TIBS 16 - S E P T E M B E R 1 9 9 1
in IFN signal transduction. A common feature of IFN signal transduction is the speed (within one minute) of the various biochemical changes induced by IFN; this makes ordering these changes difficult. At present, all the rapid changes may be thought of as early events in IFN signal transduction, particularly if they are associated with a biological activity of IFN. A single event or combination of events could lead to the establishment of one of the pleiotropic effects of IFN. The challenge is to link all these changes into a unifying hypothesis to describe IFN signal transduction.
IT WAS SHOWN in the early 1960s that ultraviolet (IN) irradiation of bacteria induced the formation of stable complexes between proteins and DNA~,3. Ten years later this finding was used to crosslink a defined protein to DNA or homopolymers 4. Photochemical crosslinking is now a powerful method for studying protein-nucleic acids interactions s. Ultraviolet light is a 'zero length' crosslinking agent, thought to crosslink proteins to nucleic acids at their contact points without additional elements that might cause conformational perturbations (this contrasts with chemical crossllnking techniques where the length of the linker may disrupt the protein-nucleic acid complex). The progress of this technique has been made by using its classic version: irradiation with conventional UV light sources for times ranging from minutes to several hours. The development of lasers as a source of a powerful beam of monochromatic UV light meant that the number of photons required for the crosslinking may be delivered in nanoor picosecond time intervals. This possibility was first demonstrated by Harrison et al. ~ when the complex between E. coli RNA polymerase and T7 phage DNA was co-
References 1 Levy,D. and Damell,J. E., Jr (1990) New Biol. 2, 923-928 2 Yap,W. H., Teo,T. S., McCoy,E. and Tal~ Y. H. (1986) Proc. Natl Acad. ,~ci. USA 83, 7765-7769 3 Yap,W. H., Teo,T. S. and Tan, Y. H. (1986) Science 234, 355-358 4 McCoy,E. E. and Strynadka,K. (1987) Prog. Clin. BioL Resp. 246, 221-230 5 Heifer, L. M., Strulovici, B. and Saltiel, A. R. (1990) Proc. Natl Acad. Sci. USA 87, 6537-6541 6 Cataldi,A. et al. (1990) FEBS Lett. 269, 465-468 7 Mehmet, H., Morris, C. M. G., TaylorPapadimitriov,J. and Rozengurt,E. (1987) Biochem. Biophys. Res. Commun. 145, 1026-1032 8 Faltynek,C. R. et al. (1989) ./. Biol. Chem. 264, 14305-14311
9 Sekiguchi,K. et aL (1987) Biochem. Biophys. Res. Commun. 145, 797-802 10 Csermely,P., Balint, E., Grimley,P. M. and Aszalos,A. (1990)J, Interferon Res. 10, 605-611 11 Dale,T. C., Imam,A. M., Kerr, I. M. and Stark, G. R. (1989) Proc. Natl Acad. Sci. USA 86, 1203-1207 12 Reich, N. C. and Pfeffer, L. M. (1990) Proc. NaU Acad. ScL USA 87, 8761-8765 13 Sehgal,P. B., Walther,Z. and Tamm, I. (1987) Proc. Natl Acad. Sci. USA 84, 3663-3667 14 Constantinescu,S. N. et aL (1990) J. Interferon Res. 10, 589-597 15 Guy,G. R. et aL J. BioL Chem. (in press) 16 Higuchi,T. et al. (1990) J. Interferon Res. 10, 413-423 17 Baeuerle, P. A. and Baltimore,D. (1988) Science 242, 540-546 18 Roy,C. and Lebleu,B. (1990) Nucleic Acids Res. 18, 2125-2131
Crosslinking proteins to nucleic acids by ultraviolet laser ' irradiation
Ultraviolet (UV) irradiation can initZote complex formation between proteins and DNA or RNA and so can be used to study such interactions. However, crosslink formation by standard UV light sources can take up to several hours. More recently, a beam of monochromatic UV light from a laser has been used to initiate crosslinking in nano- and picosecond time intervals. As noted in an earlier TIBS article 'the advantages of short pulse times and high-energy fluxes should make this a valuable technique in the future '1. In this review we ,",aracterize laser-induced crosslinking and explore the applications of t= method.
