I H, I-A action mixtures in lanes are identical to those for lanes A-E, ex- denaauraiion M-Q 11 renaOtration I cept that pOCE12 DNA was linearized previously by digestion with A T HindI. The higher molecular weight bands in these lanes are the G C AATT >- CA G C HindIII-EcoRI fragments of 354 and 387 bp. Positions of migration for TA G C the 33- and 66-bp fragments are indicated. Lanes F and L contain a 40- T -TTAA !A^T -A bp "ladder" used as a molecular weight marker (gift of J. R. Sadler). FIG. 1. Sequence and expected structures ofthe 66-bp palindromic DNA sequence. (A) The 66-bp lac operator palindrome and surround- DNA torsional tension (22). A crosslink in one arm of the cru- ing DNA sequences (23, 29, 30). Including the EcoRI sites and the ciform should effectively stabilize both arms because the lin- flanking AT:TA base pairs, the complete palindromic sequence is 76 ear form could not reform. Alternatively, an interstrand cross- In are a bp. pOCE13 there two 66-bp palindromes making perfect 142- link in the linear palindromic sequence should prevent transition bppalindromic sequence. The arrow between bases 35 and36 shows the center of twofold rotational symmetry. (B) Cruciform configuration of to the cruciform. the 76-bp palindromic DNA sequence. Although this sequence is per- To test this, varied numbers of Me3psoralen crosslinks were fectly palindromic, presumably there is a short 3- to 4-base nonpaired introduced into supercoiled DNA. The crosslinked plasmid loop at the ends of the arms (31). The EcoRI sites are at the base of the cruciform arms. (C) Denaturation and renaturationproducts of66- and 33-bp fragments resulting from EcoRI digestion of the linear and cru- structures.
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