Princeton University

DEPARTMENT OF BIOCHEMICAL SCIENCES

FRICK CHEMICAL LABORATORY

PRINCETON, NEW JERSEY 08540

March 23, 1971

Dr. Arthur Kornberg
Department of Biochemistry
Stanford University School of Medicine
Stanford, California 94304

Dear Arthur,

Just a note to let you know of the progress of our work on the

E. coli "32-protein'' here. Nolan Sigal has about 1 mg of the pure protein
and has characterized it by DNA denaturation, stoichiometry of single-
stranded DNA binding, and detailed structure by electron microscopy of the
complexes. Except for the fact that he has yet to detect catalysis of
- renaturation, and that its MW on SDS-gels is 20,000 daltons, it looks just
like 32-protein so far. We have arranged for Tom (Kornberg) to test its
effect on DNA polymerases I, 11, and I11 from E. coli. For this purpose,
Nolan will spend the latter part of this week at Columbia.

The pracedure Nolan uses is to make a sonicated extract from 17

liters of late log cells, remove all DNA by DNAse treatment, dialyze vs.
EDTA after a high speed spin (see our Nature paper on 32-protein), and then
load onto a 10 ml single-stranded DNA-cellulose column in 0.05 M NaC1,  0.02
M Tris, 7.4, 0.001 M EDTA, 10% glycerol, 0.001M Mercaptoethanol, After an
extensive rinse, most DNA binding proteins are eluted by addition of 1 mg/ml
of Dextran-sulfate 500 (Pharmacia) to the loading buffer. After a rinse
to remove dextran sulfate, the remaining proteins are eluted with 0.6 M
and 2 M NaCl rinses. Eluting between 0.6 and 2M are 4 main proteins, of
MW 75,70,20, and 10 thousand daltons respectively. The 20,000 protein is
obtained by DEAE chromatography (elutes 0.2-3 M NaCl), followed by dialysis
vs. 10% glycerol, 1 mM mercaptoethanol, 0.04 M KP04 pH7 where it precipi-
tates pure. It ks stored frozen in 10% glycerol, 2 mM Tris 8.1, 10 mM NaC1,
1 mM mercaptoethanol at abnut 1 mg/ml.
the total soluble protein.

It is estimated to be 0.05-0.1% of

Your student Scheckman phoned. He has probably been working with
                                  We don't know what

the 10,000 MW proteing which is not of 32-protein type.
it is; Nolan has that homogeneous also, and will give some to Tom to test.

Dr. Kornberg - 2

March 23, 1971

It seemed to me to make much more sense for us to do the polymerase work
with Tom rather than Scheckman, since Tom is not only conveniently located,
but has 3 polymerases available to test.

I hope that this plan meets with your approval.

Best regards,

Bruce Alberts

BA/ ew