valently linked by a single 20 ns laser pulse. The advantage of extremely short crosslinking times was explored in yon Hippel's lab, where complexes and dynamic protein-DNA interactions such as the T4 DNA replication system were studied 7. These two applications stimulated us to develop a procedure for photocrosslinking proteins to DNA in oivo by laser irradiation of nuclei and whole cells and subsequent isolation of the covalently linked comI. G. Pashev and S. I. Dimitrov are at the plexes 8. Crosslinking of protein-RNA Institute of Molecular Biology. D. Angelov is complexes with nanosecond and at the Institute of Solid State Physics, picosecond laser pulses have also been Bulgarian Academy of Sciences, 1113 Sofia, reported 9.~°. Bulgaria. © 1991,ElsevierSciencePublishers,(UK)0376--5067/91/$02.00
The use of lasers as an alternative to standard UV light sources offers important advantages. Besides the extremely short time of irradiation, the laser-induced reactions proceed via higher excited states of the nucleotide bases, which sharply increases the efficiency of crosslinking 9. In this review we summariae the peculiarities of the laser-induced crosslinking as wel] as the applications of the method in studying protein-nucleic acids interactions. The recent review by yon Hippel and co-workers is also an excellent guide to the practical applications of this promising technique n. 323
TIBS 1 6 - SEPTEMBER 1 9 9 1
generated by this method are the monophotonically induced excited states S] and T~ and the cation radicals, links between proteins and nucleic generated via the biphotonic mechacids induced by UV light are anism. With increasing light intensity, covalent ~z. They are formed through the number of both S~ and T~ states reactions of excited, highly reactive decreases and the yield of cation radstates of the nucleotide bases with icals increases until saturation is amino acid residues. The conve~tional reached ~3. The involvement of the two types of UV light source (light intensity below 10TM photons cm -2 s -~) generates triplet intermediates in the crosslinking reacexcited states (TI) through a monopho- tion can be tested by following the tonic process; these are likely to be the crosslinking quantum yield with intermediates of the crosslinking reac- increasing light intensity. If crosslinking tions s. The higher intensity UV light is a monophotonic process, the quanproduced from lasers (1023-1027 pho- tum yield should be constant in the tons cm -2 s -~) induces many excited absence of any biphotonic component bases in singlet (S~) and triplet (T~) or should decrease if such a component states, thus increasing the possibility exists but does not participate in for absorption of a second photon and, crosslinking, in the case of biphotonic respectively, the transition to the higher crosslinking, the quantum yield versus excited states 7", and S, with an energy intensity should exhibit a linear depenof 8-9 eV (Ref. 13). This energy exceeds dence at low intensity followed by a the ionization potential of the bases in saturation at increased intensities. There is contradictory evidence consolution and leads to generation of pyrimidine and purine cation radicals ~3. cerning the character of the photoproThus, the first intermediate species cesses involved in crosslinking. While
Characterizationof laser-induced crosslinking Photochemistff of the reactions. The cross-
the data of Harrison et ai. 6 and Hockensmith et al. 7 suggest a monophotonic mechanism, our results 8, as well as those of Budowski et al. 9 and Dobrov et al.~°, indicate that a biphotonic process operates in crosslinking, thus supporting earlier conclusions (for review see Ref. 13) for two-step photoreactions upon laser irradiation of nucleic acids. This is supported by the finding that laser induces crosslinks that cannot arise at all via lower excitation states 9. A reason for these disagreements might be that the range of intensities used by Hockensmith et al. 7 correspond to the region where the quantum yield for crosslinking is saturated 8,~3. The exact mechanisms of the covalent bond formation are not clear. It seems that the actual intermediates are the radicals, mainly cationic radicals, if biphotonic processes are taking place. Induction of crosslinks directly via highly excited states (as is thought by some author~ 9) seems unlikely since these states have very short life times (approximately 10-~3s). Assuming that
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TIBS 16 - SEPTEMBER 1991 the favorable mutual disposition of the two reactants at one moment is a prerequisite for crosslinki:~g, such a short time is obviously insufficient. The rate of cresslink fomlatlon is determined by the time required for base excitation and the duration of the subsequent chemical part of the reaction. While base excitation takes several nano- or picoseconds, the timespan of the chemical reaction is not precisely known, it is possible that the lifetimes of the free nucleotide radicals exceed the microsecond range"; if such longlived intermediates are involved in crosslinking, they could link molecules that have not been in contact at the precise instant of the pulse. Long-lived radicals (lifetimes more than 25 ns) are thought to be efficiently quenched by 5mM 2-mercaptoethanol~. Using this agent reduced crosslinking efficiencies of 18-50% have been reported0-9. However, until the exact mechanism of crosslink formation is known, these points should he treated with caution. According to the recent experiments and calculations of Hockensmith et aL u, the chemical part of the crosslinking process is completed in less than one microsecond. With these data in mind and considering the lifetimes of the non-quenched radicals, as well as the rates of the microconformational transitions in the macromolecules (more than 100 microseconds, see Ref. 15), it appears that the laser pulse can freeze molecules undergoing movements in one particular conformation. Specificity of crossllnklng. Initial studies with conventional UV light reported a lack of base specificity4 but also linking to pyrimidines only 7. The base specificity of crosslinking upon laser excitation has b e e n studied by Hockensmith et al. 7 it has been most effective with the pyrimidine-containing oligonucleotides, thymidine being by far the most efficient crosslinker at all wavelengths. Crosslinking through purine nucleotides has only been reported at 204 nm. However, all these data have been obtained with singlestranded homooligonucleotides;the situation with double-stranded DNA is not clear. It is generally assumed, from a structural po|nt of view, that all amino acids are potential candidates for involvement in a covalent linkage with DNA or RNAS,12. Efficiency of cresslinking. As UV crosslinking is direct, the links are formed only between DNA/RNA and proteins
