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Bleomycin-mediated DNA cross-links are dependent on closed-circular molecules with superhelical turns
Chem.-Biol. Interactions, 34 (1981) 39---46 © Elsevier/North-Holland Scientific Publishers Ltd.
39
BLEOMYCIN-MEDIATED D N A CROSS-LINKS ARE D E P E N D E N T ON C L O S E D - C I R C U L A R M O L E C U L E S WITH S U P E R H E L I C A L T U R N S
R.S. LLOYD*, D.L. ROBBERSON and C.W. HAIDLE**
Department o f Molecular Biology, Division of~Biology, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, TX 77030 (U.S.A.)
(Received June 30th, 1980) (Revision received September 24th, 1980) (Accepted September 28th, 1980) SUMMARY
N o n ~ o v a l e n t intermolecular DNA cross-links are created by reaction with the antitumor antibiotic, bleomycin. The cross-links are observed only when the reactant covalently closed circular duplex DNA contains either positive or negative superhelical turns. Multiple sites of cross-linking often extend over 500 base-pairs or more in length.
INTRODUCTION
The bleomycins are a group of glycopeptide antitumor antibiotics which are currently used in the treatment of various carcinomas and lymphomas. The antibiotic is known to react with DNA and we have demonstrated previously that, in addition to producing single-strand breaks, release of f~ee bases and direct double,strand breaks at specific sites, bleomycin also mediates a time
40 M A T E R I A L S AND METHODS
DNA preparation. Bacteriophage PM2 DNA was prepared by the procedure of Salditt et al. [2] as modified b y Strong and Hewitt [ 3 ] . Hpa H restriction endonuclease digestion. A total of 20/~g of form I PM2 DNA (20/~g/ml) in 0.01 M MgC12, 6 mM KC1, 1.0 mM dithiothreitol, 0.01 M Tris (pH 7.4) was reacted with 30 units o f the Hpa II restriction enzyme for 3 h at 37°C. After incubation, the solution was dialyzed for 48 h against three changes, 1 1 each, of 15 mM NaC1, 1.5 mM Na3 citrate. A total of 2 /~g of the Hpa II-cleaved PM2 DNA (19 /~g/ml) was then treated with 0.5 pg/ml of bleomycin in a reaction volume of 0.105 ml containing 25 mM 2-mercaptoethanol, 0.29 mM CaCl2, 21 mM Tris (pH 7.8) for 10 min at 37°C. The reaction was terminated by addition of an equal volume of 20 mM EDTA, 50 mM Tris (pH 8.0). DNAase I treatment. A total of 50 pg of form I PM2 DNA (437/~g/ml) in 0.115 ml o f a solution containing 9.6 mM MgC12, 0.96 mM dithiothreitol, 5.7 mM KC1, 9.6 mM Tris (pH 7.4) was treated with DNAase I (0.035 pg/ml) for 12.5 min at 37°C. The reaction was terminated by the addition of 0.115 ml of 20 mM EDTA, 50 mM Tris (pH 7.8). The total reaction mixture was dialyzed for 48 h against t w o changes, 3 1 each, o f 15 mM NaC1, 1.5 mM Na3 citrate. A total of 2 pg of this DNAase I-treated PM2 DNA (21 pg/ml) was reacted at 37°C with 0.5 pg/ml bleomycin in a 0.105 ml reaction volume containing 25 mM 2-mercaptoethanol, 0.29 mM CaC12, 21 mM Tris (pH 7.8) for the times indicated. Reactions were terminated by the addition of an equal volume o f 20 mM EDTA, 50 mM Tris (pH 8.0). All samples were prepared for electron microscopy as described. Form I ° DNA preparation. F o r m I ° PM2 DNA was prepared by treatm e n t of form I bacteriophage PM2 DNA with the calf t h y m u s nickingclosing enzyme. Form I ° DNA was reacted for the indicated times with 0.5/~g/ml of bleomycin with the reaction conditions described in the legend to Fig. 1. Frequencies of cross-linked DNA were determined by electron microscopy. RESULTS AND DISCUSSION
Form I bacteriophage PM2 DNA (50/~g/ml) was reacted with 0.5 pg/ml of bleomycin for 2.5, 5, 15 and 30 min, respectively, at 37°C and the DNA was prepared for electron microscopy by a modification [4] of the aqueous Kleinschmidt technique described by Davis et al. [5]. After 30 min of reaction, approx. 16% of the total mass o f the DNA (Table I) was f o u n d to occur in cross-linked structures as shown in Fig. 1. The mass fractions of each form of bleomycin-reacted DNA that remain uncross-linked (monomeric forms) as a function of time parallel the proportions of these different forms observed in the cross-linked structures [1]. Most of the cross-linked DNA molecules were associated over a very short region of point contact, b u t many also were observed to be associated over regions of 500 base-pairs
41 TABLE I FREQUENCIES OF DIFFERENT FORMS OF PM2 DNA CROSS-LINKED AS A FUNCTION OF REACTION TIME WITH BLEOMYCIN Reactant DNA
Treatment time (min) a
Mass fraction cross°linked b
Native Native Native Native Native
0 2.5 5.0 15.0 30.0
0.027 0.042 0.111 0.164 0.157
Hpa H form III Hpa II form HI
0 10.0
0.018 ± 0.010 0.014 ± 0.008
DNAase I forms II and III DNAase I forms II and III DNAase I forms II and III
0 5.0 15.0
0.044 ± 0.015 0.034 ± 0.013 0.050 -+ 0.016
Form I ° Form I ° Form I °
0 5.0 15.0
0.042 + 0.014 0.028 ± 0.012 0.042 ± 0.014
form form form form form
Ic Ic Ic Ic Ic
± 0.012 ± 0.014 -+ 0.022 + 0.026 ± 0.026
a All zero time points indicated refer to reactions in which all components except bleomycin had been added. b Mass fractions of cross-linked DNA determined by electron microscopic examination of 750 molecules with an indicated sampling error at 95% confidence limits. c Form I PM2 DNA was treated with 0.5 /zg/rnl bleomycin as described in the legend t o Fig. 1.
