Proc. Nati Acad. Sci. USA Vol. 79, pp. 4594-4598, August 1982 Biochemistry

Site-directed crosslinking of DNA (mercurated pyrimidines/restriction /DNA damage/photochemistry) WILMA A. SAFFRAN., MERRLL GOLDENBERG, AND CHARLES R. CANTOR Department of-Human 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, ), 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). 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, , 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 /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 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- (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). At the optimal ethidium bro- dGTP with 100 ,g of nicked plasmid per ml. In addition, the mide concentration, about halfofthe supercoiled substrate was samples without mercury contained 100 ,M dCTP and 100 ,M nicked (converting it to the relaxed circle) and half was cut, dTTP; the samples with mercury contained 100 ,M HgdCTP and 100 AM HgdUTP (Boehringer Mannheim). Samples were A allowed to react for 5-10min at 16°C with Escherichia coli DNA polymerase I (50 units/ml; New England BioLabs) and then 0 DNA 0 were extracted with 1 vol ofphenol. They were dialyzed twice Bam HI -I- Polymerase I \ + against 10 mM Tris'HCI, pH 8.0/10 mM EDTA/100 mM NaCl ethidium > H9dCTP, HgdUfP and. once against 10 mM Tris HCl, pH 8.0. bromide 32P-Labeled DNAwas nick-translated by the same procedure except that the reaction mixture included 10-50 ,Ci (1 Ci = 0 0 3.7 X 1010 becquerels) of [32P]dATP (2,000 Ci/mmol). Aliquots B 4psoralen were assayed after dialysis, to determine [32P]dATP incorpo- ration. The incorporation was 8-12 mol of [32P]dATP per mol denaturation > of pBR322 for samples without mercury and 6-8 for samples 0irradiation containing mercury. ) Crosslinking. Nick-translated DNA, at 100 ,ug/ml, was in- cubated overnight at room temperature with 0.25 mM SSP in C the presence of 0.1 mM mercaptoethanol to reduce the disul- fide, forming reduced SSP (V). The DNA was extracted with -C 1 vol of phenol to remove nonspecifically bound SSP and then Pat I > denaturation > was ethanol precipitated; the pellet was washed once with 80% + + ethanol. The sample was resuspended in 10 mM Tris, pH 8.0/ 100 mM NaCl and degassed for 5 min with N2 gas. All steps up -r to this point were carried out under red light and samples were protected from light to, prevent random crosslinking by non- specifically bound SSP. Crosslinks were produced by irradiat- FnIG. 2. Preparation of SSP crosslinks. (A)- Mercuration at the ing the. samples with the apparatus described by Isaacs et al. BamHI site. (B) Crosslinking with SSP. (C) Cutting with Pst I. Downloaded by guest on September 25, 2021 4596 Biochemistry: Saffran et al. Proc. Nad Acad. Sci. USA 79 (1982)

yielding linear DNA. The two strands ofthe plasmid are nicked mercurated DNA, but the linear DNA, which contained little with approximately equal frequency. mercury, was almost completely uncrosslinked. The double- Next, a short stretch ofmercurated nucleotides was inserted stranded DNA seen after alkalidenaturation was due to psoralen near-the BamHI site by nick-translation with E. coli DNA poly- crosslinking rather than to interstrand Hg-S bonds between merase I (Fig. 2A). The mercury-substituted pyrimidine nu- mercurated nucleotides and monoadducted SSP, because in- cleotides 5-mercurideoxycytidine triphosphate (HgdCTP) and cubation with 50 mM dithiothreitol failed to reduce the inten- 5-mercurideoxyuridine triphosphate (HgdUTP) are good sub- sity ofthis band (data not shown). *strates for DNA polymerase I and were incorporated nearly as The extent.of crosslinking was quantitated by incorporating well as the unmodified nucleatides (15). The modified nicked 32P-labeled nucleotides along with the mercurated nucleotides circular DNA molecules produced had a short patch of mer- during nick-translation and measuring the radioactivity in the curated bases starting at the site of the original nick and ex- gel bands. As expected, the nicked circles had a much higher tending 20-40 bases in the 3"direction, along that strand. Any specific activity than the linear molecules. The fraction ofcross- single molecule had mercury on only one strand, but.the