that are bound to them. The role of some parameters of the complex formation, such as binding affinities and the geometry of the interactions (in terms of proper positioning to thymidine) in the efficiency of crosslinking was studied by yon Hippel's group". Their data have been obtained with model systems using short single-stranded or double-stranded oligomers. However, the very process of excitation of a base in the native DNA polymer might differ from that when a free base or short synthetic homooligonucleotide is irradiated in solution ]3. An important characteristic of the crosslinking procedure is its efficiency, defined as yield of crosslinked product. This has two aspects: (I) the maximum amount of complexes that can be crosslinked, and (2) the quantum yield of the crosslinking reaction, defined as the number of DNA-bound proteins that are crosslinked per absorbed photon. According to the data so far reported, 5--20% of irradiated complexes have been crosslinked~'~2. As for the quantum yield, maximum values are in the order of I0-~I0 3 (Refs 11,13) depending on both the nature of the nucleoprotein complexes used and the laser pulse duration.
lasers induce more single-strand breaks in DNA and RNA than does the UV lamp. The rate of break formation in DNA upon picosecond and nanosecond laser irradiation is comparable to that of crosslinking. In RNA-protein complexes, however, more breaks than crosslinks have been induced by both laser regimes. The rate of formation of interstrand DNA-DNA crosslinks varied with the intensity of irradiation and was at least an order of magnitude lower than the rate of crosslinking, in spite of the source of the UV light ~3. In most of the papers o~ UV light crosslinking, photodestruction of proteins has been commented on rather than studied5.608.The first details were recently presented by Hockensmith et al. u Using a laser, they observed the maximum photodestruction at high photon flux (I0 ~7photons at 254 nm) in the range 220-240 nm, where the peptide bonds absorb. Indeed, at higher wavelengths (266 nm), which are often used for crosslinking proteins to DNA by laser irradiation, there is no detectable damage of proteins ~. Stability of the linkage. Protein-nucleic acids crosslinks, introduced by a lowintensity UV lamp, are resistant to both heat and alkali4 but are unstable in acid. Photochemical reactions other than Treatment with 1 M acetic acid or 6 M cresslinking. UV irradiation can cause HCI for 15 min at 25°C results in a comintramolecular alterations such as plete breakdown of the complexes~E pyrimidine dimer formation, breaks of Data for stability of the laser-induced sugar phosphate chains of DNA and linkage are not available. RNA, interstrand DNA-DNA crosslinks and generation of local denatured sites Laser crosslinking and protein-nucleic acid in DNA, base destructions and photo- interactions The ability of the laser to 'freeze' prolesions in proteins. All these effects are of primary importance for the subse- tein-nucleic acid complexes generated quent analysis of the crosslinked com- in very rapid reactions, the relatively plexes. The frequencies of these high yield of crosslinks and the applicacompeting reactions are determined bility of the method for in vivo studies means that a large number of problems by their quantum yields. Estimation of the quantum yields could be addressed by this technique. depends on the sources of the UV light And yet, publications on the use of the (low intensity UV lamp at 254nm, UV laser for crosslinking proteins to nanosecond UV laser at 248 nm and 266 nucleic acids are scarce. Cresslinking in model systems. The intronm and picosecond UV laser at 266 nm), duction of the laser variant of UV light the conditions of the irradiation (dose and intensity of the light, the absorp- crosslinking and the study of its basic tion of the solution and the presence of characteristics were made possible by radical scavangers) and the experimen- using the following model systems: tal system used. There do, however, complexes formed by defined protein(s) appear to be some general tendencies ~3. and either native nucleic acids or short Dimer formation upon low-intensity UV synthetic oligonucleotides. These syslamp and nanosecond laser irradiation tems also allowed investigation of difproceeds at rate about 200-400 times ferent aspects of protein-nucleic acids higher than that of crosslinking, while interactions, for example bindingin the case of high-intensity picosecond constant measurements I~, determiirradiation, the rates of the two pro. nation of the size of protein-nucleic cesses are comparable. High-intensity acid binding site" and interactions of
325
TIBS 16 - S E P T E M B E R 1 9 9 1
macromolecules consisting ol several proteins with DNAs," or RNA9. In some cases, laser crosslinking provides unique information, which is demonstrated well with the DNA-ATPase complex of the T4 DNA replication system. Once formed, this complex is greatly weakened when ATP is present, so that the DNAse I footprinting does not work. Laser crosslinking, however, is fast enough to trap the transient complex, so allowing analysis of the protein-DNA contacts".
histones upon active transcription of the ribosomal genes. It was found that the coding sequences, as well as the enhancer-promoter elements of the ribosomal spacer, were associated with histones in spite of the level of expression of the ribosomal gene (Ref. 19, manuscript submitted).