or m o r e in l e n g t h (Fig. 1, i n d i c a t e d b y a r r o w s ) as d e s c r i b e d p r e v i o u s l y [ 1 ] . In o u r initial a t t e m p t s t o d e t e r m i n e w h e t h e r t h e regions o f extensive cross-linking o c c u r r e d a t specific sites o n t h e PM2 g e n o m e , D N A was digested with t h e r e s t r i c t i o n e n z y m e H p a II f o l l o w e d b y r e a c t i o n with b l e o m y c i n f o r 10 m i n a t 37°C. Since t h e r e exists o n l y o n e H p a II r e s t r i c t i o n e n z y m e site o n t h e PM2 g e n o m e [ 6 ] , cleavage with this e n z y m e will yield full-length d u p l e x D N A . A l t h o u g h t h e b l e o m y c i n f r a g m e n t a t i o n r e a c t i o n d o e s o c c u r a n d is e v i d e n t as d o u b l e , s t r a n d breakage o f t h e H p a II-cleaved f o r m I I I m o l e c u l e s t o p r o d u c e m o l e c u l e s less t h a n u n i t l e n g t h , b l e o m y c i n cross-linked s t r u c t u r e s were n o t d e t e c t e d (Table I). Since b o t h f o r m I I a n d f o r m I I I D N A s were p r e v i o u s l y o b s e r v e d in crosslinked s t r u c t u r e s [ 1 ] , the possibility r e m a i n e d t h a t f o r m II D N A c o u l d p a r t i c i p a t e in initial cross-linking events. T o test this possibility, a m i x t u r e o f f o r m II a n d I I I PM2 D N A s was p r o d u c e d b y D N A a s e I t r e a t m e n t , p u r i f i e d a n d t h e n r e a c t e d with b l e o m y c i n as described in Materials a n d M e t h o d s . B l e o m y c i n cross-linked s t r u c t u r e s were n o t d e t e c t e d b y e l e c t r o n m i c r o s c o p y (Table I). Finally, t h e r e was a possibility t h a t cross-linking c o u l d o c c u r in the a b s e n c e o f superhelical t u r n s if all p h o s p h o d i e s t e r b o n d s were initially c o v a l e n t l y closed. T o test this l a t t e r possibility, f o r m I ° D N A ( c o v a l e n t l y
Fig. 1. Electron micrographs illustrating the appearance of bleomycin cross-linked oligomers containing two or more PM2 DNA molecules. The form I DNA (50 ug/ml) was treated with 0.5 ~g/ml of bleomycin in a solution (27 ~l) containing 0.27 mM CaC12, 25 mM 2-mercaptoethanol, 20 mM Tris (pH 7.8). The stock solution of PM2 DNA was in 0.1 × SSC so that addition of 5 ul to the bleomycin reaction solution brought the final ionic strength to 0.017 M. The reaction mixture was incubated for 30 min at 37°C and the reaction terminated by the addition of 30 ~l of 20 mM Na 2 EDTA, 50 mM Tris (pH 7.8). Samples of bleomycin-treated PM2 DNA were prepared for electron microscopy by the aqueous Kleinschmidt technique described [4,5]. The grids were rotary-shadowed with Pt-Pd (80 : 20) and examined in a Philips 300 electron microscope. Bar length = 1 ~m.
