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Bioconjugate Chem. 2000, 11, 876−879
Dimethyl Suberimidate Cross-Linking of Oligo(dT) to DNA-Binding Proteins Mark S. Dodson* Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721-0088. Received May 9, 2000; Revised Manuscript Received September 2, 2000
Dimethyl suberimidate is a bifunctional reagent that is used for cross-linking the protein components of oligomeric macromolecules. In this report, dimethyl suberimidate is shown to specifically crosslink oligo(dT) of varying lengths to the DNA-binding subunits of a multimeric helicase-primase encoded by herpes simplex virus type 1. This result indicates that dimethyl suberimidate and other imidoester cross-linking reagents may be useful for characterizing the interaction of oligo(dT) with proteins that bind single-stranded DNA.
INTRODUCTION
Chemical cross-linking reagents are commonly used to stabilize nucleoprotein complexes for analysis by a variety of experimental techniques (1-9). Most crosslinking reagents function to covalently join reactive groups on the polypeptide moieties of a nucleoprotein complex. Covalent joining of proteins to the nucleic acid component of nucleoprotein complexes is generally less efficient since the necessary reactive groups are fewer in number and may not be exposed. Currently only a few reagents, such as aldehydes, methylene blue, and difluorodinitrobenzene, have been used to directly cross-link protein to DNA (9-11). Thus, additional reagents that cross-link protein to DNA would be useful. Herpes simplex virus type 1 (HSV-1) encodes a DNA helicase-primase that consists of three different polypeptides, UL5, UL52, and UL8 (12). Sequence homology and mutagenesis studies indicate that UL5 and UL52 confer the helicase and primase activities, respectively (13-15). The UL8 subunit does not bind DNA (16). A heterodimeric subassembly consisting of UL5 and UL52 (UL5/52) exhibits all of the activities associated with the holoenzyme, including an ATPase activity that is potently activated by oligo(dT) (17-19). The minimal length of oligo(dT) that effectively binds to UL5/52 and activates its intrinsic ATPase activity is about 16 nucleotides (19). Here it is reported that the bifunctional protein crosslinking reagent dimethyl suberimidate (DMS) can specifically cross-link oligo(dT) to the UL5 and UL52, but not to the UL8, subunits of the HSV-1 helicase primase. This result indicates that imidoesters may serve as useful reagents for characterizing the interaction of DNAbinding proteins with single-stranded DNA ligands. MATERIALS AND METHODS
Reagents. The UL5/52 subassembly and UL8 were purified as described (18, 19). Deoxythymidine oligonucleotides were obtained from Midland Certified Reagent Co. (Midland, TX). [γ-32P]ATP at 6000 Ci/mmol, bis-acrylamide, ammonium persulfate, and N,N,N′,N′tetramethylenediamine (TEMED) were from Amersham * To whom correspondence should be addressed. Phone: (520) 621-6123. Fax: (520) 621-9288. E-mail: dodson@ biosci.arizona.edu.
Life Sciences (Piscataway, NJ). T4 polynucleotide kinase and acrylamide were from Life Technologies (Gaithersburg, MD). High-range molecular weight protein standards consisting of myosin, β-galactosidase, phosphorylase b, bovine serum albumin (BSA), and ovalbumin were from Bio-Rad (Hercules, CA). Triethanolamine and dimethyl sulfoxide (DMSO) were from Sigma (St. Louis, MO). DMS was from Pierce (Rockford, IL). Cross-Linking Assays. Reaction mixtures (10 µL) contained 25 mM N-(2-hydroxyethyl)piperazine-N′-(3ethanesulfonic acid) (HEPES), pH 7.5, 3.5 mM MgCl2, 75 mM NaCl, 10% glycerol, 2 mM dithiothreitol (DTT), and the indicated amounts of end-labeled oligo(dT), UL5/ 52 subassembly, and UL8. The mixtures were incubated for 20 min at 22 °C followed by the addition of 1.5 µL of freshly prepared 180 mM DMS dissolved in either DMSO or 0.5 M triethanolamine buffer, pH 8.5. The mixtures were incubated for 15 min, and another 1.5 µL of DMS solution was added followed by an additional 15 min of incubation. The mixtures were then precipitated by the addition of trichloroacetic acid to a concentration of 10% followed by centrifugation at 12000 × g at 4 °C. The protein pellets were washed with 100 µL of acetone, dissolved in 12 µL of loading buffer consisting of 200 mM Tris-HCl, 10% (v/v) glycerol, 20 mM DTT, and 0.6% SDS, and the cross-linked products were then separated on a denaturing SDS-7.5% polyacrylamide gel. The gels were autoradiographed using an intensifying screen for 12 h at -80 °C followed by staining with Coomassie Brilliant Blue. A Molecular Dynamics PhosphorImager was used to quantitate the results. RESULTS