Conclusions and perspectives Unlike the conventional UV light, the UV laser delivers high-power monochromatic light in a rapid and well Crosslinking by irradiation of nuclei and cells. controlled manner. When used for Following the in vitro crosslinking of crosslinking proteins to DNA or RNA, E. coli RNA polymerase to T7 DNA6 and these characteristics of the laser reflect T4-coded gene 32 protein to synthetic an important property: a possibility to oligonucleotides 7 by a single nanosec- fix labile protein-nucleic acid complexes ond laser pulse, a procedure was devel- in amounts sufficient for further oped in our laboratory for crosslinking analysis. These advents of the UV laser chromosomal proteins to DNA by could be used for both in vitro (model picosecond laser irradiation of nuclei systems) and in v i v o (nuclei and and whole cells s. The extremely short whole cells) studies. A combination time of irradiation makes the redistribu- with the stop-flow method would greatly tion of DNA-bound protein molecules facilitate investigations of rapid and unlikely, thus allowing one to 'freeze' in dynamic binding equilibria in model vivo existing interactions, and to isolate systems. In the latter case, the use of the complexes (from nuclei) and charac- immunochemical procedures enables terize co-existing complexes in the the search for binding of selected prowhole cells. All histones, as well as the teins to specific DNA sequences. A prohigh mobility group 1 protein, were cedure could be designed to map profound crosslinked to DNA8. Further, tein-DNA contact points; a flow cuvette covalent linkages between core histones with a built-in thermocouple would and DNA was accomplished solely via allow irradiation of cells at defined time the amino-terminal domains ~7,~s. This intervals immediately after heat shock. peculiar property of the crosslinking The laser version of the UV light reaction was exploited to demonstrate crosslinking method is not popular yet, that hyperacetylation of the amino- but its obvious potential as a terminal histone tails did not release protein-nucleic acid crosslinking agent them from interactions with DNA~7. will hopefully stimulate many investigaFurthermore, the UV laser irradiation tors to develop the use of this techof nuclei was used to follow the fate of nique for various purposes.
Acknowledgement We are greatly indebted to Dr yon
Hippel for encouraging us to write this review and for the preprint of his methodological review. References 1 Welsh,J. and Cantor,C. R. (1984) Trends Biochem. Sci. 9, 505-507 2 Smith, K. C. (1962) Biochem. Biophys. Res. Commun. 8, 157-163 3 Alexander,P. and Moroson,H. (1962) Nature 194, 882-883 4 Markovitz,A. (1972) Biochim. Biophys. Acta 281, 522-534 5 SheUar,M. D. (1980) in Photochem. PhotobioL Rev. VoL 5 (Smith,K. C., ed.), pp. 105-197, Plenum 6 Harrison,C. A., Turner,D. H. and Hinkle, D. C. (1982) Nucleic Acids Res. 10, 2399-2414 7 Hockensmith,J. W., Kubasek,W. L., Voracnek, W. R. and von Hippel,P. H. (1986) J. Biol. Chem. 261, 2512-2518 8 Angelov,D. et al. (1988) Nucleic Acids Res. 16, 4525-4538 9 Budovsky,E. I. et aL (1986) Eur. J. Biochem. 159, 95-101 10 Dobrov,E. N. et al. (1989) Photochem. Photobiol. 49, 595-598 11 Hockensmith,J. W. et al. in Methods in Enzymelogy(in press) 12 Smith,K. C. (1970) Biochem. Biophys. Res. Commun. 39, 1011-1016 13 Nikogosyan,D. N. (1990) Int. J. Rad. BioL 57, 233-299 14 Bensasson,R. V., Land,E. J. and Trescott,T. G. (1983) in Rash Photolysis and Pulse Radiolysis, pp. 121-134, Pergamon 15 Cared,G., Fasella,P. and Gratton,E. (1975) CRC Crit. Rev. Biochem. 3, 141-164 16 Parsdiso,P. R., Nakashima,Y. and Konigsberg,W. (1979) J. BioL Chem. 254, 4739-4744 17 Stefanovsky,V. Y. et al. (1989) Nucleic Acids Res. 17, 10069-10061 18 Stefanovsky,V. Y., Dimitrov,S. I., Angelov,D. and Pashev,I. (1989) Blochem. Biophys. Res. Commun. 164, 304-310 19 Dimitrov,S. I. et al. (1990) Nucleic Acids Res. 18, 6393-6397
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in IFN signal transduction. A common feature of IFN signal transduction is the speed (within one minute) of the various biochemical changes induced by IFN; this makes ordering these changes difficult. At present, all the rapid changes may be thought of as early events in IFN signal transduction, particularly if they are associated with a biological activity of IFN. A single event or combination of events could lead to the establishment of one of the pleiotropic effects of IFN. The challenge is to link all these changes into a unifying hypothesis to describe IFN signal transduction.