b~
43 closed circular DNA w i t h o u t superhelical turns) was also used as reactant D N A to test for bleomycin cross-linking. No bleomycin cross-linking was detected (Table I), although both single-strand and double-strand breakage occurred as reported previously [7]. Form I ° DNA contains an average o f zero superhelical turns and consists of a population o f molecules containing zero, +1, +2 turns etc. with the exact form of the Gaussian distribution determined b y the ionic strength and temperature of the nicking-closing reaction [8,9] (37°C and 0.2 M NaC1). When this DNA is transferred to the lower ionic strength of the bleomycin reaction (0.01 M NaC1), the molecules will shift to become a population largely containing positive superhelical turns which remain discretely distributed. It would thus appear that closed-circular duplex molecules containing zero or only a few positive superhelical turns are n o t cross-linked in the bleomycin reaction. To determine the effect of altering the n u m b e r of superhelical turns in PM2 DNA on the overall e x t e n t of bleomycin cross-linking, PM2 DNAs with superhelical densities o f + 0 . 0 0 6 , - 0 . 0 4 1 , - 0 . 1 1 5 and-0.212, determined with an error of + 0 . 0 0 4 by the ethidium bromide-CsC1 density gradient procedure [10] (photograph inset in Fig. 2) were reacted with 0.5 pg/ml bleomycin for 30 min at 37°C and then examined by electron microscopy. Form I ° DNA prepared by treatment of native form I PM2 D N A with calf t h y m u s nicking-closing enzyme at 37°C in 0.2 M NaC1, was also examined by electron microscopy after reaction with bleomycin. In the ionic strength of the bleomycin reaction mixtures and at the 37°C reaction temperature, these different closed-circular DNAs are calculated [11] to have superhelix densities of + 0 . 0 3 3 , - 0 . 0 1 4 , - 0 . 0 8 8 , - 0 . 1 8 1 and + 0 . 0 1 0 (form I°}, respectively. This calculation assumes the published estimates of the effects u p o n superhelix density of changing counterions from Cs ÷ t o Na ÷ [12] decreasing the salt concentration from 2.8 to 0.1 M [13] calculated as described [11] and then extrapolating to an ionic strength of 0.017 M. The counterion change and ionic strength reduction from 2.8 M to 0.1 M results in the addition of + 0.018 to the superhelix densities [11]. The effect of temperature on superhelix densities was corrected with the data presented by Wang [12]. Substantial mass fractions o f cross-linked DNA were detected when closed-circular DNA with either positive or negative superhelical turns was reacted with bleomycin (Fig. 2). The single negative value for mass fraction of cross-linked DNA presented in Fig. 2 derives from having subtracted 3.4% of the PM2 bacteriophage DNA mass that occurs in topologically bonded, catenated dimers (D.L. Robberson, unpublished). For the t w o points nearest zero superhelix density (Fig. 2), it is evident that no cross-linking occurs, although bleomycin fragmentation continues and, in these samples, clearly reduces the frequencies of catenated dimers. These c o m b i n e d results indicate that superhelical turns in the reactant D N A provide a necessary condition for the bleomycin-mediated cross-linking reaction to occur. Even as few as 37-+ 4 positive, superhelical turns per
44
0.12 "0
/ / /
"~ 0 . 0 8 ~ o~ o~
/
i
/ tO
~
0.04
I
I I
(x} U_ u) u) cO
I
l
~ ~
form I I - form I ~
I I
0.0
-0.04 , +0.04
!
, k / 0.0
,
!
/
, , -0.04
,
,
h ,--0.08
Superhelix
,
, , -0.12
. . . . . . . . -0.16
-0.20
Density
Fig. 2. Dependence of bleomycin-mediated cross-links on superhelix density of the reactant DNA. PM2 DNAs (8 ~g/ml) with superhelix densities indicated were separately treated with 0.5 ug/ml bleomycin for 30 min at 37°C in a total reaction volume of 150 ul containing 0.4 mM CaCl:, 6.7 mM NaCl, 0.07 mM EDTA, 47 mM Tris (pH 7.8). Reactions were terminated by the addition of an equal volume of 20 mM EDTA, 50 mM Tris (pH 8.0). All samples were examined by electron microscopy for determination of the mass fraction of cross-linked forms. Samples of different negative superhelix densities were prepared using E. coli ligase treatment of form II DNA in the presence of ethidium bromide as reported in Ref. 11. The single negative value for mass fraction of cross-linked DNA derives from having subtracted 3.4% of the PM2 bacteriophage DNA mass that occurs in topologically bonded, catenated dimers. Inset photographs demonstrate positions (marked by lines) of closed circular DNAs differing in superhelix density relative to positions of native (form I) and form II PM2 DNAs. Ultracentrifugation of a 3 ml column of ethidium bromide-CsCi solution was conducted at 38 K rev./min essentially as described [ 10,11 ]. m o l e c u l e o n the average (superhelix d e n s i t y o f + 0 . 0 3 3 , u n d e r these l o w ionic s t r e n g t h c o n d i t i o n s ) p e r m i t t h e cross-linking o f 7.8% o f the D N A mass (Fig. 2). A l t h o u g h t h e r e is a gradual increase in the mass f r a c t i o n o f D N A cross-linked as o n e goes t o h i g h e r negative superhelix densities, m o s t o f t h e D N A has already been cross-linked (12.7% o f the mass) a t the l o w e s t negative superhelix d e n s i t y e x a m i n e d ( - 0 . 0 8 8 ) . Since m o s t native PM2 D N A m o l e c u l e s with a superhelix d e n s i t y o f - 0 . 0 8 8 have been cross-linked after o n l y 5 rain o f r e a c t i o n , we c o n s i d e r it very unlikely t h a t the l o w levels o f cross-linking for t h e l o w superhelix d e n s i t y D N A s r e f l e c t a kinetic l i m i t a t i o n in the 30-rain r e a c t i o n s p e r f o r m e d . B l e o m y c i n m a y be b o u n d at single-stranded or w e a k l y h y d r o g e n b o n d e d regions o n separate f o r m I D N A m o l e c u l e s a n d the c o m p l e x e s t h e n associate
