6160 Nucleic Acids Research, Vol. 18, No. 20
Nucleic Acids Research VoL 18, No. 20
Transfer-RNA interferes with the uniform cleavage pattern of DNA by hydroxyl radicals Haralabos Zorbas, Theo Rein and Ernst-Ludwig Winnacker Institut fur Biochemie, Universitat Munchen, KarlstraBe 23, D-8000 Munchen 2, FRG Submitted August 13, 1990 Hydroxyl radicals have been used as a footprinting agent in protection (1, 2) and missing contacts analysis (3, 4) in a variety of systems. Hydroxyl radicals react with DNA in a conformation sensitive manner, which allows one to predict DNA secondary structures (5, 6). For missing contacts analysis of NF I (7) and NF-xB (8) with their cognate DNA, we modified the labeled DNAs in the absence of protein by hydroxyl radical treatment, and the cleavage products were analysed on a sequencing gel, as described (6). There we noticed with both DNA substrates regions of strong compression, which we first interpreted as profound secondary structures (Figure 1, panel A, lane 3; panel B, lane 1). However, because of the similar pattern and the consistent position of the compressions at a distance of about 80 and 90 nucleotides from the labeled ends, we considered the possibility that the aberrant migration of the bands in this region originates simply from the displacement by the co-migrating tRNA, which is usually included in the ethanol concentration step of hydroxyl radical treated DNA (9). Because the aberrant migration could not be eliminated in an alkaline gel system (HydroLink-system, AT Biochem) we used a different ethanol precipitation carrier, linear polyacrylamide, as described (10). As can be seen in figure 1, panel A, lane 4 and panel B, lane 2, the aberrant DNA migration disappeared entirely from the hydroxyl radical cleavage ladder. For obtaining unimpaired DNA cleavage patterns in sequencing gels, we therefore recommend the use of linear polyacrylamide as precipitation carrier, since even minute amounts of tRNA (here about 100 ng/slot) can lead to false interpretations concerning DNA secondary structure. Interestingly, the hydroxyl radical cleaved DNA, which we first coprecipitated with tRNA, was inactive in the interaction with NF-xB, due to specific NF-xB displacement by tRNA at 22 ,ug/20 ,!d. In contrast, polyacrylamide did not impair the concomitant interaction with the proteins (data not shown). Glycogen, an uncharged polymer like polyacrylamide which may be used as ethanol precipitation carrier, also interferes with protein/DNA interactions (10). Thus, polyacrylamide in fact appears for reasons of both unimpaired protein/DNA interaction and reliability in the analysis of the latter in sequencing gels to be the only useful alternative to tRNA or glycogen.
REFERENCES 1. Tullius,T.D. and Dombroski, B.A. (1986) Proc. Natl. Acad. Sci. USA 83, 5469-5473. 2. Schickor,P., Metzger,W., Werel,W., Lederer,H. and Heumann,H. (1990) EMBO J. 9, 2215-2220. 3. Chalepakis,G. and Beato,M. (1989) Nucl. Acids Res. 17, 1783. 4. Hayes,J. J. and Tullius,T.D. (1989) Biochemistry 28, 9521-9527. 5. Burkhoff,A.M. and Tullius,T.D. (1987) Cell 48, 935-943.
6. Zorbas,H., Rogge,L., Meisterernst,M. and Winnacker,E.-L. (1989) Nucl. Acids Res. 17, 7735-7748. 7. Nagata,K., Guggenheimer,R. A., Enomoto,T., Lichy, J. H. and Hurwitz,J. (1982) Proc. Natl. Acad. Sci. USA 79, 6438-6442. 8. Sen,R. and Baltimore,D. (1986) Cell 46, 705-716. 9. Tullius,T.D., Dombroski,B.A., Churchill, M.E.A. and Kam,L. (1987) Methods Enzymol. 155, 537-558. 10. Gaillard,C. and Strauss,F. (1990) Nucl. Acids Res. 18, 378. z
z
0- a-.z
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A'
3' A-,
c(l
C T
~
TM
C
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'c A
jG
!GA
i3
,G
0
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~
.~v..
-C T
eq.
5' 234~~~~~~~~~~~~~~~~~~~~~~~~~~* 5 1234,
A .B Figure 1: Hydroxyl radical cleavage of the NF-xB (A) and the NF I (B) DNA recognition sequences. The consensus sequences are indicated by vertical lines. Purines (Pu) and pyrimidines (Py) sequencing ladders are shown in lanes 1, 2 (panel A) and 3 and 4 (panel B). Hydroxyl radical cleavage products, that were precipitated by tRNA are in lanes 3 (panel A) and I (panel B), and cleavage products, that were precipitated by linear polyacrylamide are shown in lanes 4 (panel A) and 2 (panel B). Untreated DNA run in lane 5 (panel A).
