Berneman, A.

Robert-Gero, M.

Vigier, P.

Elsevier - Publisher Connector 

Volume 89, number 1 FEBS LETTERS May 1978 
DNA METHYLASE ACTIVITY ASSOCIATED WITH ROUS SARCOMA VIRUS 
A. BERNEMAN and M. ROBERT-GERO 
Institut de Chimie des Substances NatureIles, CNRS, 91190 Gif-w-Yvette 
and 
P. VIGIER 
Institut du Radium, 91400 Orsay, France 
Received 24 February 1978 
1. Introduction 
More than 15 enzyme activities associated with 
purified preparations of C-type RNA tumor viruses 
have been reported [I] . Some of these enzymes 
function in synthesis or in modification of proteins, 
while others are involved in nucleic acid synthesis. 
However, the only one definitely known to be coded 
for by the viral genome is RNA-directed DNA poly- 
merase (reverse transcriptase) associated with nuclease 
H [2] . In contrast, most of the other virus-associated 
enzymes are presumably coded for by the host cell. 
We have shown recently that the Nz-methylguanine 
transferase associated with avian myeloblastosis virus 
(AMV) and Rous sarcoma virus (RSV) which methyl- 
ates tRNA of Escherichia coli, presumably originates 
from the host cell [3]. An RSV-associated tRNA 
methylase which incorporates a small amount of 
radioactivity from S-adenosyl-methionine (SAM) into 
E. coli DNA was described [4] . 
S-Adenosyl-homocysteine (SAH) the demethylated 
derivative of SAM is the natural inhibitor of nearly 
all transmethylases in vitro [5,6] . Some synthetic 
analogs of SAH inhibit nearly completely RSV- 
induced cell transformation and concomitant virus 
replication [7,8] , although SAH has no such activity, 
due to its rapid intracellular degradation [9]. The 
ability of synthetic methylase inhibitors to block 
both the production of RSV and cell transformation 
by this virus, has prompted us to further study the 
Elsevier/North-Holland Biomedical Press 
RSV-associated DNA methylase. The function of this 
enzyme might be to methylate viral cDNA. This 
methylation might be required for subsequent steps 
of viral replication and cell transformation and might 
be inhibited by the analogs. Our results show that the 
activity of the RSV-associated DNA methylase is 
dependent on the presence of detergent, and is 
distinct from the virus-associated tRNA methylase. 
The enzyme, which appears to be located in the 
outer envelope or in the surface projection of the 
virus does not methylate cDNA, and the synthetic 
SAH analogs which inhibit virus replication and cell 
transformation have only a weak aftinity for this 
methylase. 
2. Materials and methods 
Different DNAs were obtained from the following 
commercial sources: Micrococcus lysodeikticus, Miles 
Laboratories Inc., USA; calf thymus, Choay, France; 
E. coli B and salmon sperm, Sigma, USA. DNA from 
normal and RSV-transformed chick embryo fibroblasts 
(CEF) was extracted as in [IO]. cDNA from the same 
cells was prepared with reverse transcriptase as in [ 111 
in final vol. 500 ~1 (extraction as above). 
2.1. Chemicals 
Compounds 2,3,4 listed in table 3 were synthesised 
in our laboratory [8]. SAH and SIBA came from 
33 
Volume 89, number 1 FEBS LETTERS May 1978 
Sefochem Fine Chemicals, Israel. Unlabelled deoxy- 
ribonucleotides came from Sigma, USA. [3H]- 
Thymidine5’triphosphate (spec. act. 10 or 30 Ci/mM) 
and S-adenosyl [methyl-3H]methionine (spec. act. 
10 Ci/mM) came from Amersham, England. The non- 
ionic detergent Nonidet P 40 (NP 40) was from Shell, 
France. 
2.2. Cell cultures and virus production 
Infection of CEF by RSV strain SRD and virus 
purification were performed as in [ 71. 
2.3. Separation of viral components 
Virus protein concentration was adjusted to 
3 mg/ml, estimated as in [ 121 . The purified virus 
preparation was then incubated with various con- 
centrations of NP 40 at 37°C for 30 min. 500 pg were 
