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Crosslinking of methylated DNA by moderate heating at neutral pH
BIOCHIMICA ET BIOPHYSICA ACTA
449
BBA 97192
CROSSLINKING OF M E T H Y L A T E D DNA BY MODERATE H E A T I N G AT N E U T R A L pH
J. B U R N O T T E
AND W. G. V E R L Y
Department o[ Biochemistry, University o[ Montreal, Montreal (Canada) (Received N o v e m b e r ISth, 1971)
SUMMARY
Interstrand crosslinks are formed in DNA methylated by methyl methanesulfonate, a monofunctional alkylating agent, after moderate heating at neutral pH. The extent of crosslinking is proportional to depurination: at pH 6.8, there is about I interstrand crosslink per 14o apurinic sites.
Three kinds of alterations have been described in DNA treated with a monofunctional alkylating agent such as methyl methanesulfonate: alkylations (of bases or phosphates), depurinations and single-strand breaks (see for example, ref. I). We report here a fourth consequence of treating DNA with methyl methanesulfonate: the formation of DNA interstrand crosslinks. The DNA was isolated from T 7 coliphage by phenol extraction2,3; it was homogeneous on ultracentrifugation with a molecular weight of about 25 ooo ooo. Methylated DNA was prepared by mixing I vol. of the DNA stock solution (420/~g DNA/ml, 15o mM NaC1, 15 mM EDTA, pH 7.5) with I vol. of a fresh methyl methanesulfonate solution (24 mM methyl methanesulfonate, 12o mM phosphate buffer, pH 6.8). After an incubation of I h at 37 °, the solution was cooled in ice and dialyzed against 15 ° mM NaC1, 15 mM EDTA, pH 6.8, at 4 °. Methylated DNA was heated at 50 ° in the NaC1-EDTA buffer, pH 6.8, for 4 h. All other conditions being identical, three controls were prepared, i.e. unheated methylated DNA and non methylated DNA heated respectively for 4 and 8 h. All samples were submitted to a formamide denaturation-renaturation cycle following the method of Brakier and Verly* and analyzed by analytical ultracentrifugation in a CsCI density gaadient. Fig. I shows that, while the unheated methylated DNA remained completely denatured after the formamide treatment (Curve b), 37 % of the heated methylated DNA renatured (Curve a); renaturation also occurred to a small extent for heated non methylated DNA (6 % renaturation afteI a 8-h-heating period) (Curves c and d). Spontaneous renaturation of the DNA after a transient exposure to denaturing conditions indicates the presence of covalent interstrand crosslinks in the moleculesS-~; therefore, DNA treatment with the monofunctional alkylating agent methyl methanesulfonate greatly enhances the formation of interstrand crosslinks upon moderate heating at neutral pH. Biochirn. Biophys. Acta, 262 (1972) 449--452
450
j. BURNOTTE, W. G. VERLY
A
b
C
\ ,-
~
~
~.~
~
,.~
d Buoyont density (g/cm 3 )
Fig. i. Interstrand crosslinks in heated DNA increased by a preliminary treatment by methyl methanesulfonate. (a) T 7 phage I)NA was methylated by methyl methanesulfonate as explained in the text; it was then heated during 4 h at 5°° in 15o mM: NaC1, 15 m2¢[ EDTA, pH 6.8, then cooled in ice, before being submitted to a denaturation-renaturation cycle (2-h exposure at 37 ° to 95 % freshly distilled formamide, followed by exhaustive dialysis at o ° against 15 mM NaC1, 15 mM EDTA, pH 7.0; final DNA concentration: 3/~g/ml). CsC1 was then dissolved until n D = 1.4ooo and the solution centrifuged at 4° ooo rev./min for 17 h at 20 ° in Spinco Model E ultracentrifuge equipped with a photoelectric scanner. The graph gives the absorbance at 265 nnl as a function of the buoyant density. Sarcina lutea denatured DNA served as a density marker (1.74 ° g/cm3); the densities of the native (1.71o g/cm8) and denatured (1.723 g/cm8) T 7 DNA_ are also indicated. (b) Control not treated by methyl methanesulfonate and not heated. (c) DNA not treated by methyl methanesulfonate, but heated 4 h at 