Cross-Linking of Oligo(dT) to the HSV-1 Helicase-Primase. In this study, mixtures containing UL5/ 52 were incubated with radiolabeled oligo(dT)16, treated either with DMSO solvent alone or with DMSO containing DMS, and the products were then separated by SDSPAGE. The gel was then subjected to autoradiography followed by staining with Coomassie Blue to visualize the UL5 and UL52 subunits (Figure 1). No difference was observed in the Coomassie-stained bands between the untreated control and the DMS cross-linked sample (Figure 1A, lanes 1 and 2, respectively). This result indicated that DMS did not cross-link UL5 and UL52
10.1021/bc000049u CCC: $19.00 © 2000 American Chemical Society Published on Web 10/21/2000
DNA−Protein Chemical Cross-Linking
Bioconjugate Chem., Vol. 11, No. 6, 2000 877
Figure 1. DMS cross-links oligo(dT)16 to both UL5 and UL52. Mixtures containing 1.1 µM 32P-labeled oligo(dT)16 and 1.4 µM UL5/52 or molecular weight protein standards were assembled and cross-linked with DMS and then separated by SDS-PAGE as described under Materials and Methods. (A) Coomassiestained gel. Lanes: 1, UL5/52, no DMS; 2, UL5/52 plus DMS; 3, molecular weight standards plus DMS. Molecular weights of the standards are indicated. (B) Autoradiograph of the same gel. Table 1. Relative Extents of DMS Cross-Linking of Oligo(dT)16 to Proteins polypeptide
relative fraction cross-linkedb
UL5 UL52 myosin (200 kDa) β-galactosidase (116 kDa) phosphorylase (97 kDa) BSA (66 kDa) ovalbumin (45 kDa)
1.00 0.64 0.08 0.05 0.03 0.14 0.02
a The cross-linked products shown in lanes 2 and 3 of Figure lB were quantitated using a Phosphorlmager. b The fraction of probe that was cross-linked to each protein relative to the amount of probe that was cross-linked to UL5 is shown.
together under the conditions used. However, in the autoradiograph, two prominent bands were observed in the lane containing the DMS-treated sample that were not observed in the lane containing the untreated sample (Figure 1B, lanes 1 and 2). The mobilities of these two bands corresponded to the mobilities of UL5 (99 kDa) and UL52 (114 kDa). This result indicates that the oligo(dT)16 probe was cross-linked to UL5 and UL52. Quantitation of the cross-linked products indicated that 0.14 and 0.09% of the probe were cross-linked to UL5 and UL52, respectively. Similar results were also obtained using labeled oligo(dT)20 as the probe and triethanolamine as the solvent for DMS (data not shown). The amount of probe that was cross-linked to UL5 was consistently higher than the amount that was cross-linked to UL52, with the ratio being 1.43 ( 0.08 to 1. DMS Cross-Linking of Oligo(dT)16 to UL5 and UL52 Is Specific. The specificity of DMS cross-linking of oligo(dT)16 to UL5 and UL52 was assessed in the above experiment by comparing the ability of DMS to crosslink the DNA to the control proteins used for molecular weight standards (Figure 1A, lane 3). Only faint traces of the probe were cross-linked to the molecular weight standards (Figure lB, lane 3). The extent of cross-linking to UL5, UL52, and the control proteins was quantitated and compared (Table 1). Cross-linking of the DNA to the control proteins ranged from 5-fold (BSA) to 50-fold (ovalabumin) less than that observed for UL5 and UL52. Effect of the Length of Oligo(dT) on DMS CrossLinking of UL5/52. The DMS cross-linking of oligo(dT) to UL5/52 was repeated in the presence and absence of a 3-fold molar excess of UL8 using oligo(dT) species ranging from 12 to 28 nucleotides in length. An excess of UL8 was used to ensure that the heterotrimeric
Figure 2. Effect of oligo(dT) length and of UL8 on cross-linking of DNA to UL5/52. Mixtures containing 0.8 µM 32P-labeled oligo(dT) of the indicated length and 0.6 µM UL5/52 were crosslinked with DMS in the presence or absence of 1.8 µM UL8, and then separated by SDS-7.5% PAGE as described in the Materials and Methods. The left two lanes of each panel show a Coomassie-stained gel containing UL5/52 alone or UL5/52 and UL8. The right two lanes of each panel show the autoradiogram of the gel. The mobilities of the subunits are indicated on the first panel.