45 to produce a non~ovalent cross-link [1]. PM2 DNA containing even very few negative superhelical turns has been shown to be sensitive to cleavage with the single-strand specific A l t e r o m o n a s endonuclease [11]. PM2 DNA containing a large number of positive superhelical turns is also sensitive to cleavage by the enzyme [11]. These results suggest that single-stranded or weakly hydrogen-bonded regions exist in negative as well as highly positive superhelical DNAs that provide substrates for the enzyme. Furthermore, in the case of the single-strand specific nulceases, closed circular duplex DNA substrates with higher negative superhelix densities have a greater sensitivity to cleavage [6,11,14]. By analogy, there are also increased levels of bleomycin cross-linked forms when PM2 DNAs with higher negative superhelix densities are examined (Fig. 2). The cleavage of positive superhelical DNA with the Alteromonas nuclease does not occur until superhelix densities greater than +0.150 are attained [11]. The observed bleomycin-mediated cross-linking reaction for PM2 DNA with the low superhelix density of +0.033 may therefore reflect binding to a different secondary structure of covalently closed circular DNA rather than the single-stranded or weakly hydrogen-bonded regions detected by A lteromonas nuclease digestions [ 11 ]. ACKNOWLEDGEMENTS
We are especially grateful to Drs. H.B. Gray, Jr. and Paul Lau for the generous gift of PM2 DNA samples differing in superhelix densities. We also thank Janie Finch and Janet Naquin for help in preparation of the manuscript. This investigation was supported in part, by Grants CA-16527 and CA-13246 awarded by the National Cancer Institute, D.H.E.W. and G-441 from the Robert A. Welch Foundation. R.S.L. was a Postdoctoral Fellow with The Robert A. Welch Foundation. REFERENCES 1 R.S. Lloyd, C.W. Haidle and D.L. Robberson, Non-covalent intermolecular crosslinks are produced by bleomycin reaction on duplex DNA, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 2674. 2 M. Salditt, S.N. Braunstein, R.D. Camerini-Otero and R.M. Franklin, Structure and synthesis of a lipid-containing bacteriophage, Virology, 48 (1972) 259. 3 J.E. Strong and R.R. Hewitt, Investigation of human DNases by DNA polyacrylamide gel electrophoresis, in: C. Markert (Ed.), Isoenzymes, Vol. 3, Academic Press, New York, 1975, pp. 473--483. 4 D.L. Robberson, Y. Altoni and J. Attardi, Electron microscopic visualization of mitochondria RNA-DNA hybrids, J. Mol. Biol., 55 (1971) 267. 5 R.W. Davis, M. Simon and N. Davidson, Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, in: L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. XXI, Academic Press, New York, 1971, pp. 413--428. 6 J.C. Wang, Interactions between twisted DNAs and enzymes: The effects of superhelical turns, J. Mol. Biol., 87 (1974) 797.
46 7 R.S. Lloyd, C.W. Haidle and D.L. Robberson, Bleomyein-specific fragmentation of double-stranded DNA, Biochemistry, 17 (1978) 1890. 8 R.E. Depew and J.C. Wang, Conformational fluctuations of DNA helix, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4275. 9 D.E. Pulleyblank, M. Shure, D. Tang, J. Vinograd and H-P. Vosberg, Action of nicking-closing enzyme in supercoiled and nonsupercoiled closed circular DNA: F o r m a t i o n o f a Boltzmann distribution o f topological isomers, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4280. 10 H.B. Gray, W.B. Upholt and J. Vinograd, A b u o y a n t method for the determination of the superhelix density o f closed circular DNA, J. Mol. Biol., 62 (1971) 1. 11 P_P. Lau and H.B. Gray, Extracellular nucleases of Alteromonas espejiana BAL 31, IV. The single-strand specific deoxyriboendonuclease activity as a probe for regions o f altered secondary structure in negatively and positively supercoiled closed circular DNA, Nucleic Acids Res., 6 (1979) 331. 12 J.C. Wang, Variation of the average rotation angle of the DNA helix and the superhelical turns o f covalently closed circular ~, DNA, J. Mol. Biol., 43 (1969) 25. 13 W.B. Upholt, H.B. Gray and J. Vinograd, Sedimentation velocity behavior of closed circular SV40 DNA as a function o f superhelix density, ionic strength, counterion and temperature, J. Mol. Biol., 61 (1971) 21. 14 M. Woodworth-Gutai and J. Lebowitz, Introduction of interrupted secondary structure in supercoiled DNA as a function of superhelix density. Consideration of hairpin structures in superhelical DNA, J. Virol., 18 (1976) 195.