5°0 in the conditions described in (a). (d) DNA not treated by methyl methanesulfonate, but heated 8 h at 5o°. D N A crosslinking resulting from u l t r a v i o l e t radiations s a n d bifunctional a l k y l a t i n g agents such as m u s t a r d gas 9 is q u i t e well understood. T h e chemical n a t u r e of crosslinking occurring, however, during D N A heating, near t h e m e l t i n g point, in acidic m e d i u m 1°, during or after exposure of D N A to nitrous acid n,lz, and, as shown in this work, d u r i n g m o d e r a t e h e a t i n g of th e D N A at n e u t r a l p H , p a r t i c u l a r l y after a m e t h y l m e t h a n e s u l f o n a t e t r e a t m e n t , is t o t a l l y u n k n o w n ; all these l a t t e r causes of crosslinking h a v e t h e f o r m a t i o n of apurinic sites in c o m m o n 13-16. In order to see w h e t h e r th e r e was a correlation b e t w e e n crosslinking an d dep u r i n a t i o n in h e a t e d m e t h y l a t e d D N A , t h e e x p e r i m e n t s were r e p e a t e d using t r i t i a t e d m e t h y l m e t h a n e s u l f o n a t e (specific a c t i v i t y 6. 7 m C / m m o l e ) . T h e m e t h y l a t e d D N A was o b t a i n e d as p r e v i o u s l y described a n d t h e n i n c u b a t e d at 50°; samples were withd r a w n after 0 (control), 0.5, 2 a n d 4 h of i n c u b a t i o n an d cooled in ice. F o r each sample, the D N A specific r a d i o a c t i v i t y was d e t e r m i n e d a n d t h e fraction of i n t e r s t r a n d crosslinked molecules was g iv e n b y th e ratio R / R + D , where D an d R were t h e r e s p e c t i v e areas u n d e r t h e d e n a t u r e d a n d r e n a t u r e d D N A peaks of t h e b a n d i n g p a t t e r n after a f o r m a m i d e d e n a t u r a t i o n - r e n a t u r a t i o n cycle followed b y an isopycnic u l t r a c e n t r i f u gation. T h e n u m b e r of a lk y l a t io n s p e r D N A molecule an d also t h e n u m b e r of depuriBiochim. Biophys. Acta, 262 (1972) 449-452
CROSSLINKING OF METHYLATED
451
DNA
nations occurring during the course of the incubation were calculated from the DNA specific radioactivities. The mean number Z of crosslinks per molecule was calculated from the fraction of interstrand crosslinked molecules using Poisson's equation; assuming the occurrence of crosslinks in a monodisperse population of DNA molecules to be random and the renaturation to be a one-hit process: D R+D
-
~e--Z
The value of Z calculated in this way is somewhat erroneous because of singlestrand breaks produced in DNA as a result of the methyl methanesulfonate treatment1; the presence of these breaks is attested b y a shift of the renatured DNA peak towards a higher buoyant density (Fig. I, Curve a). A DNA molecule with one inteistrand crosslink and one single-strand break yields, after a denaturation-renaturation cycle, a bihelical fragment with a single-stranded tail and a single-stranded fragment17; the former has a higher buoyant density than typical double stranded DNA, while the latter increases the area of the denatured DNA peak, thereby leading to an underestimation of the mean number of crosslinks per DNA molecule. No a t t e m p t was made, however, in this work to correct the Z values obtained from the above equation. Table I records the extent of the depurination of the methylated bases and the mean number of crosslinks per DNA molecule as a function of the incubation time at 5 °0 of the methylated DNA which contained 299 methyl groups per molecule at the beginning of the incubation. Fig. 2 shows that the increase of the interstrand crosslinkages is proportional to the extent of depurination; from the slope of the curve it can be calculated that there is about I crosslink per 14o depurinations. These findings tend to support a hypothesis proposed in 1964 b y Freese and CasheU ° to explain the formation of crosslinks in DNA heated near the melting point in acid medium: according to these authors, the depurination caused b y heating DNA at low p H produces an aldehyde function at the carbon I of the deoxyribose which subseTABLE