Figure 3. Relative extents of cross-linking of oligo(dT) of varying lengths to UL5 and UL52 in the presence and absence of UL8. The data from Figure 2 were quantitated using a PhosphorImager. The ratio of DNA cross-linked to UL5 versus UL52 is plotted for oligo(dT)s of the indicated lengths. Data are the averages of two separate experiments. Filled symbols, UL8 absent; open symbols, UL8 present.
holoenzyme would be stably formed (20). Again, crosslinked products that corresponded to the mobilities of UL5 and UL52 were observed for all lengths of oligo(dT) that were used (Figure 2). No species that correlated to cross-linking of the DNA to UL8 were observed. The relative extents of cross-linking of the oligonucleotides to UL5 and UL52 were quantitated by determining the ratio of the amount of UL5-cross-linked DNA to UL52cross-linked DNA (Figure 3). This ratio is unaffected by variations in the specific activities of the labeled probes or by variations in the amount of sample loaded on the gel. For oligo(dT)s of 16 or more nucleotides in length, about twice as much DNA was cross-linked to UL5 as was cross-linked to UL52. UL8 had little effect on this ratio. However, this ratio was changed substantially when oligo(dT)12 was used as the ligand. In the absence of UL8, about four times as much DNA was cross-linked to UL5 as to UL52. In the presence of UL8 this ratio increased to about 10 to 1.
878 Bioconjugate Chem., Vol. 11, No. 6, 2000
Figure 4. Proposed mechanism for DMS cross-linking of oligo(dT) to protein. (A) The imidoester reaction scheme; (B) hypothesized opening of the thymine ring to yield a primary amine that can participate in the imidoester reaction. DISCUSSION
Because of its lack of secondary structure, oligo(dT) is frequently used for characterizing the interactions of helicases with DNA ligands (1, 8, 19, 21-23). We had originally intended to use DMS as a reagent for determining if oligo(dT) induces oligomerization of the HSV-1 helicase-primase. Under the conditions used for DMS cross-linking, we were unable to detect the formation of oligo(dT)-induced oligomers of the HSV-1 helicase-primase. However, we observed that the DNA became covalently cross-linked to UL5 and UL52. This result was unexpected since oligo(dT) lacks the functional groups that are necessary to react with imidoester-based crosslinking reagents. The studies presented in this report indicate that DMS-mediated cross-linking of oligo(dT) to UL5 and UL52 is specific since several non-DNA-binding proteins are cross-linked far less effectively to oligo(dT). The modest amount of cross-linking of oligo(dT) to BSA can be accounted for by a previous observation that BSA weakly binds DNA (24). The specificity of cross-linking of oligo(dT) to UL5 and to UL52 is further demonstrated by the failure of DMS to cross-link oligo(dT) to the nonDNA-binding UL8 subunit of the holoenzyme. This covalent cross-linking of oligo(dT) to protein by DMS is a novel result that has not been previously observed. To demonstrate the utility of DMS as a tool for characterizing nucleoprotein complexes, we used DMS cross-linking to compare the ability of UL5 and UL52 to bind oligo(dT) ligands of varying lengths. For oligo(dT) ligands longer than 16 nucleotides, the ratio of the amount of DNA that is cross-linked to UL5 and UL52 is fairly constant, even in the presence of UL8. Shorter ligands are not cross-linked to either protein as efficiently as the longer DNA species. In the presence of UL8, the amount of oligo(dT)12 that is cross-linked to UL5 is 10fold greater than for UL52. The poor cross-linking of oligo(dT)12 to UL5/52 agrees with previous nitrocellulose filter binding and enzymatic activation studies which showed that oligo(dT) ligands shorter than 16 nucleotides are poorly bound by UL5/52 and are also poor activators of the DNA-dependent ATPase activity of UL5/52 (19). Our cross-linking results demonstrate that the minimal length of the DNA-binding site in UL5 is shorter than in UL52 and that the UL8 subunit affects the interaction of short oligonucleotides with the UL5/52 subassembly. DMS cross-links primary amines on proteins by an imidoester reaction (Figure 4A) (25). Since there are no primary amines on deoxythymidine, the observation that DMS cross-links oligo(dT) to UL5 and UL52 is puzzling. One explanation could be that the oligonucleotides become trapped by a “caging” effect in which the DNA