pop- linked molecules was calculated as the ratio of counts in the ulation. ofnicked circles had mercury on both strands, lying on double-stranded nicked circular band with and without alkali both sides of the BamHI site. Because the double-strand cuts denaturation. SSP, irradiation, and mercuration all were re- were staggered, the linearproducts ofBamHI cutting had cohe- quired for crosslinking. In the absence of any one of these fac- sive ends with 5' overhangs which served as templates for nu- tors there was no crosslinking (Table 1); with. all three, >40% cleotide addition by DNA polymerase I. The 3' ends were filled of the mercurated DNA became crosslinked. SSP plus light in, producing'blunt-ended linear molecules. alone efficiently crosslinked either mercurated or unmercur- The modified DNA was then incubated with SSP. The psor- ated DNA if the step of extracting nonspecifically bound SSP alen moiety bound readily to double-stranded DNA by inter- *was omitted. In this case, all three forms of DNA were cross- calation. In addition SSP bound tightly and specifically to the linked, rather than the nicked circles alone. *mercurated bases through a Hg-S bond. The noncovalently Crosslink Location. Crosslinked, plasmid molecules were bound psoralen was completely removed by phenol extraction. examined by electron microscopy. Denaturation of nicked cir- Theplasmid was then irradiated with near-UV light to produce cular DNA with one or more crosslinks located near the nick crosslinks at the site of modification (Fig. 2B). should result in-molecules shaped as diagrammed in Fig. 2B. Measurement of Crosslinking. Psoralen crosslinking was This species was seen only in samples that contained crosslinks, detected by denaturing the DNA with alkali and analyzing it as detected on agarose gels. Electron micrographs of such mol- are in 4A. The contour ofthe lin- on a nondenaturing agarose gel. Molecules.with crosslinks rap- ecules presented Fig. lengths to whereas noncrosslinked mol- ear and circular portions were found to correspond to the full idly renatured double strands, length of unmodified denatured pBR322. ecules remained single-stranded afterdenaturation and ran with The crosslinks were located close to the site of the nick; the higher mobility on the gel. Interstrand crosslinking was specific two parts ofthe figure were joined at the very end ofthe linear to mercurated plasmid. All the DNA nick-translated with un- modified nucleotides remained single-stranded after alkali de- part. Extending the patch of modified bases by increasing the duration of the nick-translation reaction resulted in the ap- naturation (Fig. 3). In contrast, a portion of the sample nick- pearance ofmolecules with crosslinks some distance in from the back to. a dou- translated with mercurated nucleotides snapped end of the linear portion. ble-strand form. Crosslinks were seen in. the nicked circular The site of crosslinking was confirmed by cutting the SSP- treated DNA at a second unique restriction site with Pst I. The No Hg With Hg linearized molecules should then appear Y-shaped after dena- turation (Fig. 2C), and they did in electron micrographs (Fig. 4B). These molecules had either two short or two long arms, depending on which strand was initially nicked by BamHI. We saw equal numbers ofthese two species (withinstatistical error), indicating that BamHI nicking occurs with, equal frequency on Circular the two strands. Linear The BamHI and Pst I sites are 1,125 bases apart on the 4,362- base-pair plasmid (16), or 0.258 times the unit length of the molecule.. The contour lengths of the arms of-the Y-shaped molecules were measured and the site of crosslinking was ex- ss X pressed as the ratio of the length of the short arm to the total length of the molecule. The mean (± SD) distance-of the in- Table 1. Effect of treatment steps on crosslinking Treatments Crosslinks, Hg Light SSP %* - - - 3.3 _ _ + 2.3 FIG. 3. Electrophoretic analysis of crosslinking. pBR322 nick- + + 2.4 translatedwith dCTPanddTTP (noHg) or HgdCTP and HgdUTP (with + - + 2.2 Hg) was treated with SSP plus light. Half of each sample was run + + + 43.8 without further treatment [native (Nat.)] and the other-was alkali de- natured before electrophoresis [denatured (Den.)]. Crosslinked mole- * Double-stranded relaxed circular bands were cut from agarose gels cules are those-runningwith the same mobility with andwithout alkali and assayed; % crosslinking = 100 x [32P cpm (denatured)/32P.cpm treatment. ss, Single-stranded. (native)]. Downloaded by guest on September 25, 2021 Biochemistry: Saffran et at Proc. NatL Acad. Sci. USA 79 (1982) 4597