IT WAS SHOWN in the early 1960s that ultraviolet (IN) irradiation of bacteria induced the formation of stable complexes between proteins and DNA~,3. Ten years later this finding was used to crosslink a defined protein to DNA or homopolymers 4. Photochemical crosslinking is now a powerful method for studying protein-nucleic acids interactions s. Ultraviolet light is a 'zero length' crosslinking agent, thought to crosslink proteins to nucleic acids at their contact points without additional elements that might cause conformational perturbations (this contrasts with chemical crossllnking techniques where the length of the linker may disrupt the protein-nucleic acid complex). The progress of this technique has been made by using its classic version: irradiation with conventional UV light sources for times ranging from minutes to several hours. The development of lasers as a source of a powerful beam of monochromatic UV light meant that the number of photons required for the crosslinking may be delivered in nanoor picosecond time intervals. This possibility was first demonstrated by Harrison et al. ~ when the complex between E. coli RNA polymerase and T7 phage DNA was co-
References 1 Levy,D. and Damell,J. E., Jr (1990) New Biol. 2, 923-928 2 Yap,W. H., Teo,T. S., McCoy,E. and Tal~ Y. H. (1986) Proc. Natl Acad. ,~ci. USA 83, 7765-7769 3 Yap,W. H., Teo,T. S. and Tan, Y. H. (1986) Science 234, 355-358 4 McCoy,E. E. and Strynadka,K. (1987) Prog. Clin. BioL Resp. 246, 221-230 5 Heifer, L. M., Strulovici, B. and Saltiel, A. R. (1990) Proc. Natl Acad. Sci. USA 87, 6537-6541 6 Cataldi,A. et al. (1990) FEBS Lett. 269, 465-468 7 Mehmet, H., Morris, C. M. G., TaylorPapadimitriov,J. and Rozengurt,E. (1987) Biochem. Biophys. Res. Commun. 145, 1026-1032 8 Faltynek,C. R. et al. (1989) ./. Biol. Chem. 264, 14305-14311
9 Sekiguchi,K. et aL (1987) Biochem. Biophys. Res. Commun. 145, 797-802 10 Csermely,P., Balint, E., Grimley,P. M. and Aszalos,A. (1990)J, Interferon Res. 10, 605-611 11 Dale,T. C., Imam,A. M., Kerr, I. M. and Stark, G. R. (1989) Proc. Natl Acad. Sci. USA 86, 1203-1207 12 Reich, N. C. and Pfeffer, L. M. (1990) Proc. NaU Acad. ScL USA 87, 8761-8765 13 Sehgal,P. B., Walther,Z. and Tamm, I. (1987) Proc. Natl Acad. Sci. USA 84, 3663-3667 14 Constantinescu,S. N. et aL (1990) J. Interferon Res. 10, 589-597 15 Guy,G. R. et aL J. BioL Chem. (in press) 16 Higuchi,T. et al. (1990) J. Interferon Res. 10, 413-423 17 Baeuerle, P. A. and Baltimore,D. (1988) Science 242, 540-546 18 Roy,C. and Lebleu,B. (1990) Nucleic Acids Res. 18, 2125-2131
Crosslinking proteins to nucleic acids by ultraviolet laser ' irradiation
Ultraviolet (UV) irradiation can initZote complex formation between proteins and DNA or RNA and so can be used to study such interactions. However, crosslink formation by standard UV light sources can take up to several hours. More recently, a beam of monochromatic UV light from a laser has been used to initiate crosslinking in nano- and picosecond time intervals. As noted in an earlier TIBS article 'the advantages of short pulse times and high-energy fluxes should make this a valuable technique in the future '1. In this review we ,",aracterize laser-induced crosslinking and explore the applications of t= method.
