Dimethyl Suberimidate Cross-Linking of Oligo(dT) to DNA-BindingProteins
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 componentsof oligomeric macromolecules. In this report, dimethyl suberimidate is shown to specifically cross-link oligo(dT) of varying lengths to the DNA-binding subunits of a multimeric helicase-primase encodedby herpes simplex virus type 1. This result indicates that dimethyl suberimidate and other imidoestercross-linking reagents may be useful for characterizing the interaction of oligo(dT) with proteins thatbind single-stranded DNA.
INTRODUCTION
Chemical cross-linking reagents are commonly used tostabilize nucleoprotein complexes for analysis by a va-riety of experimental techniques (1-9). Most cross-linking reagents function to covalently join reactivegroups on the polypeptide moieties of a nucleoproteincomplex. Covalent joining of proteins to the nucleic acidcomponent of nucleoprotein complexes is generally lessefficient since the necessary reactive groups are fewerin number and may not be exposed. Currently only a fewreagents, such as aldehydes, methylene blue, and difluo-rodinitrobenzene, have been used to directly cross-linkprotein to DNA (9-11). Thus, additional reagents thatcross-link protein to DNA would be useful.
Herpes simplex virus type 1 (HSV-1) encodes a DNAhelicase-primase that consists of three different polypep-tides, UL5, UL52, and UL8 (12). Sequence homology andmutagenesis studies indicate that UL5 and UL52 conferthe helicase and primase activities, respectively (13-15).The UL8 subunit does not bind DNA (16). A het-erodimeric subassembly consisting of UL5 and UL52(UL5/52) exhibits all of the activities associated with theholoenzyme, including an ATPase activity that is potentlyactivated by oligo(dT) (17-19). The minimal length ofoligo(dT) that effectively binds to UL5/52 and activatesits intrinsic ATPase activity is about 16 nucleotides (19)-. Here it is reported that the bifunctional protein cross-linking reagent dimethyl suberimidate (DMS) can specif-ically cross-link oligo(dT) to the UL5 and UL52, but notto the UL8, subunits of the HSV-1 helicase primase. Thisresult indicates that imidoesters may serve as usefulreagents for characterizing the interaction of DNA-binding proteins with single-stranded DNA ligands.
MATERIALS AND METHODS
Reagents. The UL5/52 subassembly and UL8 werepurified as described (18, 19). Deoxythymidine oligo-nucleotides were obtained from Midland Certified Re-agent Co. (Midland, TX). [γ-32P]ATP at 6000 Ci/mmol,bis-acrylamide, ammonium persulfate, and N,N,N′,N′-tetramethylenediamine (TEMED) were from Amersham
Life Sciences (Piscataway, NJ). T4 polynucleotide kinaseand acrylamide were from Life Technologies (Gaithers-burg, MD). High-range molecular weight protein stan-dards consisting of myosin, â-galactosidase, phosphory-lase b, bovine serum albumin (BSA), and ovalbumin werefrom Bio-Rad (Hercules, CA). Triethanolamine and di-methyl 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′-(3-ethanesulfonic 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 incubatedfor 20 min at 22 °C followed by the addition of 1.5 µL offreshly prepared 180 mM DMS dissolved in either DMSOor 0.5 M triethanolamine buffer, pH 8.5. The mixtureswere incubated for 15 min, and another 1.5 µL of DMSsolution was added followed by an additional 15 min ofincubation. The mixtures were then precipitated by theaddition of trichloroacetic acid to a concentration of 10%followed by centrifugation at 12000 × g at 4 °C. Theprotein pellets were washed with 100 µL of acetone,dissolved in 12 µL of loading buffer consisting of 200 mMTris-HCl, 10% (v/v) glycerol, 20 mM DTT, and 0.6% SDS,and the cross-linked products were then separated on adenaturing SDS-7.5% polyacrylamide gel. The gels wereautoradiographed using an intensifying screen for 12 hat -80 °C followed by staining with Coomassie BrilliantBlue. A Molecular Dynamics PhosphorImager was usedto quantitate the results.
RESULTS
Cross-Linking of Oligo(dT) to the HSV-1 Heli-case-Primase. In this study, mixtures containing UL5/52 were incubated with radiolabeled oligo(dT)16, treatedeither with DMSO solvent alone or with DMSO contain-ing DMS, and the products were then separated by SDS-PAGE. The gel was then subjected to autoradiographyfollowed by staining with Coomassie Blue to visualize theUL5 and UL52 subunits (Figure 1). No difference wasobserved in the Coomassie-stained bands between theuntreated control and the DMS cross-linked sample(Figure 1A, lanes 1 and 2, respectively). This resultindicated that DMS did not cross-link UL5 and UL52
* To whom correspondence should be addressed. Phone: (520)621-6123. Fax: (520) 621-9288. E-mail: [email protected].