Dodson
becomes pinned beneath cross bars of DMS that link some of the residues which form the DNA-binding sites. However, these caged structures would be expected to dissociate when denatured in the SDS-PAGE loading buffer. Alternatively, upon binding of oligo(dT) to UL5 or UL52, some thymine residues may be rendered partially susceptible to hydrolysis or damage at the 3 and 4 positions of the pyrimidine ring. This could yield a β-ureidoisobutenoic acid moiety containing a primary amino group that could participate in the imidoester cross-linking reaction (Figure 4B). The total fraction of labeled oligo(dT) that was crosslinked to protein in our study was about 0.25%. This figure is roughly comparable to the extent of halogenated oligo(dT) that can be cross-linked to UL5 and UL52 by brief exposure to long wave UV light (23). It is often necessary to test several methods in order to identify one that can be successfully used for crosslinking a specific set of macromolecules. Few methods are available for covalently cross-linking proteins to DNA. Here we have shown that imidoester cross-linking reagents such as DMS offer an alternative to these other methods. In contrast to methylene blue (10), DMS can effectively cross-link single-stranded nucleic acids to protein, and, in contrast to aldehyde-based cross-linkers, DMS can yield cross-linked species that retain enzymatic activity (1). In addition, DMS may be more appropriate than UV light for cross-linking DNA to proteins in the presence of nucleotides or other UV-absorbing compounds. Thus, the observation that imidoesters such as DMS can covalently cross-link protein to DNA expands the limited repertoire of methods that are available for studying the interaction of DNA with DNA-binding proteins. ACKNOWLEDGMENT
This research was supported in part by Grant RPG9705601NP from the American Cancer Society. The author thanks Dr. Lauren Murata and Nicoleta Constantin for helpful discussions, critical reading of the manuscript, and assistance with production of the figures. LITERATURE CITED (1) Chao, K., and Lohman, T. M. (1991) DNA-induced dimerization of the Escherichia coli Rep helicase. J. Mol. Biol. 221, 1165-1181. (2) Dean, F. B., Borowiec, J. A., Eki, T., and Hurwitz, J. (1992) The simian virus-40 T-antigen double hexamer assembles around the DNA at the replication origin. J. Biol. Chem. 267, 14129-14137. (3) Dodson, M., Roberts, J., McMacken, R., and Echols, H. (1985) Specialized nucleoprotein structures at the origin of replication of bacteriophage λ: Complexes with λ O protein and with λ O, λ P, and Escherichia coli DnaB proteins. Proc. Natl. Acad. Sci. U.S.A. 82, 4678-4682. (4) Heichman, K. A., and Johnson, R. C. (1990) The hin invertasome: protein-mediated joining of distant recombination sites at the enhancer. Science 249, 511-517. (5) Fouts, E. T., Yu, X., Egelman, E. H., and Botchan, M. R. (1999) Biochemical and electron microscopic image analysis of the hexameric El helicase. J. Biol. Chem. 274, 4447-4458. (6) Levin, M. K., and Patel, S. S. (1999) The helicase from hepatitis C virus is active as an oligomer. J. Biol. Chem. 274, 31839-31846. (7) Dong, F., Gogol, E. P., and von Hippel, P. H. (1995) The phage T4-coded DNA replication helicase (gp41) forms a hexamer upon activation by nucleoside triphosphate. J. Biol. Chem. 270, 7462-7473.