39
BLEOMYCIN-MEDIATED D N A CROSS-LINKS ARE D E P E N D E N T ON C L O S E D - C I R C U L A R M O L E C U L E S WITH S U P E R H E L I C A L T U R N S
R.S. LLOYD*, D.L. ROBBERSON and C.W. HAIDLE**
Department o f Molecular Biology, Division of~Biology, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, TX 77030 (U.S.A.)
(Received June 30th, 1980) (Revision received September 24th, 1980) (Accepted September 28th, 1980) SUMMARY
N o n ~ o v a l e n t intermolecular DNA cross-links are created by reaction with the antitumor antibiotic, bleomycin. The cross-links are observed only when the reactant covalently closed circular duplex DNA contains either positive or negative superhelical turns. Multiple sites of cross-linking often extend over 500 base-pairs or more in length.
INTRODUCTION
The bleomycins are a group of glycopeptide antitumor antibiotics which are currently used in the treatment of various carcinomas and lymphomas. The antibiotic is known to react with DNA and we have demonstrated previously that, in addition to producing single-strand breaks, release of f~ee bases and direct double,strand breaks at specific sites, bleomycin also mediates a time
40 M A T E R I A L S AND METHODS
DNA preparation. Bacteriophage PM2 DNA was prepared by the procedure of Salditt et al. [2] as modified b y Strong and Hewitt [ 3 ] . Hpa H restriction endonuclease digestion. A total of 20/~g of form I PM2 DNA (20/~g/ml) in 0.01 M MgC12, 6 mM KC1, 1.0 mM dithiothreitol, 0.01 M Tris (pH 7.4) was reacted with 30 units o f the Hpa II restriction enzyme for 3 h at 37°C. After incubation, the solution was dialyzed for 48 h against three changes, 1 1 each, of 15 mM NaC1, 1.5 mM Na3 citrate. A total of 2 /~g of the Hpa II-cleaved PM2 DNA (19 /~g/ml) was then treated with 0.5 pg/ml of bleomycin in a reaction volume of 0.105 ml containing 25 mM 2-mercaptoethanol, 0.29 mM CaCl2, 21 mM Tris (pH 7.8) for 10 min at 37°C. The reaction was terminated by addition of an equal volume of 20 mM EDTA, 50 mM Tris (pH 8.0). DNAase I treatment. A total of 50 pg of form I PM2 DNA (437/~g/ml) in 0.115 ml o f a solution containing 9.6 mM MgC12, 0.96 mM dithiothreitol, 5.7 mM KC1, 9.6 mM Tris (pH 7.4) was treated with DNAase I (0.035 pg/ml) for 12.5 min at 37°C. The reaction was terminated by the addition of 0.115 ml of 20 mM EDTA, 50 mM Tris (pH 7.8). The total reaction mixture was dialyzed for 48 h against t w o changes, 3 1 each, o f 15 mM NaC1, 1.5 mM Na3 citrate. A total of 2 pg of this DNAase I-treated PM2 DNA (21 pg/ml) was reacted at 37°C with 0.5 pg/ml bleomycin in a 0.105 ml reaction volume containing 25 mM 2-mercaptoethanol, 0.29 mM CaC12, 21 mM Tris (pH 7.8) for the times indicated. Reactions were terminated by the addition of an equal volume o f 20 mM EDTA, 50 mM Tris (pH 8.0). All samples were prepared for electron microscopy as described. Form I ° DNA preparation. F o r m I ° PM2 DNA was prepared by treatm e n t of form I bacteriophage PM2 DNA with the calf t h y m u s nickingclosing enzyme. Form I ° DNA was reacted for the indicated times with 0.5/~g/ml of bleomycin with the reaction conditions described in the legend to Fig. 1. Frequencies of cross-linked DNA were determined by electron microscopy. RESULTS AND DISCUSSION
Form I bacteriophage PM2 DNA (50/~g/ml) was reacted with 0.5 pg/ml of bleomycin for 2.5, 5, 15 and 30 min, respectively, at 37°C and the DNA was prepared for electron microscopy by a modification [4] of the aqueous Kleinschmidt technique described by Davis et al. [5]. After 30 min of reaction, approx. 16% of the total mass o f the DNA (Table I) was f o u n d to occur in cross-linked structures as shown in Fig. 1. The mass fractions of each form of bleomycin-reacted DNA that remain uncross-linked (monomeric forms) as a function of time parallel the proportions of these different forms observed in the cross-linked structures [1]. Most of the cross-linked DNA molecules were associated over a very short region of point contact, b u t many also were observed to be associated over regions of 500 base-pairs
41 TABLE I FREQUENCIES OF DIFFERENT FORMS OF PM2 DNA CROSS-LINKED AS A FUNCTION OF REACTION TIME WITH BLEOMYCIN Reactant DNA
Treatment time (min) a
Mass fraction cross°linked b
Native Native Native Native Native
0 2.5 5.0 15.0 30.0
0.027 0.042 0.111 0.164 0.157
Hpa H form III Hpa II form HI
0 10.0
0.018 ± 0.010 0.014 ± 0.008
DNAase I forms II and III DNAase I forms II and III DNAase I forms II and III
0 5.0 15.0
0.044 ± 0.015 0.034 ± 0.013 0.050 -+ 0.016
Form I ° Form I ° Form I °
0 5.0 15.0
0.042 + 0.014 0.028 ± 0.012 0.042 ± 0.014
form form form form form
Ic Ic Ic Ic Ic
± 0.012 ± 0.014 -+ 0.022 + 0.026 ± 0.026
a All zero time points indicated refer to reactions in which all components except bleomycin had been added. b Mass fractions of cross-linked DNA determined by electron microscopic examination of 750 molecules with an indicated sampling error at 95% confidence limits. c Form I PM2 DNA was treated with 0.5 /zg/rnl bleomycin as described in the legend t o Fig. 1.