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FIG. 4. Electron micrographs of denatured crosslinked pBR322. (A) Uncut. (B) Cut with Pst I. (Bar = 0.5 MAm.) tersection from the Pst I site was found to be 0.25 ± 0.04 (n DISCUSSION = 47), in good agreement with the predicted value. We con- clude that, within the resolution ofthe electron microscope, the We have placed SSP crosslinks at a chosen location in the plas- crosslinks are located near the BamHI site. mid pBR322 in a two-step reaction. The nick-translation pro- Downloaded by guest on September 25, 2021 4598 Biochemistry: Saffran et al. Proc. Natl. Acad. Sci. USA 79 (1982)

cedure used directs SSP to a small segment of the DNA mol- This work was supported by National Institutes of Health Grant Fellow- ecule rather than to a specific base. The limited resolution of GM14825 and by a National Institutes of Health Postdoctoral electron microscopic analysis indicates that the crosslinks are ship to W.A.S. located within 150 bases ofthe BamHI site, although the actual 1. Cole, R. S. (1970) Biochim. Biophys. Acta 217, 30-39. locations ofthe crosslinks may be considerably closer to the re- 2. Dall'acqua, F., Marciani, S. & Rodighiero, S. (1970) FEBS Lett. striction endonuclease site. Preliminary results indicate that 9, 121-123. 3. Song, P.-S. & Tapley, K. J. (1979) Photochem. Photobiol. 29, 20-25 nucleotides are introduced by nick-translation on each 1177-1197. side ofthe restriction site. The length of the mercurated patch 4. Parsons, B. J. (1980) Photochem. Photobiol 32, 813-821. can be controlled to some extent by the conditions ofthe nick- 5. Isaacs, S. T., Shen, C.-K., Hearst, J. E. & Rapoport, H. (1977) translation reaction, although control is limited by the proces- Biochemistry 16, 1058-1064. sivity DNA polymerase I. SSP crosslinking should prove to 6. Carlsson, J., Drevin, H. & Axen, R. (1978) Biochem. J. 173, of 723-737. be useful in a number ofareas, including DNA structure, repair, 7. Grossetti, D. R. & Murray, J. F. (1967) Arch. Biochem. Biophys. recombination, and replication. SSP may be introduced at the 119, 41-49. site of any specific nick. The crosslinking reaction also can be 8. Bolivar, F. & Boekman, K. (1980) Methods Enzymol 68, 245-267. used to identify any sites containing mercurated bases inserted 9. Wilson, G. A. & Young, F. E. (1980) Methods Enzymol. 65, 147-153. by other techniques. 10. Hayward, G. S. & Smith, M. G. (1972)J. Mol. Biol. 63, 383-395. The reactions of this psoralen derivative are extremely flex- 11. Cech, T. R. & Pardue, M. L. (1976) Proc. Natl. Acad. Sci. USA ible. SSP is a trifunctional reagent, able to react with DNA at 72, 2644-2648. its thiol group as well as at the 3,4 and 4',5' double bonds of 12. Simpson, R. B. (1961) J. Am. Chem. Soc. 83, 4711-4717. the furocoumarin. The mercury-specific binding can be easily 13. Cantor, C. R. (1980) Ann. N. Y. Acad. Sci. 346, 379-385. 14. Parker, R. C., Watson, R. M. & Vinograd, J. (1977) Proc. Natl. reversed by adding an excess of reducing agent, whereas the Acad. Sci. USA 74, 851-855. cycloaddition to pyrimidines can be photoreversed by short 15. Dale, R. M. K. & Ward, D. C. (1975) Biochemistry 14, 2458- wavelength UV light (17). The timing and extent of the pho- 2469. toreactions can be finely controlled; irradiation at 390 nm will 16. Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, can by sub- 77-90. result in monoaddition, and crosslinks be formed 17. Straub, K., Kanne, D., Hearst, J. E. & Rapoport, H. (1981) J. sequent irradiation at 360 nm (18). Other thiol-containing re- Am. Chem. Soc. 103, 2347-2355. agents, such as spectroscopic probes, could be attached to DNA 18. Chatterjee, P. K. & Cantor, C. R. (1978) Nucleic Acids Res. 5, at known sites by the same scheme as the one outlined here. 3619-3633. Downloaded by guest on September 25, 2021