Site specificity of psoralen-DNA interstrand cross-linking determined by nuclease Bal31 digestion

A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of the pi-pi electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA.     

Sequence preferences of DNA interstrand cross-linking agents: dG-to-dG cross-linking at 5'-CG by structurally simplified analogues of mitomycin C

The nucleotide sequence preferences of the DNA interstrand cross-linking agents dehydroretronecine diacetate (DHRA), 2,3-bis(acetoxymethyl)-1-methylpyrrole (BAMP), dehydromonocrotaline, and dehydroretrorsine were studied by using synthetic DNA duplex fragments and polyacrylamide gel electrophoresis (PAGE). These agents have structural features in common with the reductively activated aziridinomitosene of mitomycin C (MC). Like MC, they preferentially cross-linked DNA duplexes containing the duplex sequence 5'-CG. For DHRA and BAMP interstrand cross-linked DNA duplexes, PAGE analysis of iron(II)-EDTA fragmentation reactions revealed the interstrand cross-links to be deoxyguanosine to deoxyguanosine (dG-to-dG), again analogous to DNA cross-links caused by MC. Unlike MC, DHRA could be shown to dG-to-dG cross-link a 5'-GC sequence. Furthermore, the impact of flanking sequence on the efficiency of interstrand cross-linking at 5'-CG was reduced for BAMP, with 5'-TCGA and 5'-GCGC being equally efficiently cross-linked. Possible origins of the 5'-CG sequence recognition common to all of the agents are discussed. A model is presented in which the transition state for the conversion of monoadducts to cross-links more closely resembles ground-state DNA at 5'-CG sequences.     

Anomalous cross-linking by mechlorethamine of DNA duplexes containing C-C mismatch pairs

Nitrogen mustards such as mechlorethamine have previously been shown to covalently cross-link DNA through the N7 position of the two guanine bases of a d[GXC].d[GYC] duplex sequence, a so-called 1,3 G-G-cross-link, when X-Y = C-G or T-A. Here, we report the formation of a new mechlorethamine cross-link with the d[GXC].d[GYC] fragment when X-Y is a C-C mismatch pair. Mechlorethamine cross-links this fragment preferentially between the two mismatched cytosine bases, rather than between the guanine bases. The cross-link also forms when one or both of the guanine bases of the d[GCC].d[GCC] fragment are replaced by N7-deazaguanine, and, more generally, forms with any C-C mismatch, regardless of the flanking base pairs. Piperidine cleavage of the cross-link species containing the d[GCC].d[GCC] sequence gives DNA fragments consistent with alkylation at the mismatched cytosine bases. We also provide evidence that the cross-link reaction occurs between the N3 atoms of the two cytosine bases by showing that the formation of the C-C cross-link is pH dependent for both mechlorethamine and chlorambucil. Dimethyl sulfate (DMS) probing of the cross-linked d[GCC].d[GCC] fragment showed that the major groove of the guanine adjacent to the C-C mismatch is still accessible to DMS. In contrast, the known minor groove binder Hoechst 33258 inhibits the cross-link formation with a C-C mismatch pair flanked by A-T base pairs. These results suggest that the C-C mismatch is cross-linked by mechlorethamine in the minor groove. Since C-C pairs may be involved in unusual secondary structures formed by the trinucleotide repeat sequence d[CCG]n, and associated with triplet repeat expansion diseases, mechlorethamine may serve as a useful probe for these structures.     

Phased psoralen cross-links do not bend the DNA double helix

Although the chemical reaction of psoralens with nucleic acids is well understood, the structure of psoralen-DNA cross-linked products is still not clear. Model building studies base on the crystal structure of the psoralen-thymine monoadduct suggest that each cross-link bends the DNA double helix by 46.5 degrees [Pearlman, D. A., Holbrook, S. R., Pirkle, D. H., & Kim, S.-H. (1985) Science (Washington, D.C.) 227, 1304-1308]. On the other hand, Sinden and Hagerman [Sinden, R. R., & Hagerman, P. J. (1984) Biochemistry 23, 6299-6303] find that, in solution, psoralen cross-linked DNA is not bent. Here we use gel electrophoresis to test the validity of the current models. We have synthesized a series of DNA fragments (21-24 base pairs in length), each containing one unique T-A site for 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) cross-linking. Because of an estimated 28 degrees unwinding of the helix by HMT [Wiesehahn, G., & Hearst, J. E. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2703-2707], one expects that the 22-bp cross-linked fragment will be repeated nearly in phase with the average helical screw when multimerized. In that sequence ligation will maximally amplify any deformation to the double helix. We find that the ligated multimers of cross-linked DNA migrate close to the multimers of non-cross-linked DNA on polyacrylamide gels. Our observations place an upper limit of 10 degrees on DNA bending induced by psoralen cross-linking and indicate unwinding by about 1 bp, as well as stiffening of the double helix. These properties are not unexpected for classical intercalators.     