valently linked by a single 20 ns laser pulse. The advantage of extremely short crosslinking times was explored in yon Hippel's lab, where complexes and dynamic protein-DNA interactions such as the T4 DNA replication system were studied 7. These two applications stimulated us to develop a procedure for photocrosslinking proteins to DNA in oivo by laser irradiation of nuclei and whole cells and subsequent isolation of the covalently linked comI. G. Pashev and S. I. Dimitrov are at the plexes 8. Crosslinking of protein-RNA Institute of Molecular Biology. D. Angelov is complexes with nanosecond and at the Institute of Solid State Physics, picosecond laser pulses have also been Bulgarian Academy of Sciences, 1113 Sofia, reported 9.~°. Bulgaria. © 1991,ElsevierSciencePublishers,(UK)0376--5067/91/$02.00
The use of lasers as an alternative to standard UV light sources offers important advantages. Besides the extremely short time of irradiation, the laser-induced reactions proceed via higher excited states of the nucleotide bases, which sharply increases the efficiency of crosslinking 9. In this review we summariae the peculiarities of the laser-induced crosslinking as wel] as the applications of the method in studying protein-nucleic acids interactions. The recent review by yon Hippel and co-workers is also an excellent guide to the practical applications of this promising technique n. 323
TIBS 1 6 - SEPTEMBER 1 9 9 1
generated by this method are the monophotonically induced excited states S] and T~ and the cation radicals, links between proteins and nucleic generated via the biphotonic mechacids induced by UV light are anism. With increasing light intensity, covalent ~z. They are formed through the number of both S~ and T~ states reactions of excited, highly reactive decreases and the yield of cation radstates of the nucleotide bases with icals increases until saturation is amino acid residues. The conve~tional reached ~3. The involvement of the two types of UV light source (light intensity below 10TM photons cm -2 s -~) generates triplet intermediates in the crosslinking reacexcited states (TI) through a monopho- tion can be tested by following the tonic process; these are likely to be the crosslinking quantum yield with intermediates of the crosslinking reac- increasing light intensity. If crosslinking tions s. The higher intensity UV light is a monophotonic process, the quanproduced from lasers (1023-1027 pho- tum yield should be constant in the tons cm -2 s -~) induces many excited absence of any biphotonic component bases in singlet (S~) and triplet (T~) or should decrease if such a component states, thus increasing the possibility exists but does not participate in for absorption of a second photon and, crosslinking, in the case of biphotonic respectively, the transition to the higher crosslinking, the quantum yield versus excited states 7", and S, with an energy intensity should exhibit a linear depenof 8-9 eV (Ref. 13). This energy exceeds dence at low intensity followed by a the ionization potential of the bases in saturation at increased intensities. There is contradictory evidence consolution and leads to generation of pyrimidine and purine cation radicals ~3. cerning the character of the photoproThus, the first intermediate species cesses involved in crosslinking. While
Characterizationof laser-induced crosslinking Photochemistff of the reactions. The cross-
the data of Harrison et ai. 6 and Hockensmith et al. 7 suggest a monophotonic mechanism, our results 8, as well as those of Budowski et al. 9 and Dobrov et al.~°, indicate that a biphotonic process operates in crosslinking, thus supporting earlier conclusions (for review see Ref. 13) for two-step photoreactions upon laser irradiation of nucleic acids. This is supported by the finding that laser induces crosslinks that cannot arise at all via lower excitation states 9. A reason for these disagreements might be that the range of intensities used by Hockensmith et al. 7 correspond to the region where the quantum yield for crosslinking is saturated 8,~3. The exact mechanisms of the covalent bond formation are not clear. It seems that the actual intermediates are the radicals, mainly cationic radicals, if biphotonic processes are taking place. Induction of crosslinks directly via highly excited states (as is thought by some author~ 9) seems unlikely since these states have very short life times (approximately 10-~3s). Assuming that
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TIBS 16 - SEPTEMBER 1991 the favorable mutual disposition of the two reactants at one moment is a prerequisite for crosslinki:~g, such a short time is obviously insufficient. The rate of cresslink fomlatlon is determined by the time required for base excitation and the duration of the subsequent chemical part of the reaction. While base excitation takes several nano- or picoseconds, the timespan of the chemical reaction is not precisely known, it is possible that the lifetimes of the free nucleotide radicals exceed the microsecond range"; if such longlived intermediates are involved in crosslinking, they could link molecules that have not been in contact at the precise instant of the pulse. Long-lived radicals (lifetimes more than 25 ns) are thought to be efficiently quenched by 5mM 2-mercaptoethanol~. Using this agent reduced crosslinking efficiencies of 18-50% have been reported0-9. However, until the exact mechanism of crosslink formation is known, these points should he treated with caution. According to the recent experiments and calculations of Hockensmith et aL u, the chemical part of the crosslinking process is completed in less than one microsecond. With these data in mind and considering the lifetimes of the non-quenched radicals, as well as the rates of the microconformational transitions in the macromolecules (more than 100 microseconds, see Ref. 15), it appears that the laser pulse can freeze molecules undergoing movements in one particular conformation. Specificity of crossllnklng. Initial studies with conventional UV light reported a lack of base specificity4 but also linking to pyrimidines only 7. The base specificity of crosslinking upon laser excitation has b e e n studied by Hockensmith et al. 7 it has been most effective with the pyrimidine-containing oligonucleotides, thymidine being by far the most efficient crosslinker at all wavelengths. Crosslinking through purine nucleotides has only been reported at 204 nm. However, all these data have been obtained with singlestranded homooligonucleotides;the situation with double-stranded DNA is not clear. It is generally assumed, from a structural po|nt of view, that all amino acids are potential candidates for involvement in a covalent linkage with DNA or RNAS,12. Efficiency of cresslinking. As UV crosslinking is direct, the links are formed only between DNA/RNA and proteins
that are bound to them. The role of some parameters of the complex formation, such as binding affinities and the geometry of the interactions (in terms of proper positioning to thymidine) in the efficiency of crosslinking was studied by yon Hippel's group". Their data have been obtained with model systems using short single-stranded or double-stranded oligomers. However, the very process of excitation of a base in the native DNA polymer might differ from that when a free base or short synthetic homooligonucleotide is irradiated in solution ]3. An important characteristic of the crosslinking procedure is its efficiency, defined as yield of crosslinked product. This has two aspects: (I) the maximum amount of complexes that can be crosslinked, and (2) the quantum yield of the crosslinking reaction, defined as the number of DNA-bound proteins that are crosslinked per absorbed photon. According to the data so far reported, 5--20% of irradiated complexes have been crosslinked~'~2. As for the quantum yield, maximum values are in the order of I0-~I0 3 (Refs 11,13) depending on both the nature of the nucleoprotein complexes used and the laser pulse duration.
