marker N6-methyladenine. However, under the experimental conditions, it would not have been possible to distinguish between deaminated and nitrosated ...
Vol. 34, No. 1
JOURNAL OF VIROLOGY, Apr. 1980, p. 277-279 0022-538X/80/04-0277/03$02.00/0
Specificity of the Bacteriophage Mu mom+-Controlled DNA Modification STANLEY HATTMANt University of Rochester, Department ofBiology, Rochester, New York 14627
Bacteriophage Mu DNA was labeled after induction in the presence of [8-3H]adenine. Purified DNA was enzymatically digested, and the 3H-labeled dinucleotides were isolated. Approximately 15 to 20% of the adenine residues were modified to a new form, A., as observed previously (S. Hattman, J. Virol. 32:468-475, 1979) in bulk DNA. Paper electrophoretic analysis revealed that only two dinucleotide species contain A., namely, (A.,C) and (A.,G). The observation that only C and G are the nearest neighbors of A. is consistent with the proposal of Kahmann and Kamp (R. Kahmann and D. Kamp, J. Mol. Biol., in press) that modification of Mu DNA occurs at the A residue within the pentanucleotide sequence, 5'.. .&)-A-(G)-N-Py... .3'. I have shown previously (3) that bacteriophage Mu DNA contains an unusual modified adenine residue, A1; approximately 15% of the adenine bases are modified. The occurrence of this modification is dependent on the expression of the phage mom' gene and the host dam' gene (7, 8); these results indicate that the A. modification is responsible for protecting Mu DNA against in vivo and in vitro restriction (1, 6; R. Kahmann and D. Kamp, J. Mol. Biol., in press). The molecular structure of A. is not yet defined, but the added moiety contains a free carboxyl group (3). In that study, it was erroneously concluded that the modification does not occur at the N6-amino group of adenine; this conclusion was based on electrophoretic analyses of control and nitrous acid-treated 3H-labeled A1 and marker N6-methyladenine. However, under the experimental conditions, it would not have been possible to distinguish between deaminated and nitrosated bases. Therefore, it is still possible that A1 modification occurs at the N6-amino group. Herein I report the results of an analysis of the nearest neighbors to the A1 residue. I show that only C and G are adjacent to A., indicating that the A1 modification process involves specific nucleotide sequence recognition. Furthermore, the nature of the nearest neighbors is in accord with the modification sequence deduced by Kahmann and Kamp (in press) from studies on the protection against in vitro cleavage by site-specific nucleases; the proposed sequence is 5'...(8)-A-(G)-N-Py ..3', where A is the site of modification. t Present address: Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112.
After thermal induction in a dam' host, phage Mu was labeled in the presence of [8-3H]adenine. The DNA was isolated from purified phage and enzymatically digested; the resulting 5'-monoand dinucleotides were then fractionated by ionexchange chromatography (4, 5). Both monoand dinucleotides contained approximately 15% modification of adenine to A.; this was demonstrated by paper chromatographic analysis of bases liberated by acid hydrolysis (data not shown). The same degree of modification has been observed after hydrolysis of total DNA (3). To investigate which bases are the nearest neighbors of A., the dinucleotides were subjected to paper electrophoresis. Because A. contains an acidic group, A.-containing dinucleotides should migrate further toward the anode than the corresponding A-containing dinucleotides. Thus, A.-containing dinucleotides should be recognized as radioactive species which do not migrate with the major peaks of A-containing dinucleotides. After electrophoresis at pH 1.9, a minor radioactive peak is observed migrating ahead of the dinucleotide, (A,G) (Fig. 1). At pH 3.5, this species is again observed to migrate ahead of the (A,G) marker (Fig. 2a). After acid hydrolysis and paper chromatography (2), 85 to 95% of the 3H radioactivity was observed in A., and the remainder was observed in adenine. Based on its electrophoretic behavior, I conclude that the chemical composition of this dinucleotide is (A., G). At pH 1.9, (A1,G) was the only A.-containing dinucleotide evident (Fig. 1); however, when each of the three major peaks was analyzed for the presence of A., I observed that the (A,G) region also contained about 25% [3H]A1, but no [3H]A. was observed in (A,T) or in (A,A)/(A,C)
277
278 NOTES (data not shown). Because of its electrophoretic mobility, this species cannot be (A.,G); the most likely dinucleotide composition has to be either (A.,C) or (A.,A). To distinguish between these two compositions, the sample was subjected to paper electrophoresis at pH 3.5, where (A,C), (A,A), and (A,G) are al separated from one another. As shown in Fig. 2b, the 3H label was divided into two regions: 75% of the 3H was in the (A,G) region {due to the authentic [3H](A,G)), and 25% of the label migrated with the (A,A) marker. Because [3H](A.,A) should migrate further than (A,A), the composition of (A.,A) has to be ruled out. To support the notion that the dinucleotide is (A.,C), advantage was taken of the observation that the mononucleotides pG and pA. have similar electrophoretic mobilities (data not shown). Thus, [3H](A.,C) and marker (G,C) would be expected to coelectrophorese; as can be seen in Fig. 2c, this was observed to occur. The results presented above show that A. is found only in the dinucleotides, (A.,C) and (A.,G); from the data in Fig. 1 and 2, I calculate