Sequence specificity of psoralen photobinding to DNA: a quantitative approach.

The effects of different DNA sequences on the photoreaction of various furocoumarin derivatives was investigated from a quantitative point of view using a number of self-complementary oligonucleotides. These contained 5'-TA and 5'-AT residues, having various flanking sequences. The furocoumarins included classical bifunctional derivatives, such as 8-methoxy- and 5-methoxypsoralen, as well as monofunctional compounds, such as angelicin and benzopsoralen. Taking into an account the thermodynamic constant for noncovalent binding of each psoralen to each DNA sequence, the rate constants for the photobinding process to each fragment were evaluated. The extent of photoreaction is greatly affected by the DNA sequence examined. While sequences of the type 5'-(GTAC)n are quite reactive towards all furocoumarins, 5'-TATA exhibited a reduced rate of photobinding using monofunctional psoralens. In addition terminal 5'-TA groups were the least reactive with 5- and 8-methoxypsoralen, but not with angelicin or benzopsoralen. Also 5'-AT-containing fragments exhibited remarkably variable responses toward monofunctional or bifunctional psoralen derivatives. As a general trend the photoreactivity rate of the former is less sequence-sensitive, the ratio between maximum and minimum being less than 2 for the examined fragments. The same ratio is about 3.4 for 8-methoxypsoralen and 6.2 for 5-methoxypsoralen. This approach, in combination with footprinting studies, appears to be quite useful for a quantitative investigation of the process of covalent binding of psoralens to specific sites in DNA.     

DNA base composition determines the specificity of UvrABC endonuclease incision of a psoralen cross-link

The sequences flanking a psoralen interstrand cross-link may determine how it is repaired. Our comparison of the Escherichia coli UvrABC endonuclease incision of a variety of specific cross-link sequences in a single natural DNA fragment showed that DNA base composition determines which of two cross-linked DNA strands will be incised. G/C enrichment of the region 6-12 bases 5' of the modified T on the furan-side strand results in preferential incision of the furan-side strand. When the G/C-rich region is on the 3' side, or on neither side, incisions occur on either strand. These effects of DNA base composition suggest that UvrAB can bind in two ways to a psoralen cross-link.     

Structure of the DNA interstrand cross-link of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) cross-links a 5' TpA or a 5' ApT site by photoreacting with one thymine moiety in each DNA strand. We are interested in whether psoralen interstrand cross-links all share one structure or whether there are significant differences. In this paper, we employed a rapid method for probing the structure of the cross-link by making a series of TMP cross-linked duplexes containing specific base-pair mismatches. The relative stability provided by a base pair can be correlated with neighboring base pairs by comparing the extents of gel retardation when base-pair mismatches happen in each position. From our studies, we infer that with respect to the furan-side strand, the 5'T.A base pair of the two T.A base pairs in the TpA site is not hydrogen bonded. Immediately on each side of the cross-linked TpA site is a highly stabilized base pair. Next, a region of decreased stability occurs in each arm of a cross-linked duplex and these base pairs of least stability are located farther away from the cross-linked thymines as the lengths of the arms of the cross-linked helix increase. Finally, even in 7 M urea at 49 degrees C the cross-linked helix is hydrogen bonded at both ends of a duplex of 22 base pairs. We propose that the structures of interstrand cross-links in DNA vary appreciably with the DNA sequence, the length of the DNA duplex, and the structures of the DNA cross-linking agents.     

Sequence-selective recognition of duplex DNA through covalent interstrand cross-linking: kinetic and molecular modeling studies with pyrrolobenzodiazepine dimers