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together under the conditions used. However, in theautoradiograph, two prominent bands were observed inthe lane containing the DMS-treated sample that werenot observed in the lane containing the untreated sample(Figure 1B, lanes 1 and 2). The mobilities of these twobands corresponded to the mobilities of UL5 (99 kDa) andUL52 (114 kDa). This result indicates that the oligo(dT)16probe was cross-linked to UL5 and UL52. Quantitationof the cross-linked products indicated that 0.14 and 0.09%of the probe were cross-linked to UL5 and UL52, respec-tively. Similar results were also obtained using labeledoligo(dT)20 as the probe and triethanolamine as thesolvent for DMS (data not shown). The amount of probethat was cross-linked to UL5 was consistently higherthan the amount that was cross-linked to UL52, with theratio being 1.43 ( 0.08 to 1.
DMS Cross-Linking of Oligo(dT)16 to UL5 andUL52 Is Specific. The specificity of DMS cross-linkingof oligo(dT)16 to UL5 and UL52 was assessed in the aboveexperiment by comparing the ability of DMS to cross-link the DNA to the control proteins used for molecularweight standards (Figure 1A, lane 3). Only faint tracesof the probe were cross-linked to the molecular weightstandards (Figure lB, lane 3). The extent of cross-linkingto UL5, UL52, and the control proteins was quantitatedand compared (Table 1). Cross-linking of the DNA to thecontrol 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 Cross-Linking of UL5/52. The DMS cross-linking of oligo(dT)to UL5/52 was repeated in the presence and absence ofa 3-fold molar excess of UL8 using oligo(dT) speciesranging from 12 to 28 nucleotides in length. An excessof UL8 was used to ensure that the heterotrimeric
holoenzyme would be stably formed (20). Again, cross-linked products that corresponded to the mobilities ofUL5 and UL52 were observed for all lengths of oligo(dT)that were used (Figure 2). No species that correlated tocross-linking of the DNA to UL8 were observed. Therelative extents of cross-linking of the oligonucleotidesto UL5 and UL52 were quantitated by determining theratio of the amount of UL5-cross-linked DNA to UL52-cross-linked DNA (Figure 3). This ratio is unaffected byvariations in the specific activities of the labeled probesor by variations in the amount of sample loaded on thegel. For oligo(dT)s of 16 or more nucleotides in length,about twice as much DNA was cross-linked to UL5 aswas cross-linked to UL52. UL8 had little effect on thisratio. However, this ratio was changed substantiallywhen oligo(dT)12 was used as the ligand. In the absenceof UL8, about four times as much DNA was cross-linkedto UL5 as to UL52. In the presence of UL8 this ratioincreased to about 10 to 1.
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 µMUL5/52 or molecular weight protein standards were assembledand cross-linked with DMS and then separated by SDS-PAGEas described under Materials and Methods. (A) Coomassie-stained gel. Lanes: 1, UL5/52, no DMS; 2, UL5/52 plus DMS;3, molecular weight standards plus DMS. Molecular weights ofthe standards are indicated. (B) Autoradiograph of the samegel.
Table 1. Relative Extents of DMS Cross-Linking ofOligo(dT)16 to Proteins
polypeptiderelative fraction
cross-linkedb
UL5 1.00UL52 0.64myosin (200 kDa) 0.08â-galactosidase (116 kDa) 0.05phosphorylase (97 kDa) 0.03BSA (66 kDa) 0.14ovalbumin (45 kDa) 0.02
a The cross-linked products shown in lanes 2 and 3 of FigurelB were quantitated using a Phosphorlmager. b The fraction ofprobe that was cross-linked to each protein relative to the amountof probe that was cross-linked to UL5 is shown.
Figure 2. Effect of oligo(dT) length and of UL8 on cross-linkingof DNA to UL5/52. Mixtures containing 0.8 µM 32P-labeled oligo-(dT) of the indicated length and 0.6 µM UL5/52 were cross-linked with DMS in the presence or absence of 1.8 µM UL8,and then separated by SDS-7.5% PAGE as described in theMaterials and Methods. The left two lanes of each panel showa Coomassie-stained gel containing UL5/52 alone or UL5/52 andUL8. The right two lanes of each panel show the autoradiogramof the gel. The mobilities of the subunits are indicated on thefirst panel.
Figure 3. Relative extents of cross-linking of oligo(dT) ofvarying lengths to UL5 and UL52 in the presence and absenceof UL8. The data from Figure 2 were quantitated using aPhosphorImager. The ratio of DNA cross-linked to UL5 versusUL52 is plotted for oligo(dT)s of the indicated lengths. Data arethe averages of two separate experiments. Filled symbols, UL8absent; open symbols, UL8 present.