or m o r e in l e n g t h (Fig. 1, i n d i c a t e d b y a r r o w s ) as d e s c r i b e d p r e v i o u s l y [ 1 ] . In o u r initial a t t e m p t s t o d e t e r m i n e w h e t h e r t h e regions o f extensive cross-linking o c c u r r e d a t specific sites o n t h e PM2 g e n o m e , D N A was digested with t h e r e s t r i c t i o n e n z y m e H p a II f o l l o w e d b y r e a c t i o n with b l e o m y c i n f o r 10 m i n a t 37°C. Since t h e r e exists o n l y o n e H p a II r e s t r i c t i o n e n z y m e site o n t h e PM2 g e n o m e [ 6 ] , cleavage with this e n z y m e will yield full-length d u p l e x D N A . A l t h o u g h t h e b l e o m y c i n f r a g m e n t a t i o n r e a c t i o n d o e s o c c u r a n d is e v i d e n t as d o u b l e , s t r a n d breakage o f t h e H p a II-cleaved f o r m I I I m o l e c u l e s t o p r o d u c e m o l e c u l e s less t h a n u n i t l e n g t h , b l e o m y c i n cross-linked s t r u c t u r e s were n o t d e t e c t e d (Table I). Since b o t h f o r m I I a n d f o r m I I I D N A s were p r e v i o u s l y o b s e r v e d in crosslinked s t r u c t u r e s [ 1 ] , the possibility r e m a i n e d t h a t f o r m II D N A c o u l d p a r t i c i p a t e in initial cross-linking events. T o test this possibility, a m i x t u r e o f f o r m II a n d I I I PM2 D N A s was p r o d u c e d b y D N A a s e I t r e a t m e n t , p u r i f i e d a n d t h e n r e a c t e d with b l e o m y c i n as described in Materials a n d M e t h o d s . B l e o m y c i n cross-linked s t r u c t u r e s were n o t d e t e c t e d b y e l e c t r o n m i c r o s c o p y (Table I). Finally, t h e r e was a possibility t h a t cross-linking c o u l d o c c u r in the a b s e n c e o f superhelical t u r n s if all p h o s p h o d i e s t e r b o n d s were initially c o v a l e n t l y closed. T o test this l a t t e r possibility, f o r m I ° D N A ( c o v a l e n t l y
Fig. 1. Electron micrographs illustrating the appearance of bleomycin cross-linked oligomers containing two or more PM2 DNA molecules. The form I DNA (50 ug/ml) was treated with 0.5 ~g/ml of bleomycin in a solution (27 ~l) containing 0.27 mM CaC12, 25 mM 2-mercaptoethanol, 20 mM Tris (pH 7.8). The stock solution of PM2 DNA was in 0.1 × SSC so that addition of 5 ul to the bleomycin reaction solution brought the final ionic strength to 0.017 M. The reaction mixture was incubated for 30 min at 37°C and the reaction terminated by the addition of 30 ~l of 20 mM Na 2 EDTA, 50 mM Tris (pH 7.8). Samples of bleomycin-treated PM2 DNA were prepared for electron microscopy by the aqueous Kleinschmidt technique described [4,5]. The grids were rotary-shadowed with Pt-Pd (80 : 20) and examined in a Philips 300 electron microscope. Bar length = 1 ~m.