I
DEPURINATION AND CROSSLINKAGE IN HEATED METHYLATED D N A T7 D N A (2Io/~g/ml) in 75 m M NaC1, 7.5 m M E D T A , 60 m M s o d i u m p h o s p h a t e buffer, p H 6.8, w a s t r e a t e d b y 12 mlV[ [ a l l ] m e t h y l m e t h a n e s u l f o n a t e for I h a t 37°; t h e solution w a s cooled a n d dialyzed a g a i n s t cold buffer (15o mlV[ lX!aC1, 15 mlVf E D T A , p H 6.8). D e p u r i u a t i o n w a s o b t a i n e d a f t e r t r a n s f e r of t h e dialysis b a g s into t h e s a m e w a r m b u f f e r (50 °) for v a r i o u s periods of t i m e . T h e specific a c t i v i t y of t h e D N A s a m p l e s w a s m e a s u r e d a f t e r five e t h a n o l p r e c i p i t a t i o n s following r e d i s s o l u t i o n s in t h e s a m e buffer. Z was c a l c u l a t e d f r o m t h e CsC1 b a n d i n g p a t t e r n of D N A after a f o r m a m i d e d e n a t u r a t i o n - r e n a t u r a t i o n cycle (see t e x t a n d Fig. i); t h e s t a t i s t i c a l s t a n d a r d error is a b o u t ± o . 0 3 . Incubation time at 5 °0 (h):
o
o.5
2.o
4.o
Specific a c t i v i t y of t h e D N A (dpm//~g) M e t h y l g r o u p s per D N A m o l e c u l e M e t h y l a t e d b a s e s h y d r o l y z e d (depurin a t i o n s ) per D N A m o l e c u l e d u r i n g incubation iVfean n u m b e r of crosslinks, Z, p e r D N A molecule (uncorrected)
4494 299
4023 273
3751 258
3164 232
o
26
41
67
o
o.13
o.31
o.47
Biochim. Biophys. Acta, 262 (1972) 449-452
452
J. BURNOTTE, W. G. VERLY
0.~
0.2/ 2'o
io
6'o
so
motes of depurinated bases/mote of DNA
Fig. 2. Relation b e t w e e n crosslinkage a n d d e p u r i n a t i o n in D N A t r e a t e d b y a m o n o f u n c t i o n a l alkylating agent. T 7 p h a g e D N A w a s m e t h y l a t e d b y m e t h y l m e t h a n e s u l f o n a t e , t h e n incubated at 50 °, p H 6.8; s a m p l e s were t a k e n after o, o.5, 2 a n d 4 h of incubation. The e x p e r i m e n t a l conditions are described in Table I. The slope of the curve indicates i i n t e r s t r a n d crosslink for a b o u t 14o depurinations.
sequently reacts with a chemical group in the opposite DNA strand creating an interstrand crosslink. Depurination could also be responsible for the DNA crosslinking caused by nitrous acid as we have suggested previouslym: deamination, like alkylation, increases the depufination rate. ACKNOWLEDGEMENT
The present investigation was supported by a grant from the Medical Research Council of Canada. REFERENCES
i B.
Strauss, 1V[. Coyle and M. Robbins, Ann. N . Y . Acad. Sci., 163 (1969) 765 . 2 P. F, D a v i s o n and D. Freilfelder, J. Mol. Biol., 5 (1962) 635. 3 P. F. D a v i s o n and D. Freilfelder, J. Mol. Biol., 5 (1962) 643. 4 L . Brakier a n d W. G. Verly, Biochim. Biophys. Acta, 213 (197 o) 296. 5 E . P. Geiduschek, Proc. Natl. Acad. Sci. U.S., 47 (1961) 95 o. 6 E . P. Geiduschek, J. Mol. Biol., 4 (1962) 467 . 7 D • Luzzati, Biochem. Biophys. Res. Commun., 9 (1962) 5 °8. 8 V . R. Gligin a n d t s. Doty, Biochim. Biophys. Acta, 142 (I967) 314 . 9 P. D. Lawley and P. Brookes, J. Mol. Biol., 25 (1967) 143. lO E. Freese and H. Cashel, Biochim. Biophys. Acta, 91 (1964) 167. I I E. F. Becker Jr., B. K. Z i m m e r m a n and E. P. Geiduschek, J. Mol. Biol., 8 (1964) 337. 12 J. B u r n o t t e and W. G. Verly, J. Biol. Chem., 246 (1971) 5914. 13 S. Greer and S. Zamenhof, J. Mol. Biol., 4 (1962) 123. 14 H. Schuster, Z. Naturforsch., I5b (196o) 298. 15 H. S. Shapiro a n d E. Chargaff, Biochemistry, 5 (1966) 3o12. 16 P. D. Lawley a n d P. Brookes, Biochem. J., 89 (1963) 127. 17 w . C. S u m m e r s a n d W. Szybalski, J. Mol. Biol., 26 (1967) lO 7.