Members of a homologous series of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers with C8-O-(CH(2))(n)-O-C8' diether linkages (n = 3-6 for 2a-d, respectively) have been studied for their ability to interact with oligonucleotide duplexes containing potential target binding sites. The results confirm earlier predictions that the n = 3 analogue (2a, DSB-120) will covalently bind to a 5'-Pu-GATC-Py sequence by cross-linking opposite-strand guanines separated by 2 bp. Preference for this DNA sequence is shown using oligonucleotides with altered bases between and/or flanking these guanines. The more extended PBD dimer 2c (n = 5) can span an extra base pair and cross-link the 5'-Pu-GA(T/A)TC-Py sequence. The ability of each homologue to cross-link linear plasmid DNA has been determined, with a rank order that correlates with the reported order of in vitro cytotoxicity: n = 3 (2a) > n = 5 (2c) > n = 6 (2d) > n = 4 (2b). The n = 3 homologue (2a) is >300-fold more efficient at cross-linking DNA than the clinically used cross-linking agent melphalan under the same conditions. Kinetic studies reveal that the n = 3 and 5 dimers achieve faster cross-linking to plasmid DNA (108 and 81% cross-linking h(-1) microM(-1) at 37 degrees C, respectively), whereas the n = 4 and 6 homologues are significantly less efficient at 10.3 and 23% cross-linking h(-1) microM(-1), respectively. Alternating activity for the odd n and even n dimers is probably due to configurational factors governed by the spatial separation of the PBD subunits and the flexible character of the tethering linkage. Molecular modeling confirms the order of cross-linking reactivity, and highlights the role of linker length in dictating sequence recognition for this class of DNA-reactive agent.     

Optimization of the efficiency of cross-linking PtII oligonucleotide phosphorothioate complexes to complementary oligonucleotides.

We have investigated the efficiency with which PtII complexes cross-link phosphorothioates of oligonucleotides to complementary DNA targets. The A and G residues 2-5 bases downstream from the 5'-phosphorothioate group are preferred sites for cross-linking. Replacement of residues in this part of the target by T residues results in greatly decreased cross-linking when cis platinum diammine dichloride (cisPtII) or potassium platinous chloride (K2PtCl4) are used. Trans platinum diammine dichloride (transPtII) forms cross-links with T residues if A and G residues are absent from the susceptible region of the target. Oligomers containing an internal phosphorothioate group can also be linked to their templates with transPtII, but not with cisPtII or K2PtCl4. Cross-linking via an internal phosphorothioate group tends to be less efficient than cross-linking via a 5'-terminal phosphorothioate. The Sp isomers of internal phosphorothioates are cross-linked more efficiently than the Rp isomers. Preliminary experiments suggest that the efficiency of cross-linking to RNA targets will prove similar to that found for DNA targets.     

Alkaline gel electrophoresis of deoxyribonucleic acid photoreacted with trimethylpsoralen: rapid and sensitive detection of interstrand cross-links

Restriction fragments of phage lambda and phi X174 deoxyribonucleic acid (DNA) were photoreacted with 4,5',8-trimethylpsoralen to various extents, and the amount of covalent cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The DNA was then analyzed by electrophoresis in alkaline agarose gels. A single cross-link in a DNA molecule produced a large decrease in its electrophoretic mobility. With DNA fragments 0.3--4 kilobase pairs in size, the apparent Mr (molecular weight) of the cross-linked DNA was 2.0 +/- 0.1 times and Mr of the unreacted, single-stranded DNA. A single cross-link in a larger DNA molecule resulted in an even greater increase in apparent Mr. Further cross-linking produced a decrease in the apparent Mr of the DNA, reaching a plateau at a value of 1.4 +/- 0.1 times the Mr of the unreacted, single-stranded DNA over a large range of fragment sizes (0.6--10 kilobase pairs). The apparent Mr of the cross-linked DNA was weakly dependent on the percentage of agarose in the gel. Although highly sensitive to interstrand cross-links the electrophoretic mobilities appeared to be unaffected by low levels of monoadducts (trimethylpsoralen covalently bound to one strand of the DNA). The DNA bandwidths increased by as much as 4-fold at low extents of cross-linking, presumably due to heterogeneity in the locations of the cross-links in the DNA molecules. The bands became sharp again at high levels of reaction. These observations from the basis of a new assay for interstrand DNA cross-links that is both more sensitive and more convenient than previous methods.     

Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase

The bifunctional reagents para-phenyldimaleimide and maleimidobenzoyl-N-hydroxysuccinimide ester were used to chemically cross-link the subunits of the transducin and cGMP phosphodiesterase (PDE) complexes of bovine rod photoreceptor cells. The cross-linked products were identified by Western immunoblotting using antisera against purified subunits of transducin (T alpha and T beta gamma) and PDE. Oligomeric cross-linked products of transducin subunits as large as (T alpha beta gamma)3 were observed in the latent form of transducin with bound GDP. In addition to the expected T alpha beta and T beta gamma cross-linked products, a (T alpha gamma)2 structure was detected. The close proximity of T alpha and T gamma suggests that T gamma may play a role in conferring the specificity of the interaction between T alpha and rhodopsin. Most of the oligomeric cross-linked structures between T alpha and T beta gamma were diminished in the activated form of transducin, with guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp(NH)p) bound. However, cross-linking between T beta and T gamma was not altered. These results suggest that transducin exists as an oligomer in solution which dissociates upon the binding of Gpp(NH)p. To identify the possible interacting domains between the T alpha, T beta, and T gamma subunits, the cross-linked products were subjected to limited tryptic proteolysis. Several cross-linked tryptic peptides of transducin subunits were found and include the cross-linked products of the N terminus 15-kDa fragment of T beta and the C terminus 5-kDa fragment of T alpha, T gamma and the 12-kDa fragment of T alpha, T gamma and the 15-kDa as well as the 23-kDa fragments of T beta, and an intra-T alpha cross-linked product of the 2- and 21-kDa fragments. These results have allowed the construction of a topographical model for the transducin subunits. The organization of the subunits of PDE (P alpha, P beta, and P gamma) was also studied. The formation of the high molecular size cross-linked products of PDE resulted in the concurrent loss of the P beta and P gamma subunits, suggesting that they are in close proximity. Finally, the interaction between transducin and PDE was examined by chemical cross-linking of transducin-Gpp(NH)p and PDE. Two additional cross-linked products of 180 and 210 kDa were obtained which could be due to the cross-linking of T alpha or T beta with P alpha beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence-specific DNA interstrand cross-linking by an aziridinomitosene in the absence of exogenous reductant

The aziridinomitosene derivative (1S,2S)-6-desmethyl(methylaziridino)mitosene (4) was shown to alkylate plasmid DNA at pH 7.4 in the absence of a reducing agent [Vedejs, E., Naidu, B. N., Klapars, A., Warner, D. L., Li, V. -s., Na, Y., and Kohn, H. (2003) J. Am. Chem. Soc. 125, 15796-15806], an activity not found in the parent mitomycins. We sought to evaluate aziridinomitosene 4 for the presence of DNA interstrand cross-linking activity using nonreductive reaction conditions. Radiolabeled DNA treated with 4 was analyzed by denaturing polyacrylamide gel electrophoresis (DPAGE), a technique that readily separates the less mobile cross-linked ds DNA from the more mobile ss DNA products. Nonreduced 4 produced an interstrand cross-link (ICL) in duplex DNA containing 5'-d(CG) sites, and the yield of ICL was comparable to that obtained from reduced MC under similar conditions. A ds DNA having the central tetranucleotide 5'-d(ACGT) provided the greatest ICL yield from both nonreduced 4 and reduced MC. Substitution of 5'-d(CG) with the inverted sequence 5'-d(GC) completely abolished interstrand cross-linking by 4, revealing 5'-d(CG) as its specific site of ICL formation. Replacement of dG at 5'-d(CG) with 2'-deoxyinosine (dI), which lacks the exocyclic C2 amino group present in dG, also prevented DNA ICL formation by 4, revealing an essential role for the dG C2 amino group in the interstrand cross-linking reaction between 4 and duplex DNA. This report directly demonstrates the presence of bifunctional alkylating activity in a nonreduced aziridinomitosene and clearly shows that unreduced 4 alkylates residues in the minor groove of ds DNA, cross-linking with the same 5'-d(CG) sequence specificity displayed by reduced MC.     

DNA sequence specificity of mitomycin cross-linking

Using a gel electrophoresis assay, we show that the target DNA sequence cross-linked by N-methylmitomycin A, its aziridinomitosene, and mitomycin C is CpG, in strong preference over GpC. The yield per CpG site increases as the number of successive CpG sequences increases. Molecular modeling reveals no systematic difference between the energies of mitomycin cross-links at CpG in comparison with GpC. However, the distance between guanine amino groups in CpG sequences is nearly the same as the distance in the cross-linked adduct, whereas the amino group separation at GpC sites is substantially larger in the starting DNA than in the adduct. We suggest that the favorable placement of the second reaction center in CpG greatly accelerates the second step in the cross-linking reaction. As shown by a competition assay, mitomycins bind A-T and G-C sequences noncovalently equally well, even though the only sequence that yields appreciable cross-linking is CpG. N-Methylmitomycin A and its aziridinomitosene are found to be better cross-linking agents than is mitomycin C.     

DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footprinting.