b~
43 closed circular DNA w i t h o u t superhelical turns) was also used as reactant D N A to test for bleomycin cross-linking. No bleomycin cross-linking was detected (Table I), although both single-strand and double-strand breakage occurred as reported previously [7]. Form I ° DNA contains an average o f zero superhelical turns and consists of a population o f molecules containing zero, +1, +2 turns etc. with the exact form of the Gaussian distribution determined b y the ionic strength and temperature of the nicking-closing reaction [8,9] (37°C and 0.2 M NaC1). When this DNA is transferred to the lower ionic strength of the bleomycin reaction (0.01 M NaC1), the molecules will shift to become a population largely containing positive superhelical turns which remain discretely distributed. It would thus appear that closed-circular duplex molecules containing zero or only a few positive superhelical turns are n o t cross-linked in the bleomycin reaction. To determine the effect of altering the n u m b e r of superhelical turns in PM2 DNA on the overall e x t e n t of bleomycin cross-linking, PM2 DNAs with superhelical densities o f + 0 . 0 0 6 , - 0 . 0 4 1 , - 0 . 1 1 5 and-0.212, determined with an error of + 0 . 0 0 4 by the ethidium bromide-CsC1 density gradient procedure [10] (photograph inset in Fig. 2) were reacted with 0.5 pg/ml bleomycin for 30 min at 37°C and then examined by electron microscopy. Form I ° DNA prepared by treatment of native form I PM2 D N A with calf t h y m u s nicking-closing enzyme at 37°C in 0.2 M NaC1, was also examined by electron microscopy after reaction with bleomycin. In the ionic strength of the bleomycin reaction mixtures and at the 37°C reaction temperature, these different closed-circular DNAs are calculated [11] to have superhelix densities of + 0 . 0 3 3 , - 0 . 0 1 4 , - 0 . 0 8 8 , - 0 . 1 8 1 and + 0 . 0 1 0 (form I°}, respectively. This calculation assumes the published estimates of the effects u p o n superhelix density of changing counterions from Cs ÷ t o Na ÷ [12] decreasing the salt concentration from 2.8 to 0.1 M [13] calculated as described [11] and then extrapolating to an ionic strength of 0.017 M. The counterion change and ionic strength reduction from 2.8 M to 0.1 M results in the addition of + 0.018 to the superhelix densities [11]. The effect of temperature on superhelix densities was corrected with the data presented by Wang [12]. Substantial mass fractions o f cross-linked DNA were detected when closed-circular DNA with either positive or negative superhelical turns was reacted with bleomycin (Fig. 2). The single negative value for mass fraction of cross-linked DNA presented in Fig. 2 derives from having subtracted 3.4% of the PM2 bacteriophage DNA mass that occurs in topologically bonded, catenated dimers (D.L. Robberson, unpublished). For the t w o points nearest zero superhelix density (Fig. 2), it is evident that no cross-linking occurs, although bleomycin fragmentation continues and, in these samples, clearly reduces the frequencies of catenated dimers. These c o m b i n e d results indicate that superhelical turns in the reactant D N A provide a necessary condition for the bleomycin-mediated cross-linking reaction to occur. Even as few as 37-+ 4 positive, superhelical turns per
44
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Fig. 2. Dependence of bleomycin-mediated cross-links on superhelix density of the reactant DNA. PM2 DNAs (8 ~g/ml) with superhelix densities indicated were separately treated with 0.5 ug/ml bleomycin for 30 min at 37°C in a total reaction volume of 150 ul containing 0.4 mM CaCl:, 6.7 mM NaCl, 0.07 mM EDTA, 47 mM Tris (pH 7.8). Reactions were terminated by the addition of an equal volume of 20 mM EDTA, 50 mM Tris (pH 8.0). All samples were examined by electron microscopy for determination of the mass fraction of cross-linked forms. Samples of different negative superhelix densities were prepared using E. coli ligase treatment of form II DNA in the presence of ethidium bromide as reported in Ref. 11. The single negative value for mass fraction of cross-linked DNA derives from having subtracted 3.4% of the PM2 bacteriophage DNA mass that occurs in topologically bonded, catenated dimers. Inset photographs demonstrate positions (marked by lines) of closed circular DNAs differing in superhelix density relative to positions of native (form I) and form II PM2 DNAs. Ultracentrifugation of a 3 ml column of ethidium bromide-CsCi solution was conducted at 38 K rev./min essentially as described [ 10,11 ]. m o l e c u l e o n the average (superhelix d e n s i t y o f + 0 . 0 3 3 , u n d e r these l o w ionic s t r e n g t h c o n d i t i o n s ) p e r m i t t h e cross-linking o f 7.8% o f the D N A mass (Fig. 2). A l t h o u g h t h e r e is a gradual increase in the mass f r a c t i o n o f D N A cross-linked as o n e goes t o h i g h e r negative superhelix densities, m o s t o f t h e D N A has already been cross-linked (12.7% o f the mass) a t the l o w e s t negative superhelix d e n s i t y e x a m i n e d ( - 0 . 0 8 8 ) . Since m o s t native PM2 D N A m o l e c u l e s with a superhelix d e n s i t y o f - 0 . 0 8 8 have been cross-linked after o n l y 5 rain o f r e a c t i o n , we c o n s i d e r it very unlikely t h a t the l o w levels o f cross-linking for t h e l o w superhelix d e n s i t y D N A s r e f l e c t a kinetic l i m i t a t i o n in the 30-rain r e a c t i o n s p e r f o r m e d . B l e o m y c i n m a y be b o u n d at single-stranded or w e a k l y h y d r o g e n b o n d e d regions o n separate f o r m I D N A m o l e c u l e s a n d the c o m p l e x e s t h e n associate