lasers induce more single-strand breaks in DNA and RNA than does the UV lamp. The rate of break formation in DNA upon picosecond and nanosecond laser irradiation is comparable to that of crosslinking. In RNA-protein complexes, however, more breaks than crosslinks have been induced by both laser regimes. The rate of formation of interstrand DNA-DNA crosslinks varied with the intensity of irradiation and was at least an order of magnitude lower than the rate of crosslinking, in spite of the source of the UV light ~3. In most of the papers o~ UV light crosslinking, photodestruction of proteins has been commented on rather than studied5.608.The first details were recently presented by Hockensmith et al. u Using a laser, they observed the maximum photodestruction at high photon flux (I0 ~7photons at 254 nm) in the range 220-240 nm, where the peptide bonds absorb. Indeed, at higher wavelengths (266 nm), which are often used for crosslinking proteins to DNA by laser irradiation, there is no detectable damage of proteins ~. Stability of the linkage. Protein-nucleic acids crosslinks, introduced by a lowintensity UV lamp, are resistant to both heat and alkali4 but are unstable in acid. Photochemical reactions other than Treatment with 1 M acetic acid or 6 M cresslinking. UV irradiation can cause HCI for 15 min at 25°C results in a comintramolecular alterations such as plete breakdown of the complexes~E pyrimidine dimer formation, breaks of Data for stability of the laser-induced sugar phosphate chains of DNA and linkage are not available. RNA, interstrand DNA-DNA crosslinks and generation of local denatured sites Laser crosslinking and protein-nucleic acid in DNA, base destructions and photo- interactions The ability of the laser to 'freeze' prolesions in proteins. All these effects are of primary importance for the subse- tein-nucleic acid complexes generated quent analysis of the crosslinked com- in very rapid reactions, the relatively plexes. The frequencies of these high yield of crosslinks and the applicacompeting reactions are determined bility of the method for in vivo studies means that a large number of problems by their quantum yields. Estimation of the quantum yields could be addressed by this technique. depends on the sources of the UV light And yet, publications on the use of the (low intensity UV lamp at 254nm, UV laser for crosslinking proteins to nanosecond UV laser at 248 nm and 266 nucleic acids are scarce. Cresslinking in model systems. The intronm and picosecond UV laser at 266 nm), duction of the laser variant of UV light the conditions of the irradiation (dose and intensity of the light, the absorp- crosslinking and the study of its basic tion of the solution and the presence of characteristics were made possible by radical scavangers) and the experimen- using the following model systems: tal system used. There do, however, complexes formed by defined protein(s) appear to be some general tendencies ~3. and either native nucleic acids or short Dimer formation upon low-intensity UV synthetic oligonucleotides. These syslamp and nanosecond laser irradiation tems also allowed investigation of difproceeds at rate about 200-400 times ferent aspects of protein-nucleic acids higher than that of crosslinking, while interactions, for example bindingin the case of high-intensity picosecond constant measurements I~, determiirradiation, the rates of the two pro. nation of the size of protein-nucleic cesses are comparable. High-intensity acid binding site" and interactions of
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TIBS 16 - S E P T E M B E R 1 9 9 1
macromolecules consisting ol several proteins with DNAs," or RNA9. In some cases, laser crosslinking provides unique information, which is demonstrated well with the DNA-ATPase complex of the T4 DNA replication system. Once formed, this complex is greatly weakened when ATP is present, so that the DNAse I footprinting does not work. Laser crosslinking, however, is fast enough to trap the transient complex, so allowing analysis of the protein-DNA contacts".
histones upon active transcription of the ribosomal genes. It was found that the coding sequences, as well as the enhancer-promoter elements of the ribosomal spacer, were associated with histones in spite of the level of expression of the ribosomal gene (Ref. 19, manuscript submitted).