Biochim. Biophys. Acta, 262 (1972) 449-452
449
BBA 97192
CROSSLINKING OF M E T H Y L A T E D DNA BY MODERATE H E A T I N G AT N E U T R A L pH
J. B U R N O T T E
AND W. G. V E R L Y
Department o[ Biochemistry, University o[ Montreal, Montreal (Canada) (Received N o v e m b e r ISth, 1971)
SUMMARY
Interstrand crosslinks are formed in DNA methylated by methyl methanesulfonate, a monofunctional alkylating agent, after moderate heating at neutral pH. The extent of crosslinking is proportional to depurination: at pH 6.8, there is about I interstrand crosslink per 14o apurinic sites.
Three kinds of alterations have been described in DNA treated with a monofunctional alkylating agent such as methyl methanesulfonate: alkylations (of bases or phosphates), depurinations and single-strand breaks (see for example, ref. I). We report here a fourth consequence of treating DNA with methyl methanesulfonate: the formation of DNA interstrand crosslinks. The DNA was isolated from T 7 coliphage by phenol extraction2,3; it was homogeneous on ultracentrifugation with a molecular weight of about 25 ooo ooo. Methylated DNA was prepared by mixing I vol. of the DNA stock solution (420/~g DNA/ml, 15o mM NaC1, 15 mM EDTA, pH 7.5) with I vol. of a fresh methyl methanesulfonate solution (24 mM methyl methanesulfonate, 12o mM phosphate buffer, pH 6.8). After an incubation of I h at 37 °, the solution was cooled in ice and dialyzed against 15 ° mM NaC1, 15 mM EDTA, pH 6.8, at 4 °. Methylated DNA was heated at 50 ° in the NaC1-EDTA buffer, pH 6.8, for 4 h. All other conditions being identical, three controls were prepared, i.e. unheated methylated DNA and non methylated DNA heated respectively for 4 and 8 h. All samples were submitted to a formamide denaturation-renaturation cycle following the method of Brakier and Verly* and analyzed by analytical ultracentrifugation in a CsCI density gaadient. Fig. I shows that, while the unheated methylated DNA remained completely denatured after the formamide treatment (Curve b), 37 % of the heated methylated DNA renatured (Curve a); renaturation also occurred to a small extent for heated non methylated DNA (6 % renaturation afteI a 8-h-heating period) (Curves c and d). Spontaneous renaturation of the DNA after a transient exposure to denaturing conditions indicates the presence of covalent interstrand crosslinks in the moleculesS-~; therefore, DNA treatment with the monofunctional alkylating agent methyl methanesulfonate greatly enhances the formation of interstrand crosslinks upon moderate heating at neutral pH. Biochirn. Biophys. Acta, 262 (1972) 449--452
450
j. BURNOTTE, W. G. VERLY
A
b
C
\ ,-
~
~
~.~
~
,.~
d Buoyont density (g/cm 3 )
Fig. i. Interstrand crosslinks in heated DNA increased by a preliminary treatment by methyl methanesulfonate. (a) T 7 phage I)NA was methylated by methyl methanesulfonate as explained in the text; it was then heated during 4 h at 5°° in 15o mM: NaC1, 15 m2¢[ EDTA, pH 6.8, then cooled in ice, before being submitted to a denaturation-renaturation cycle (2-h exposure at 37 ° to 95 % freshly distilled formamide, followed by exhaustive dialysis at o ° against 15 mM NaC1, 15 mM EDTA, pH 7.0; final DNA concentration: 3/~g/ml). CsC1 was then dissolved until n D = 1.4ooo and the solution centrifuged at 4° ooo rev./min for 17 h at 20 ° in Spinco Model E ultracentrifuge equipped with a photoelectric scanner. The graph gives the absorbance at 265 nnl as a function of the buoyant density. Sarcina lutea denatured DNA served as a density marker (1.74 ° g/cm3); the densities of the native (1.71o g/cm8) and denatured (1.723 g/cm8) T 7 DNA_ are also indicated. (b) Control not treated by methyl methanesulfonate and not heated. (c) DNA not treated by methyl methanesulfonate, but heated 4 h at 5°0 in the conditions described in (a). (d) DNA not treated by methyl methanesulfonate, but