45 to produce a non~ovalent cross-link [1]. PM2 DNA containing even very few negative superhelical turns has been shown to be sensitive to cleavage with the single-strand specific A l t e r o m o n a s endonuclease [11]. PM2 DNA containing a large number of positive superhelical turns is also sensitive to cleavage by the enzyme [11]. These results suggest that single-stranded or weakly hydrogen-bonded regions exist in negative as well as highly positive superhelical DNAs that provide substrates for the enzyme. Furthermore, in the case of the single-strand specific nulceases, closed circular duplex DNA substrates with higher negative superhelix densities have a greater sensitivity to cleavage [6,11,14]. By analogy, there are also increased levels of bleomycin cross-linked forms when PM2 DNAs with higher negative superhelix densities are examined (Fig. 2). The cleavage of positive superhelical DNA with the Alteromonas nuclease does not occur until superhelix densities greater than +0.150 are attained [11]. The observed bleomycin-mediated cross-linking reaction for PM2 DNA with the low superhelix density of +0.033 may therefore reflect binding to a different secondary structure of covalently closed circular DNA rather than the single-stranded or weakly hydrogen-bonded regions detected by A lteromonas nuclease digestions [ 11 ]. ACKNOWLEDGEMENTS
We are especially grateful to Drs. H.B. Gray, Jr. and Paul Lau for the generous gift of PM2 DNA samples differing in superhelix densities. We also thank Janie Finch and Janet Naquin for help in preparation of the manuscript. This investigation was supported in part, by Grants CA-16527 and CA-13246 awarded by the National Cancer Institute, D.H.E.W. and G-441 from the Robert A. Welch Foundation. R.S.L. was a Postdoctoral Fellow with The Robert A. Welch Foundation. REFERENCES 1 R.S. Lloyd, C.W. Haidle and D.L. Robberson, Non-covalent intermolecular crosslinks are produced by bleomycin reaction on duplex DNA, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 2674. 2 M. Salditt, S.N. Braunstein, R.D. Camerini-Otero and R.M. Franklin, Structure and synthesis of a lipid-containing bacteriophage, Virology, 48 (1972) 259. 3 J.E. Strong and R.R. Hewitt, Investigation of human DNases by DNA polyacrylamide gel electrophoresis, in: C. Markert (Ed.), Isoenzymes, Vol. 3, Academic Press, New York, 1975, pp. 473--483. 4 D.L. Robberson, Y. Altoni and J. Attardi, Electron microscopic visualization of mitochondria RNA-DNA hybrids, J. Mol. Biol., 55 (1971) 267. 5 R.W. Davis, M. Simon and N. Davidson, Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, in: L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. XXI, Academic Press, New York, 1971, pp. 413--428. 6 J.C. Wang, Interactions between twisted DNAs and enzymes: The effects of superhelical turns, J. Mol. Biol., 87 (1974) 797.
46 7 R.S. Lloyd, C.W. Haidle and D.L. Robberson, Bleomyein-specific fragmentation of double-stranded DNA, Biochemistry, 17 (1978) 1890. 8 R.E. Depew and J.C. Wang, Conformational fluctuations of DNA helix, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4275. 9 D.E. Pulleyblank, M. Shure, D. Tang, J. Vinograd and H-P. Vosberg, Action of nicking-closing enzyme in supercoiled and nonsupercoiled closed circular DNA: F o r m a t i o n o f a Boltzmann distribution o f topological isomers, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 4280. 10 H.B. Gray, W.B. Upholt and J. Vinograd, A b u o y a n t method for the determination of the superhelix density o f closed circular DNA, J. Mol. Biol., 62 (1971) 1. 11 P_P. Lau and H.B. Gray, Extracellular nucleases of Alteromonas espejiana BAL 31, IV. The single-strand specific deoxyriboendonuclease activity as a probe for regions o f altered secondary structure in negatively and positively supercoiled closed circular DNA, Nucleic Acids Res., 6 (1979) 331. 12 J.C. Wang, Variation of the average rotation angle of the DNA helix and the superhelical turns o f covalently closed circular ~, DNA, J. Mol. Biol., 43 (1969) 25. 13 W.B. Upholt, H.B. Gray and J. Vinograd, Sedimentation velocity behavior of closed circular SV40 DNA as a function o f superhelix density, ionic strength, counterion and temperature, J. Mol. Biol., 61 (1971) 21. 14 M. Woodworth-Gutai and J. Lebowitz, Introduction of interrupted secondary structure in supercoiled DNA as a function of superhelix density. Consideration of hairpin structures in superhelical DNA, J. Virol., 18 (1976) 195.