Proc. Nati Acad. Sci. USA Vol. 79, pp. 4594-4598, August 1982 Biochemistry Site-directed psoralen crosslinking of DNA (mercurated pyrimidines/restriction nucleases/DNA damage/photochemistry) WILMA A. SAFFRAN., MERRLL GOLDENBERG, AND CHARLES R. CANTOR Department of-Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Communicated by Gilbert Stork, May 3, 1982 ABSTRACT A technique has-been developed for placing site- -,CH2-N), 2.4 (6H, s, CH3-N-), 2.5-2.2 (2H, s, H-N); mass spec- specific crosslinks into DNA by using a psoralen derivative con- trum (chemical ionization in methane) 177 (M + 1), 205 (M taining a sulfhydryl group. Plasmid pBR322 was nicked at the + 29), 217 (M + 41). unique BamHI restriction-site, and mercurated pyrimidines were Chloromethyltrimethylpsoralen (II). This compound was introduced by nick-translation. Then the psoralen was specifically made (with minor modifications) according to the literature (5): directed to this site in the dark through a Hg-S linkage prior to yield (not including mother liquor) 32%; mp 212-215°C (lit. irradiation to form a crosslink. The location of interstrand cross- 215-2170C); NMR (Varian T60) 8 7.5 (1H, s, phenyl), 6.17 (1H, links was examined by electron microscopy ofPst I-cut molecules s, lactone), 4.7 (2H, s, Cl-CH2-Ar), 2.5.(9H, m, methyls). *and found to be at or-near the BamHI site. Methylaminodiethoxyethanemethylaminomethyltrimethyl- psoralen (Ill). A mixture of 176 mg of II and 1.69 g of I was Interstrand crosslinks in nucleic acids are readily formed by heated with stirring in 20 ml ofdry toluene overnight. The re- reaction with psoralen or its derivatives (1, 2). Psoralens bind action was monitored by TLC on silica gel with enzene/meth- to-double-stranded DNA or RNA noncovalently in-the dark, by anol, or 1:1 (vol/vol) 95% ethanol/concentrated aqueous am- intercalation. Irradiation of this complex with 365-nm light re- monia, 4: 1, as eluants. The product (140 mg) was eluted from sults in the formation of a cycloadduct between a pyrimidine a silicagel (Baker, 60-200mesh)flashcolumnwith95% ethanol/ residue on one strand and the psoralen. This may then react concentrated aqueous ammonia, 4: 1, as a pale yellow oil: NMR photochemically with a second pyrimidine on the opposite (80 MHz) 8 7.68 (lH, s, phenyl), 6.2 (LH, s, lactone), 3.9-3.4 strand to form a crosslink. Although the relative rates ofreaction (1OH, m, -CH2-O-, -N-CH2-Ar), 3-2.2 (19H, m, -CH3 and -CH2- with various pyrimidines differ, there is little indication of any N-); mass spectrum (chemical ionization in methane) 417 (M more general sequence specificity. + 1), 445 (M + 29), 457 (M + 41). .In addition to its medical application in the treatment ofpso- 2-Pyridyldithioethylmethylamidodiethoxyethanerrthylami- riasis, the psoralen photoreaction has been used in studies of nomethyltrimethylpsoralen (IV) [site-specifwc psoralen (SSP)]. nucleic acid secondary structure, synthesis, mutagenesis, and N-Succinimidyl 3-(2-pyridyldithio)propionate (6) (SPDP; 29mg; DNA repair and recombination (3, 4). Although some of these Pharmacia) was dissolved in 200 ,ul ofdry methylene chloride. investigations have taken advantage ofthe double-strand spec- Then 35 mg ofIII dissolved in 100 ,ul ofdry methylene chloride ificity of the crosslinking photoreaction to map secondary struc- was added and the reaction mixture was stirred in the dark at ture in nucleic acids or to probe the organization ofchromatin, room temperature. The disappearance ofIll and SPDP and the the molecules under study reacted randomly with psoralen, and appearance of the product and N-hydroxysuccinimide were a heterogeneous mixture of products was produced. monitored by TLC (silica gel with methanol eluant). The re- *We describe here a method ofdirectingpsoralen crosslinking action was complete within 15 min. The productwas eluted with to a specific location in a DNA molecule by using a derivative benzene/methanol, 1:1, on a reversed-phase (silica gel 60, ofpsoralen containing a thiol group. We have prepared plasmid Merck) flash column. The fractions were analyzed on a linear pBR322 DNA molecules with interstrand crosslinks placed near KC18 plate. (On this TLC system, the RFS of the product, the unique BamHI restriction site by enzymatically incorpo- SPDP, N-hydroxysuccinimide, and Ill, were 0.24, 0.7, 0.46, rating mercurated nucleotides at this site and then directing and 0.04, respectively.) On all TLC plates the product spot ab- psoralen to the modified bases through -a Hg-S linkage. sorbs 260 nm light, fluoresces pale blue on irradiation with 370 nm light and is stained by iodine. Yield was 41 mg. The product MATERIALS AND METHODS was further characterized by UV spectroscopy. With