put on top of a 30-66% linear sucrose density 
gradient. Sedimentation was run for 16 h in a Spinco 
ultracentrifuge, in a SW 41 rotor, at 30 000 revlmin. 
Fractions of 300 /_d were then collected from the top 
to the bottom of the gradient and theA2s0 monitored. 
2.4. DNA methylase assay 
The incubation mixture in 50 ~1 had the following 
composition: 50 mM Tris-HCl, pH 8.3,60 mM NaCl, 
16 mM MgClz, 3 mM dithiothreitol, 50 pg heat dena- 
tured calf thymus DNA, 0.2% NP 40, 1.6 nCi methyl- 
labelled SAM. The mixture was incubated at 37°C 
for 1 h. Blanks were run under the same conditions, 
without DNA. The reaction was stopped by adding 
cold 5% trichloroacetic acid (TCA), then the precipitate 
was filtered on GF/C Whatman filters, thoroughly 
washed by cold 5% TCA and counted. Blank values were 
deducted in order to determine the incorporation of 
[3H]methyl from SAM into DNA; specific activity is 
expressed in pmol [3H] methyl groups incorporated 
into 50 ng DNA in 1 h at 37°C. 
2.5. Reverse transcriptase assay 
This was performed as in [ 111. RNA methylase 
was assayed as in [ 51 . 
2.6. Thin-layer chromatography 
The hydrolysate of DNA was chromatographed 
with a mixture of known bases in the following 
solvents: first dimension, ethyl acetate, methanol, 
HzO, formic acid (100 : 25 : 20 : 1); second dimen- 
34 
sion,acetonitrile,ethyl acetate, 2-propanol, 1-butanol, 
34%ammoniumhydroxide (40 : 30 : 20 : 10 : 37.5). 
The spots were detected in ultraviolet, scraped and 
counted. 
3. Results and discussion 
We reported recently that an N2-methylguanine 
transferase associated with AMV and with RSV is 
located inside the viral envelope but outside the viral 
core, contrary to reverse transcriptase which is in the 
core [3]. In order to know whether the DNA methyl- 
ase activity associated with RSV is distinct from the RNA 
methylase, we have studied its location by sucrose density 
gradient centrifugation of purified viruses in the 
presence of detergent (NP 40) under conditions 
allowing the separation of virions, viral cores and 
intermediate particles. Before performing this study, 
we checked that no methylase activity was associated 
with purified concentrated viruses. As seen in fig.1 a 
when 1% NP 40 was used, reverse transcriptase and 
RNA methylase activities remained together, at a 
density of 1 .I 5 g/cm3, while the DNA methylase 
activity sedimented at 1.16 g/cm3. With 2% NP 40, 
DNA methylase activity remained at a density of 
1.16- 1.17 g/cm3, while RNA methylase activity 
sedimented at a density of 1.23 g/cm3 and reverse 
transcriptase activity at 1.26 g/cm3, corresponding 
to that expected for viral cores. (fig. 1 b). These 
results suggest hat the two methylases are distinct 
proteins, which are not adsorbed onto the virions 
since their activity is detected only following treat- 
ment with NP 40. Furthermore, neither activity 
sediments with the viral core but at lower densities 
suggesting that the tRNA methylase may be located 
inside the outer viral envelope, whereas the DNA 
methylase may be associated with this envelope, or 
with its surface projections. Indeed, treatment of 
AMV with NP 40 releases the surface projections in 
a distinct aggregate which can be separated com- 
pletely from other virus material by density gradient 
centrifugation [ 131 . 
We also looked for the presence of a DNA methyl- 
ase activity in other RNA viruses such as Rous- 
associated leukemia virus (RAVi ) avian myeloblastosis 
virus (AMV) murine sarcoma virus (MSV) and vesic- 
ular stomatitis virus (VSV). Only RSV and RAVi 
Volume 89, number 1 FEBS LETTERS May 1978 
E 
froc t ion number fraction number 
Fig.1. Localization of enzymatic activities in disrupted viral particles. A: treatment with 1% NP 40. B: treatment with 2% NP 40. 