Nucleic Acids Research VoL 18, No. 20
Transfer-RNA interferes with the uniform cleavage pattern of DNA by hydroxyl radicals Haralabos Zorbas, Theo Rein and Ernst-Ludwig Winnacker Institut fur Biochemie, Universitat Munchen, KarlstraBe 23, D-8000 Munchen 2, FRG Submitted August 13, 1990 Hydroxyl radicals have been used as a footprinting agent in protection (1, 2) and missing contacts analysis (3, 4) in a variety of systems. Hydroxyl radicals react with DNA in a conformation sensitive manner, which allows one to predict DNA secondary structures (5, 6). For missing contacts analysis of NF I (7) and NF-xB (8) with their cognate DNA, we modified the labeled DNAs in the absence of protein by hydroxyl radical treatment, and the cleavage products were analysed on a sequencing gel, as described (6). There we noticed with both DNA substrates regions of strong compression, which we first interpreted as profound secondary structures (Figure 1, panel A, lane 3; panel B, lane 1). However, because of the similar pattern and the consistent position of the compressions at a distance of about 80 and 90 nucleotides from the labeled ends, we considered the possibility that the aberrant migration of the bands in this region originates simply from the displacement by the co-migrating tRNA, which is usually included in the ethanol concentration step of hydroxyl radical treated DNA (9). Because the aberrant migration could not be eliminated in an alkaline gel system (HydroLink-system, AT Biochem) we used a different ethanol precipitation carrier, linear polyacrylamide, as described (10). As can be seen in figure 1, panel A, lane 4 and panel B, lane 2, the aberrant DNA migration disappeared entirely from the hydroxyl radical cleavage ladder. For obtaining unimpaired DNA cleavage patterns in sequencing gels, we therefore recommend the use of linear polyacrylamide as precipitation carrier, since even minute amounts of tRNA (here about 100 ng/slot) can lead to false interpretations concerning DNA secondary structure. Interestingly, the hydroxyl radical cleaved DNA, which we first coprecipitated with tRNA, was inactive in the interaction with NF-xB, due to specific NF-xB displacement by tRNA at 22 ,ug/20 ,!d. In contrast, polyacrylamide did not impair the concomitant interaction with the proteins (data not shown). Glycogen, an uncharged polymer like polyacrylamide which may be used as ethanol precipitation carrier, also interferes with protein/DNA interactions (10). Thus, polyacrylamide in fact appears for reasons of both unimpaired protein/DNA interaction and reliability in the analysis of the latter in sequencing gels to be the only useful alternative to tRNA or glycogen.
REFERENCES 1. Tullius,T.D. and Dombroski, B.A. (1986) Proc. Natl. Acad. Sci. USA 83, 5469-5473. 2. Schickor,P., Metzger,W., Werel,W., Lederer,H. and Heumann,H. (1990) EMBO J. 9, 2215-2220. 3. Chalepakis,G. and Beato,M. (1989) Nucl. Acids Res. 17, 1783. 4. Hayes,J. J. and Tullius,T.D. (1989) Biochemistry 28, 9521-9527. 5. Burkhoff,A.M. and Tullius,T.D. (1987) Cell 48, 935-943.
6. Zorbas,H., Rogge,L., Meisterernst,M. and Winnacker,E.-L. (1989) Nucl. Acids Res. 17, 7735-7748. 7. Nagata,K., Guggenheimer,R. A., Enomoto,T., Lichy, J. H. and Hurwitz,J. (1982) Proc. Natl. Acad. Sci. USA 79, 6438-6442. 8. Sen,R. and Baltimore,D. (1986) Cell 46, 705-716. 9. Tullius,T.D., Dombroski,B.A., Churchill, M.E.A. and Kam,L. (1987) Methods Enzymol. 155, 537-558. 10. Gaillard,C. and Strauss,F. (1990) Nucl. Acids Res. 18, 378. z
z
0- a-.z
-""
A'
3' A-,
c(l
C T
~
TM
C
I'1
'c A
jG
!GA
i3
,G
0
~
~
.~v..
-C T
eq.
5' 234~~~~~~~~~~~~~~~~~~~~~~~~~~* 5 1234,
A .B Figure 1: Hydroxyl radical cleavage of the NF-xB (A) and the NF I (B) DNA recognition sequences. The consensus sequences are indicated by vertical lines. Purines (Pu) and pyrimidines (Py) sequencing ladders are shown in lanes 1, 2 (panel A) and 3 and 4 (panel B). Hydroxyl radical cleavage products, that were precipitated by tRNA are in lanes 3 (panel A) and I (panel B), and cleavage products, that were precipitated by linear polyacrylamide are shown in lanes 4 (panel A) and 2 (panel B). Untreated DNA run in lane 5 (panel A).