a molar Synthesis of Site-Specific PsorAlen. The sequence. is shown extinction coefficient of6,884 (at 280 nm) for psoralen (5), 5,100 in Fig. 1. (at 280 nm) for 2-pyridyl disulfide, and (after reduction of the 1,2-Bis(2-nethylaminoethoxy)ethane (I). Twenty grams of disulfide with dithiothreitol) 8,080 (at 345 nm) for 2-thiopyri- 1,2-bis(2-chloroethoxy)ethane (Eastman) and 165 g of methyl- done (7), it was determined that the molar ratio of2-pyridyldi- amine (40% in water) (Matheson, Coleman, and Bell) were sulfide to psoralen was 1:1: mass spectrum (chemical ionization placed in a pressure bottle and, heated at 85°C for 3 days. The in methane) 614 (M + 1), 628 (M + 15), 642 (M + 29). solution was made basic with sodium hydroxide and the product Preparation of SSP Crosslinks in Plasmid DNA. Nick-trans- was extracted into chloroform, and dried over anhydrous so- lation with mercurated nucleotides. Plasmid pBR322 was iso- dium sulfate. After rotoevaporation ofthe solvent, the product lated by the method of Bolivar and Boekman (8). BamHI was (76% yield) was purified by vacuum distillation: bp at 12 mm prepared by the method ofWilson and Young-(9). Plasmid was .Hg 112-114'C; NMR(C2HC13, Varian T60).8 3.6 (8H, s, su- nicked with BamHI in the presence ofethidium bromide under perimposed on t, J = 5 Hz, -CH2-O-), 2.75 (4H, t, J = 8 Hz, the following conditions: 100 ,ug ofDNA per ml, 150 mM NaCl, 6 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 0.5 mg ofbovine serum The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: SSP, site-specific psoralen; SPDP, N-succinimidyl 3-(2- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. pyridyldithio)propionate. 4594 Downloaded by guest on September 25, 2021 Biochemistry: Saffran et al. Proc. Natl. Acad. Sci. USA 79 (1982) 4595 CH3 CH3 (5). Irradiation time was generally 10 min. H-N-(CH2)2-0-(CH2)-O0-CH2)Z-N-H R-Cl Crosslinks were detected by electrophoresis of native and I 1 denatured DNA on 1.2% agarose in Tris-acetate/EDTA pH 7.6 buffer (10). Equal aliquots ofsample, one native and one dena- tured in 0.2 M NaOH, were run in adjacent lanes. Denatured, 1000, toluene noncrosslinked DNA ran as single strands, with higher mobility than the double-stranded species; the rapidly renaturing cross- CH3 CH3 linked molecules had the same mobility as the native DNA. The H-N-(CH2)2-0-(CH2)2-O-(CH2)27N-R amount of crosslinked DNA in 32P-labeled samples was mea- m sured by cutting out the ethidium bromide-stained gel bands, SPDP, CH2CI2 dissolving them in 1 ml of formamide for 1 hr at 500C, and then assaying them in 10 ml of scintillation fluid. DNA was dena- O CH3 CH3 tured and spread by the method of Cech and Pardue (11). (S-S-(CH ),7C-N-'C`H2)2-O-(CH2)270-(CH )i-N-R N DTT RESULTS Synthesis and Properties of SSP. This psoralen derivative 0 CH3 CH3 was constructed to optimize the-chances ofsite-specific reaction HS-(CH2)2-C-N-(CH2)2-0-(CH2)2°--(CH2)2-N-R near mercurated bases in DNA. It has the following properties: (i) a disulfide group that is readily reduced to a thiol [this moiety CH3 binds specifically and tightly (Kd = 10-16 M) to mercury in the CH2 dark (12)]; (ii) is soluble in water; (iii) is positively charged, which R= increases the affinity for DNA; and (iv) has a long and flexible CH 0 0 linker between the furocoumarin and the thiol, which enables CH3 the psoralen moiety to intercalate into DNA while attached to a mercurated base through the sulfur at its other end. This FIG. 1. Synthesis of site-specific psoralen (SSP). linker is 20 A long, allowing intercalation three to four bases from the site ofattachment. Cystaminylmethyltrimethylpsoralen, albumin per ml, 25 ,g of ethidium bromide per ml, and 100 another thiol-containing psoralen derivative with a shorter (5 units of BamHI per ml. After incubation for 1 hr at 37°C, the A) linker (13), failed to produce any specific interstrand reaction mixture was extracted with 1 vol of phenol and then crosslinks. ethanol precipitated. Up to this step, all procedures were car- Preparation of pBR322 Crosslinked near the BamHI Site. ried out under red light to minimize random nicking by ethi- To prepare pBR322 DNA containing mercury near or at a spe- dium bromide. The pellet was washed once with 80% ethanol cific site, intact plasmid was first nicked with BamHI in the and resuspended in 10 mM Tris (pH 8.0) at a concentration of presence of ethidium bromide (Fig. 2A). Ethidium bromide 1 mg/ml. inhibits restriction endonucleases, and this treatment results Nick-translation was carried out in 50-mM Tris HCl, pH 7.6/ in the production of single-strand cuts rather than the normal 6.7 mM MgCl2/6 mM mercaptoethanol/10 ,M dATP/100 ,M staggered double-strand cuts (14).