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FEBS 2228
Febrwry
1985
E.V. Bobst, A.M, Bobst, F.W. Ferrino+, R.R, Meyer* and D.C. Rein+
~sc~eric~ia
cdi single-strands I3NA binding protein
(SSB), essential for DNA ~e~~~at~~n,~~~~b~~~~
lion and repair, can undergo a ~~~a~~~ induced irreversible ~onform~t~o~a~ change which does not ehmi-
The
nate its biological activity, but changes the number of nucieotides it covers (binding site size) when binding
to a single-stranded nucleic acidhttice. The binding site size of native srnd conformationally changed SSB
was also found to be a function of the molecular mass of the polynucteotide, an observation which is unusual for single-stranded DNA binding proteins and will greatly affect the afftnity relationship of this protein
for nucleic acids. A radioimmunoassay used to quantitate in SSB level in cells revealed the number of SSB
tetramers to be larger than initial estimates by a factor of as much as six. All these data suggest that the
biological role of SSB and its mechanism of action is by far more complex than originally assumed,
tein [S].
2. MATERIALS
AND METHODS
FEBS LETTERS
February 1985
3. RESULTS
DISCUSSION
AND
February
FEBSLETTERS
1985
Table 1
0
12
16
20
24
28
32
36
40
44
40
p/N x 10-2
SSB
Unboiledb
(DUAP,dT),
(dT)n
(ldT,dT),
fd DNA
25
50
50
75
f 10%
f 10%
_+ 10%
+ 10%
Boiledb
45 _+ 10%
70 f 10%
_
100 f 10%
FEBS LETTERS
4-77
12
18
20
P/w
24
x
28
10-2
32
35
40
44
February
48
20
40
60
80
G4 ssDNA. ng
of 2 M NaCI.
Table 2
Media
Total soluble
protein @g/PI)
SSB in extracta
(W/cl)
c600
C600/pSR6b
M9
M9
0.24 + 0.006
0.13 f 0,006
0.12 + 0.01
1.1 tfr 0.17
C600
C6OO/pSR6
LB
LB
0.31 f 0.047
0.20 f 0.008
0.28 4: 0.032
6.1 of:0.51
w3110
M9
0.45 + 0.036
0.10 f 0.009
0.22 f 0.025
AB1157
M9
0.41 * 0.011
0.13 + 0.025
0.31 -t 0.055
Cell strain
SSBltotal protein
(ng/pg)
0.49 k 0.039
8.6 f 1.2
0.86 f 0.14
+ 2.7
31
a SSB was determined by RIA in 2 M NaCl using cleared high salt lysates prepared from sonicated
cells
b ssb plasmid 19)
136
1985
FEBS LETTERS
40 ng/ml.
AC~~~WLEDGE~ENTS
This work was supported in part by NIH grants
GM 27002 (A.M.B.) and GM 31196 (R.R.M.),
REFERENCES
[If Meyer, R.R+, Gfassberg, J. and Kurn&r~, A,
(1979) Proc. Natl. Acad. Sci. USA 76,1702-1705.
[2] Glassberg, .I., Meyer, R.R. and Kornberg, A.
(1979) .I, Bacterioi. 140, 14-19.
[3] Golup, E.I. and Low, K.B. (1983) Proc. Natf.
Acad. Sci. USA 80, 1401-1405.
[4] Whittier, R.F. and Chase, J.W. (1983) Mol. Gen.
Genet. 190, 101-111.
February
1985
137