J. VIROL.
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40 C 3 b
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500
310 1500 I
AT
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20
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FIG. 2. Paper electrophoresis analysis ofpartially purified 3H-labeled dinucleotides. After electrophoretic separation at pH 1.9 (see Fig. 1), various dinucleotide regions were eluted and taken for paper electrophoresis at pH 3.5 (2.5 h; 35 V/cm; origin, 12 cm from cathode). a, (A,,, G); b, (A, G); c, (A, G).
(AX,G)
0
3 35
10 cm
20 15 from origin
25
30 (
FIG. 1. Paper electrophoresis analysis of [8-SHIadenine-labeled dinucleotides obtained from Mu mom+*dam' DNA. [8-3H]adenine-labeled DNA was prepared (3) and enzymatically digested to 5'-mononucleotides and dinucleotides (5). The dinucleotides were purified (4, 5) and separated by filter-paper (Whatman 3 MM) electrophoresis at pH 1.9 (3.5 h; 35 V/cm; origin, 15 cm from the cathode). Authentic 5'dinucleotide markers were included in the analysis (indicated by the solid bars).
that these two species were present in nearly equal amounts. The fact that only C and G appear to be the nearest neighbors of A. leads to the conclusion that the mom' modification process involves specific nucleotide sequence recognition. Because the relative position of A. in each dinucleotide was not determined, the trinucleotide core sequence of the modification site cannot be deduced. In this regard, however, Kahmann and Kamp (in press) have obtained evidence (from in vitro studies with site-specific nucleases) for the protection of the sequence,
VOL. 34, 1980
5'...(c)-A-(G)-N-Py.. .3'.
Mu DNA (50% G-C content) should contain about 13% of its adenine residues in this sequence (assuming a random distribution of bases). In view of the fact that about 15% of the adenines are A., I propose that A. modification occurs specifically in the above sequence and that the modification protects Mu DNA against restriction nuclease action in this site. This work was supported by Public Health Service grant AI-10864. I thank my colleague K. Drlica for optimistic suggestions and R. Kahmann and D. Kamp for sharing their results and ideas before publication.
LITERATURE CMD 1. Allet, B., and A. I. Bukhari. 1975. Analysis of bacteriophage Mu and X-Mu hybrid DNAs by specific endonucleases. J. Mol. Biol. 92:529-540.
NOTES
279
2. Hattman, S. 1970. DNA methylation of T-even bacteriophages and of their nonglucosylated mutants: its role in Pl-directed restriction. Virology 42:359-367. 3. Hattman, S. 1979. Unusual modification of bacteriophage Mu DNA. J. Virol. 32:468-475. 4. Hattman, S., J. E. Brooks, and M. Masurekar. 1978. Sequence specificity of the Pl-modification methylase (M.Eco P1) and the DNA methylase (M.Eco dam) controlled by the E. coli dam-gene. J. Mol. Biol. 126: 367-380. 5. Hattman, S., H. van Ormondt, and A. deWaard. 1978. Sequence specificity of the wild-type (dam') and mutant (damh) forms of bacteriophage T2 DNA adenine methylase. J. Mol. Biol. 119:361-376. 6. Khatoon, H., and A. I. Bukhari. 1978. Bacteriophage Mu-induced modification of DNA is dependent upon a host function. J. Bacteriol. 136:423-428. 7. Toussaint, A. 1976. The DNA modification function of temperate phage Mu-1. Virology 70:17-27. 8. Toussaint, A. 1977. DNA modification of bacteriophage Mu-1 requires both host and bacteriophage functions. J. Virol. 23:825-826.