heated 8 h at 5o°. D N A crosslinking resulting from u l t r a v i o l e t radiations s a n d bifunctional a l k y l a t i n g agents such as m u s t a r d gas 9 is q u i t e well understood. T h e chemical n a t u r e of crosslinking occurring, however, during D N A heating, near t h e m e l t i n g point, in acidic m e d i u m 1°, during or after exposure of D N A to nitrous acid n,lz, and, as shown in this work, d u r i n g m o d e r a t e h e a t i n g of th e D N A at n e u t r a l p H , p a r t i c u l a r l y after a m e t h y l m e t h a n e s u l f o n a t e t r e a t m e n t , is t o t a l l y u n k n o w n ; all these l a t t e r causes of crosslinking h a v e t h e f o r m a t i o n of apurinic sites in c o m m o n 13-16. In order to see w h e t h e r th e r e was a correlation b e t w e e n crosslinking an d dep u r i n a t i o n in h e a t e d m e t h y l a t e d D N A , t h e e x p e r i m e n t s were r e p e a t e d using t r i t i a t e d m e t h y l m e t h a n e s u l f o n a t e (specific a c t i v i t y 6. 7 m C / m m o l e ) . T h e m e t h y l a t e d D N A was o b t a i n e d as p r e v i o u s l y described a n d t h e n i n c u b a t e d at 50°; samples were withd r a w n after 0 (control), 0.5, 2 a n d 4 h of i n c u b a t i o n an d cooled in ice. F o r each sample, the D N A specific r a d i o a c t i v i t y was d e t e r m i n e d a n d t h e fraction of i n t e r s t r a n d crosslinked molecules was g iv e n b y th e ratio R / R + D , where D an d R were t h e r e s p e c t i v e areas u n d e r t h e d e n a t u r e d a n d r e n a t u r e d D N A peaks of t h e b a n d i n g p a t t e r n after a f o r m a m i d e d e n a t u r a t i o n - r e n a t u r a t i o n cycle followed b y an isopycnic u l t r a c e n t r i f u gation. T h e n u m b e r of a lk y l a t io n s p e r D N A molecule an d also t h e n u m b e r of depuriBiochim. Biophys. Acta, 262 (1972) 449-452
CROSSLINKING OF METHYLATED
451
DNA
nations occurring during the course of the incubation were calculated from the DNA specific radioactivities. The mean number Z of crosslinks per molecule was calculated from the fraction of interstrand crosslinked molecules using Poisson's equation; assuming the occurrence of crosslinks in a monodisperse population of DNA molecules to be random and the renaturation to be a one-hit process: D R+D
-
~e--Z
The value of Z calculated in this way is somewhat erroneous because of singlestrand breaks produced in DNA as a result of the methyl methanesulfonate treatment1; the presence of these breaks is attested b y a shift of the renatured DNA peak towards a higher buoyant density (Fig. I, Curve a). A DNA molecule with one inteistrand crosslink and one single-strand break yields, after a denaturation-renaturation cycle, a bihelical fragment with a single-stranded tail and a single-stranded fragment17; the former has a higher buoyant density than typical double stranded DNA, while the latter increases the area of the denatured DNA peak, thereby leading to an underestimation of the mean number of crosslinks per DNA molecule. No a t t e m p t was made, however, in this work to correct the Z values obtained from the above equation. Table I records the extent of the depurination of the methylated bases and the mean number of crosslinks per DNA molecule as a function of the incubation time at 5 °0 of the methylated DNA which contained 299 methyl groups per molecule at the beginning of the incubation. Fig. 2 shows that the increase of the interstrand crosslinkages is proportional to the extent of depurination; from the slope of the curve it can be calculated that there is about I crosslink per 14o depurinations. These findings tend to support a hypothesis proposed in 1964 b y Freese and CasheU ° to explain the formation of crosslinks in DNA heated near the melting point in acid medium: according to these authors, the depurination caused b y heating DNA at low p H produces an aldehyde function at the carbon I of the deoxyribose which subseTABLE