Reverse transcriptase (o---o). RNA methylase activity (@---@). DNA methylase activity (o-o). 
showed this activity. The fact that the virus associated 
DNA methylase appears to be located on the outer 
membrane of the virus or its projections led us to 
consider, whether this enzyme might have a cellular 
origin. However, no DNA methylase activity was 
found in purified CEF cell membranes prepared as 
in [14]. 
The main characteristics of this DNA methylase 
were subsequently determined. 
The following specific activities were obtained 
with DNAs from different sources: heat-denatured 
calf thymus, 340; RSV-transformed CEF, 175 ; 
salmon sperm, 165; E. coli B, 95; normal CEF, 68; 
M. lysodeikticus, 50; RSV cDNA, 3.6. 
The best substrate is thus heat-denatured calf 
thymus DNA, whereas cDNA from RSV is not a good 
substrate. A linear relationship was obtained between 
the enzyme activity and the protein concentration up 
to 200 pg/ml and between enzyme activity and 
incubation time. The optimal pH range of the DNA 
methylase with heat-denatured calf thymus DNA was 
between 8.0 and 8.4. No enzyme activity could be 
detected without NP 40 and the optimal activity was 
obtained when the concentration of the non-ionic 
detergent was between 0.1% and 0.2% (results not 
shown). 
The products of this DNA methylase have been 
determined. After extraction [ 1.51 the [3H]methyl- 
labelled calf thymus DNA was treated with 88% 
formic acid for 2 h at 174’C. A two-dimensional 
thin-layer chromatography of the hydrolysate re- 
vealed that 40% of the radioactivity is incorporated 
into 5-methyl-cytosine and 38% into 1 -methyl- 
adenine. 22% of the radioactivity was distributed to 
about the same proportion between N-6 methyl- 
adenine, 2-methyl-adenine,N-6,iV-6dimethyl-adenine, 
N-2-methyl-guanine and 5-methyl uracil. It is rather 
unprobable that the C-5 methylation of cytosine and 
the N-l methylation of adenine are catalyzed by the 
same enzyme. The methylation of adenine by a DNA 
methylase is rather unexpected; however, l-methyl- 
adenine was a minor product of in vivo methylation 
of hamster liver DNA by the carcinogen dimethyl- 
nitrosamine [ 161. 
Since structural analogs of SAH are good inhibitors 
of cell transformation, we have tried to correlate this 
inhibition with their effect on the DNA methylase. 
Table 1 shows that all the analogs tested have low 
affinities for this enzyme in vitro. Hence, these results 
suggest hat this methylase contrary to cellular tRNA 
and protein methylases is not the main target of the 
inhibitors. 
35 
Volume 89. number 1 FEBS LETTERS May 1978 
Table 1 
Effect of SAH and some of its analogs on RSV associated 
DNA methylases 
t-i6 OH 
No. R Ki 
(ctM) 
1 SIBA 4310 
2 ISOSIBA CH,-CH,-CHS- 6000 
CH, 
3 CH,=CH-CH,S- 380 181 
4 HOH,C(HO)HC-CH,S- 500 
5 SAH HOOC CH-(CH, ),S- 1.5 
NH, 
SAH and the analogs behave like competitive inhibitors with 
respect to SAM. The Km value for SAM is 0.72 MM. The 
substrate used was heat-denatured calf thymus DNA 
Acknowledgements 
The authors gratefully acknowledge Professor E. 
Lederer for helpful discussions and critical reading of 
the manuscript. This work was supported by grants 
from the Centre National de Recherche Scientifique, 
ATP ‘Pharmacologic des Substances anticancereuses 
et immunomodulatrices’, the Institut National de la 
Sante et de la Recherche Mddicale ATP 52.77.84 
‘Pharmacologic et effets biologiques des nucleosides, 
nucleotides et substances apparentees’, and by the 
Commissariat a 1’Energie Atomique for the purchase 
of labelled compounds. 
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DNA methylase activity associated with rous sarcoma virus 

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DNA methylase activity associated with rous sarcoma virus

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