Conclusions and perspectives Unlike the conventional UV light, the UV laser delivers high-power monochromatic light in a rapid and well Crosslinking by irradiation of nuclei and cells. controlled manner. When used for Following the in vitro crosslinking of crosslinking proteins to DNA or RNA, E. coli RNA polymerase to T7 DNA6 and these characteristics of the laser reflect T4-coded gene 32 protein to synthetic an important property: a possibility to oligonucleotides 7 by a single nanosec- fix labile protein-nucleic acid complexes ond laser pulse, a procedure was devel- in amounts sufficient for further oped in our laboratory for crosslinking analysis. These advents of the UV laser chromosomal proteins to DNA by could be used for both in vitro (model picosecond laser irradiation of nuclei systems) and in v i v o (nuclei and and whole cells s. The extremely short whole cells) studies. A combination time of irradiation makes the redistribu- with the stop-flow method would greatly tion of DNA-bound protein molecules facilitate investigations of rapid and unlikely, thus allowing one to 'freeze' in dynamic binding equilibria in model vivo existing interactions, and to isolate systems. In the latter case, the use of the complexes (from nuclei) and charac- immunochemical procedures enables terize co-existing complexes in the the search for binding of selected prowhole cells. All histones, as well as the teins to specific DNA sequences. A prohigh mobility group 1 protein, were cedure could be designed to map profound crosslinked to DNA8. Further, tein-DNA contact points; a flow cuvette covalent linkages between core histones with a built-in thermocouple would and DNA was accomplished solely via allow irradiation of cells at defined time the amino-terminal domains ~7,~s. This intervals immediately after heat shock. peculiar property of the crosslinking The laser version of the UV light reaction was exploited to demonstrate crosslinking method is not popular yet, that hyperacetylation of the amino- but its obvious potential as a terminal histone tails did not release protein-nucleic acid crosslinking agent them from interactions with DNA~7. will hopefully stimulate many investigaFurthermore, the UV laser irradiation tors to develop the use of this techof nuclei was used to follow the fate of nique for various purposes.
Acknowledgement We are greatly indebted to Dr yon
Hippel for encouraging us to write this review and for the preprint of his methodological review. References 1 Welsh,J. and Cantor,C. R. (1984) Trends Biochem. Sci. 9, 505-507 2 Smith, K. C. (1962) Biochem. Biophys. Res. Commun. 8, 157-163 3 Alexander,P. and Moroson,H. (1962) Nature 194, 882-883 4 Markovitz,A. (1972) Biochim. Biophys. Acta 281, 522-534 5 SheUar,M. D. (1980) in Photochem. PhotobioL Rev. VoL 5 (Smith,K. C., ed.), pp. 105-197, Plenum 6 Harrison,C. A., Turner,D. H. and Hinkle, D. C. (1982) Nucleic Acids Res. 10, 2399-2414 7 Hockensmith,J. W., Kubasek,W. L., Voracnek, W. R. and von Hippel,P. H. (1986) J. Biol. Chem. 261, 2512-2518 8 Angelov,D. et al. (1988) Nucleic Acids Res. 16, 4525-4538 9 Budovsky,E. I. et aL (1986) Eur. J. Biochem. 159, 95-101 10 Dobrov,E. N. et al. (1989) Photochem. Photobiol. 49, 595-598 11 Hockensmith,J. W. et al. in Methods in Enzymelogy(in press) 12 Smith,K. C. (1970) Biochem. Biophys. Res. Commun. 39, 1011-1016 13 Nikogosyan,D. N. (1990) Int. J. Rad. BioL 57, 233-299 14 Bensasson,R. V., Land,E. J. and Trescott,T. G. (1983) in Rash Photolysis and Pulse Radiolysis, pp. 121-134, Pergamon 15 Cared,G., Fasella,P. and Gratton,E. (1975) CRC Crit. Rev. Biochem. 3, 141-164 16 Parsdiso,P. R., Nakashima,Y. and Konigsberg,W. (1979) J. BioL Chem. 254, 4739-4744 17 Stefanovsky,V. Y. et al. (1989) Nucleic Acids Res. 17, 10069-10061 18 Stefanovsky,V. Y., Dimitrov,S. I., Angelov,D. and Pashev,I. (1989) Blochem. Biophys. Res. Commun. 164, 304-310 19 Dimitrov,S. I. et al. (1990) Nucleic Acids Res. 18, 6393-6397
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