I
DEPURINATION AND CROSSLINKAGE IN HEATED METHYLATED D N A T7 D N A (2Io/~g/ml) in 75 m M NaC1, 7.5 m M E D T A , 60 m M s o d i u m p h o s p h a t e buffer, p H 6.8, w a s t r e a t e d b y 12 mlV[ [ a l l ] m e t h y l m e t h a n e s u l f o n a t e for I h a t 37°; t h e solution w a s cooled a n d dialyzed a g a i n s t cold buffer (15o mlV[ lX!aC1, 15 mlVf E D T A , p H 6.8). D e p u r i u a t i o n w a s o b t a i n e d a f t e r t r a n s f e r of t h e dialysis b a g s into t h e s a m e w a r m b u f f e r (50 °) for v a r i o u s periods of t i m e . T h e specific a c t i v i t y of t h e D N A s a m p l e s w a s m e a s u r e d a f t e r five e t h a n o l p r e c i p i t a t i o n s following r e d i s s o l u t i o n s in t h e s a m e buffer. Z was c a l c u l a t e d f r o m t h e CsC1 b a n d i n g p a t t e r n of D N A after a f o r m a m i d e d e n a t u r a t i o n - r e n a t u r a t i o n cycle (see t e x t a n d Fig. i); t h e s t a t i s t i c a l s t a n d a r d error is a b o u t ± o . 0 3 . Incubation time at 5 °0 (h):
o
o.5
2.o
4.o
Specific a c t i v i t y of t h e D N A (dpm//~g) M e t h y l g r o u p s per D N A m o l e c u l e M e t h y l a t e d b a s e s h y d r o l y z e d (depurin a t i o n s ) per D N A m o l e c u l e d u r i n g incubation iVfean n u m b e r of crosslinks, Z, p e r D N A molecule (uncorrected)
4494 299
4023 273
3751 258
3164 232
o
26
41
67
o
o.13
o.31
o.47
Biochim. Biophys. Acta, 262 (1972) 449-452
452
J. BURNOTTE, W. G. VERLY
0.~
0.2/ 2'o
io
6'o
so
motes of depurinated bases/mote of DNA
Fig. 2. Relation b e t w e e n crosslinkage a n d d e p u r i n a t i o n in D N A t r e a t e d b y a m o n o f u n c t i o n a l alkylating agent. T 7 p h a g e D N A w a s m e t h y l a t e d b y m e t h y l m e t h a n e s u l f o n a t e , t h e n incubated at 50 °, p H 6.8; s a m p l e s were t a k e n after o, o.5, 2 a n d 4 h of incubation. The e x p e r i m e n t a l conditions are described in Table I. The slope of the curve indicates i i n t e r s t r a n d crosslink for a b o u t 14o depurinations.
sequently reacts with a chemical group in the opposite DNA strand creating an interstrand crosslink. Depurination could also be responsible for the DNA crosslinking caused by nitrous acid as we have suggested previouslym: deamination, like alkylation, increases the depufination rate. ACKNOWLEDGEMENT
The present investigation was supported by a grant from the Medical Research Council of Canada. REFERENCES
i B.
Strauss, 1V[. Coyle and M. Robbins, Ann. N . Y . Acad. Sci., 163 (1969) 765 . 2 P. F, D a v i s o n and D. Freilfelder, J. Mol. Biol., 5 (1962) 635. 3 P. F. D a v i s o n and D. Freilfelder, J. Mol. Biol., 5 (1962) 643. 4 L . Brakier a n d W. G. Verly, Biochim. Biophys. Acta, 213 (197 o) 296. 5 E . P. Geiduschek, Proc. Natl. Acad. Sci. U.S., 47 (1961) 95 o. 6 E . P. Geiduschek, J. Mol. Biol., 4 (1962) 467 . 7 D • Luzzati, Biochem. Biophys. Res. Commun., 9 (1962) 5 °8. 8 V . R. Gligin a n d t s. Doty, Biochim. Biophys. Acta, 142 (I967) 314 . 9 P. D. Lawley and P. Brookes, J. Mol. Biol., 25 (1967) 143. lO E. Freese and H. Cashel, Biochim. Biophys. Acta, 91 (1964) 167. I I E. F. Becker Jr., B. K. Z i m m e r m a n and E. P. Geiduschek, J. Mol. Biol., 8 (1964) 337. 12 J. B u r n o t t e and W. G. Verly, J. Biol. Chem., 246 (1971) 5914. 13 S. Greer and S. Zamenhof, J. Mol. Biol., 4 (1962) 123. 14 H. Schuster, Z. Naturforsch., I5b (196o) 298. 15 H. S. Shapiro a n d E. Chargaff, Biochemistry, 5 (1966) 3o12. 16 P. D. Lawley a n d P. Brookes, Biochem. J., 89 (1963) 127. 17 w . C. S u m m e r s a n d W. Szybalski, J. Mol. Biol., 26 (1967) lO 7.
Biochim. Biophys. Acta, 262 (1972) 449-452