The technique of DNAase I footprinting has been used to investigate preferred binding sites for actinomycin D and distamycin on a 160-base-pair DNA fragment from E. coli containing the tyr T promoter sequence. Only sites containing the dinucleotide step GpC are protected by binding of actinomycin, and all such sites are protected. Distamycin recognizes four major regions rich in A + T residues. Both antibiotics induce enhanced rates of cleavage at certain regions flanking their binding sites. These effects are not restricted to any particular base sequence since they are produced in runs of A and T by actinomycin and in GC-rich sequences by distamycin. The observed increases in susceptibility to nuclease attack are attributed to DNA structural variations induced in the vicinity of the ligand binding site, most probably involving changes in the width of the helical minor groove.     

Cross-linking of the cAMP receptor protein of Escherichia coli by o-phenylenedimaleimide as a probe of conformation.

Reaction of the cAMP (cyclic adenosine 3'--5'-monophosphate) receptor protein (CRP) of Escherichia coli with the bifunctional reagent o-phenylenedimaleimide (oPDM) results in the cross-linking of the two subunits of a CRP protomer. In the presence of cAMP the rate of cross-linking increases. CRP modified with oPDM retains [3H]cAMP binding activity but loses [3H]d(I-C)n binding activity. Proteolysis of cross-linked CRP gives distinct sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns depending upon whether cAMP was present during the reaction with oPDM. CRP cross-linked in the absence of cAMP retains the same relative resistance to proteolysis as unmodified CRP. The presence of 0.1 mM cAMP during proteolysis results in the production of two fragments, one of approximately 13 000 daltons and a second of approximately 20 000 daltons. CRP cross-linked with oPDM in the presence of cAMP (then dialyzed to remove cAMP) remains sensitive to alpha-chymotrypsin digestion even in the absence of added cAMP producing only the 13 000-dalton fragment. It is suggested that the nature of the oPDM cross-link is a consequence of the conformational state of CRP.     

Characterization of recognition sites on bacteriophage HP1c1 DNA which interact with the DNA uptake system of Haemophilus influenzae Rd

The 32.4-kb genome of the Haemophilus influenzae bacteriophage HP1c1 contains at least twelve sites, each conferring high affinity for the DNA uptake system of transformable H. influenzae Rd. Five of these high-affinity sites have been located and their nucleotide sequences determined. Three sites contained a contiguous 9-bp sequence identical to the first nine residues of the 11-bp site previously identified as conferring high affinity for the H. influenzae transformation receptor to DNA fragments. The remaining two sites contained complete 11-bp sequences. In contrast, an HP1c1 restriction fragment containing a sequence identical to the final nine residues of the 11-bp uptake site exhibits only a low affinity for the DNA uptake system. An 8-bp sequence consisting of the first eight residues of the 11-bp site was 1% as active as the longer, high-affinity sites. Thus the first 9-bp of the 11-bp site are sufficient to direct high-affinity uptake, while the first 8-bp or the distal 9-bp are not. These results provide an initial assessment of the relative contributions of the individual residues constituting the 11-bp site to the apparent affinity of DNA fragments for the receptor of Haemophilus transformation.     

The effects of deoxycholate and trypsin on the cross-linking of rabbit skeletal muscle sarcoplasmic reticulum proteins.

Dithiobis (succinimidyl propionate) has been used to cross-link sarcoplasmic reticulum microsome proteins. Although the 100,000 dalton calcium stimulated ATPase and the 60,000 dalton calcium-binding protein calsequestrin were readily cross-linked to form homopolymers, no heteropolymer formation between these two proteins were detected. The 90,000 dalton protein A1 which is always observed in our preparations appeared to preferrentially form dimers on cross-linking. When calsequestrin was solubilized using 0.1 mg deoxycholate/mg protein, this protein was not cross-linked even at dithiobis(succinimidyl propionate) concentrations ten times those used to cross-link this protein in the intact membrane. In a similar manner the deoxycholate-solubilized ATPase (0.5 mg deoxycholate/mg protein) was not cross-linked by dithiobis (succinimidyl propionate). These results suggest that the state of aggregation of the sarcoplasmic reticulum proteins may be modified when solubilized in detergents such as deoxycholate. When the 100,000 dalton ATPase polypeptide was cleaved with trypsin to two fragments with molecular weights of approximately 55,000, these could be readily cross-linked. The fragments were capable of forming polymers with either other 55,000 dalton fragments or with the 100,000 dalton ATPase. The 29,000 and 22,000 dalton fragments, produced by further tryptic cleavage of the 55,000 dalton fragments, were not cross-linked at dithiobis (succinimidyl propionate) concentrations which readily cross-linked the 55,000 dalton fragments. Thus tryptic cleavage of the ATPase to fragments smaller than 55,000 dalton altered associations made by the ATPase in the membrane.