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DISCUSSION
Because of its lack of secondary structure, oligo(dT) isfrequently used for characterizing the interactions ofhelicases with DNA ligands (1, 8, 19, 21-23). We hadoriginally intended to use DMS as a reagent for deter-mining if oligo(dT) induces oligomerization of the HSV-1helicase-primase. Under the conditions used for DMScross-linking, we were unable to detect the formation ofoligo(dT)-induced oligomers of the HSV-1 helicase-pri-mase. However, we observed that the DNA becamecovalently cross-linked to UL5 and UL52. This result wasunexpected since oligo(dT) lacks the functional groupsthat are necessary to react with imidoester-based cross-linking reagents. The studies presented in this reportindicate that DMS-mediated cross-linking of oligo(dT) toUL5 and UL52 is specific since several non-DNA-bindingproteins are cross-linked far less effectively to oligo(dT).The modest amount of cross-linking of oligo(dT) to BSAcan be accounted for by a previous observation that BSAweakly binds DNA (24). The specificity of cross-linkingof oligo(dT) to UL5 and to UL52 is further demonstratedby the failure of DMS to cross-link oligo(dT) to the non-DNA-binding UL8 subunit of the holoenzyme. Thiscovalent cross-linking of oligo(dT) to protein by DMS isa novel result that has not been previously observed.
To demonstrate the utility of DMS as a tool forcharacterizing nucleoprotein complexes, we used DMScross-linking to compare the ability of UL5 and UL52 tobind oligo(dT) ligands of varying lengths. For oligo(dT)ligands longer than 16 nucleotides, the ratio of theamount of DNA that is cross-linked to UL5 and UL52 isfairly constant, even in the presence of UL8. Shorterligands are not cross-linked to either protein as efficientlyas the longer DNA species. In the presence of UL8, theamount of oligo(dT)12 that is cross-linked to UL5 is 10-fold greater than for UL52. The poor cross-linking ofoligo(dT)12 to UL5/52 agrees with previous nitrocellulosefilter binding and enzymatic activation studies whichshowed that oligo(dT) ligands shorter than 16 nucleotidesare poorly bound by UL5/52 and are also poor activatorsof the DNA-dependent ATPase activity of UL5/52 (19).Our cross-linking results demonstrate that the minimallength of the DNA-binding site in UL5 is shorter thanin UL52 and that the UL8 subunit affects the interactionof short oligonucleotides with the UL5/52 subassembly.
DMS cross-links primary amines on proteins by animidoester reaction (Figure 4A) (25). Since there are noprimary amines on deoxythymidine, the observation thatDMS cross-links oligo(dT) to UL5 and UL52 is puzzling.One explanation could be that the oligonucleotidesbecome trapped by a “caging” effect in which the DNA
becomes pinned beneath cross bars of DMS that linksome of the residues which form the DNA-binding sites.However, these caged structures would be expected todissociate when denatured in the SDS-PAGE loadingbuffer. Alternatively, upon binding of oligo(dT) to UL5or UL52, some thymine residues may be renderedpartially susceptible to hydrolysis or damage at the 3 and4 positions of the pyrimidine ring. This could yield aâ-ureidoisobutenoic acid moiety containing a primaryamino group that could participate in the imidoestercross-linking reaction (Figure 4B).
The total fraction of labeled oligo(dT) that was cross-linked to protein in our study was about 0.25%. Thisfigure is roughly comparable to the extent of halogenatedoligo(dT) that can be cross-linked to UL5 and UL52 bybrief exposure to long wave UV light (23).
It is often necessary to test several methods in orderto identify one that can be successfully used for cross-linking a specific set of macromolecules. Few methodsare available for covalently cross-linking proteins to DNA.Here we have shown that imidoester cross-linking re-agents such as DMS offer an alternative to these othermethods. In contrast to methylene blue (10), DMS caneffectively cross-link single-stranded nucleic acids toprotein, and, in contrast to aldehyde-based cross-linkers,DMS can yield cross-linked species that retain enzymaticactivity (1). In addition, DMS may be more appropriatethan UV light for cross-linking DNA to proteins in thepresence of nucleotides or other UV-absorbing com-pounds. Thus, the observation that imidoesters such asDMS can covalently cross-link protein to DNA expandsthe limited repertoire of methods that are available forstudying the interaction of DNA with DNA-bindingproteins.
ACKNOWLEDGMENT
This research was supported in part by GrantRPG9705601NP from the American Cancer Society. Theauthor thanks Dr. Lauren Murata and Nicoleta Con-stantin for helpful discussions, critical reading of themanuscript, and assistance with production of the fig-ures.
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