DNA−Protein Chemical Cross-Linking (8) Patel, S. S., and Hingorani, M. M. (1993) Oligomeric structure of bacteriophage T7 primase/helicase proteins. J. Biol. Chem. 268, 10668-10675. (9) Simonsson, S., Samulesson, T., and Elias, P. (1998) The herpes simplex virus type 1 origin binding protein. Specific recognition of phosphates and methyl groups defines the interacting surface for a monomeric DNA binding domain in the major groove of DNA. J. Biol. Chem. 273, 24633-24639. (10) Lalwani, R., Maiti, S., and Mukherji, S. (1990) Visible light induced DNA-protein cross-linking in DNA-histone complex and sarcoma-180 chromatin in the presence of methylene blue. Photochem. Photobiol., B 7, 57-73. (11) Toth, J., and Biggin, M. D. (2000) The specificity of proteinDNA cross-linking by formaldehyde: in vitro and in drosophila embryos. Nucleic Acids Res. 28, e4. (12) Crute, J. J., Tsurumi, T., Zhu, L., Weller, S. K., Olivo, P. D., Challberg, M. D., Mocarski, E. S., and Lehman, I. R. (1989) Herpes simplex virus 1 helicase-primase: a complex of three herpes encoded gene products. Proc. Natl. Acad. Sci. U.S.A. 86, 2186-2189. (13) Klinedinst, D. K., and Challberg, M. D. (1994) Helicaseprimase complex of herpes simplex type 1: a mutation in the UL52 subunit abolishes primase activity. J. Virol. 68, 36933701. (14) Dracheva, S., Koonin, E. V., and Crute, J. J. (1995) Identification of the primase active site of the herpes simplex virus type 1 helicase-primase. J. Biol. Chem. 270, 1414814153. (15) Graves-Woodward, K. L., Gottlieb, J., Challberg, M. D., and Weller, S. K. (1997) Biochemical analyses of mutations in the HSV-1 helicase-primase that alter ATP hydrolysis, DNA unwinding, and coupling between hydrolysis and unwinding. J. Biol. Chem. 272, 4623-4630. (16) Parry, M. E., Stow, N. D., and Marsden, H. S. (1993) Purification and properties of the herpes simplex virus type-1 UL8 protein. J. Gen. Virol. 74, 607-612. (17) Calder, J. M., and Stow, N. D. (1990). Herpes simplex virus helicase-primase: the UL8 protein is not required for DNA-
Bioconjugate Chem., Vol. 11, No. 6, 2000 879 dependent ATPase and DNA helicase activities. Nucleic Acids Res. 18, 3573-3578. (18) Dodson, M. S., and Lehman, I. R. (1991) Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase-primase composed of the UL5 and UL52 gene products. Proc. Natl. Acad. Sci. U.S.A. 88, 1105-1109. (19) Healy, S., You, X., and Dodson, M. (1997) Interactions of a subassembly of the herpes simplex virus type 1 helicaseprimase with DNA. J. Biol. Chem. 272, 3411-3415. (20) Tenney, D. J., Hurlburt, W. W., Micheletti, P. A., Bifano, M., and Hamatake, R. K. (1994) The UL8 component of the herpes-simplex virus helicase-primase complex stimulates primer synthesis by a subassembly of the UL5 and UL52 components. J. Biol. Chem. 269, 5030-5035. (21) Wong, I., Chao, K. L., Bujalowski, W., and Lohman, T. M. (1992) DNA-induced dimerization of the Escherichia coli Rep helicase. Allosteric effects of single-stranded and duplex DNA. J. Biol. Chem. 267, 7596-7610. (22) Dodson, M. S., and Lehman, I. R. (1993) The herpes simplex virus type-1 origin binding protein. DNA-dependent nucleoside triphosphatase activity. J. Biol. Chem. 268, 12131219. (23) Biswas, N., and Weller, S. K. (1999) A mutation in the C-terminal putative Zn2+ finger motif of UL52 severely affects the biochemical activities of the HSV-1 helicase-primase subcomplex. J. Biol. Chem. 274, 8068-8076. (24) Braun, A., and Merrick, B. (1975) Properties of the ultraviolet-light-mediated binding of bovine serum albumin to DNA. Photochem. Photobiol. 21, 243-247. (25) Davies, G. E., and Stark, G. R. (1970) Use of dimethyl suberimidate, a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc. Natl. Acad. Sci. U.S.A. 66, 651-656.
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