Download interpreted as a demonstration of a biologically significant protein

430
[32]
POLYMERASE ACCESSORY FUNCTIONS
interpreted as a demonstration of a biologically significant protein-protein
interaction. The two proteins must be shown to be involved in the same
process in vivo; ideally the interaction should be demonstrated by a functional assay or by any of several genetic techniques that can detect proteinprotein interactions. Nevertheless, protein affinity chromatography is a
useful technique, both as an initial method for detecting the potential
components of protein machines and for the purification of such components. Protein affinity chromatography provides a unique opportunity to
exploit the forces that cause protein machines to assemble to allow exploration of the composition and regulation of these complexes that act in so
many cellular processes.
[32]
Radiolabeling
of Proteins for Biochemical
Studies
By Zvi KELMAN,VYTAUTAS NAKTINIS, and MIKZ O'DONNELL
Introduction
Several processes in nucleic acid metabolism such as replication, transcription, and translation require the coordinated action of numerous proteins. This coordination is manifest through protein-protein and proteinnucleic acid interactions ranging from stabile complexes to transient
contacts. Important questions in these multicomponent systems include the
following: How much of each protein is present in protein complexes or
on the nucleic acid? Is the protein interacting with a specific nucleic acid
structure or sequence? How tightly associated are the subunits of a complex,
and how are these interactions influenced by other proteins or by ATP?
Which surfaces on a protein interact with other proteins? Answers to
questions such as these require a convergence of several experimental
techniques. Some techniques that address these subjects utilize radioactive proteins.
This chapter describes two radioactive labeling methods and illustrates
a few applications of labeled proteins in biochemical studies. One method
is reductive methylation, 1-3 which was described in this series using
[14C]formaldehyde.2 We present a modified protocol using NaB3H4 that
I G. E. Means, this series, Vol. 47, p. 469.
2 G. E. Means and R. E. Feeney, Biochemistry 7, 2192 (1968).
3 R. H. Rice and G. E. Means, J. Biol. Chem. 246, 831 (1971).
METHODS IN ENZYMOLOGY,VOL. 262
Copyright © 1995 by AcademicPress, Inc.
All rightsof reproductionin any formreserved.
[32]
RADIOLABELING OF PROTEINS
431
introduces only 1 or 2 [3H]methyl groups per protein for a specific activity
of 20 to 40 Ci/mmol (0.5 to 1.0 × 106 cpm//zg of 50-kDa protein). The
second method uses a specific protein kinase to 32p-end-label a protein
into which a 5-7 amino acid residue kinase recognition motif has been
engineered onto the N or C terminus. 4,5 This procedure results in a specific
activity up to 3000 Ci/mmol (>60 × 106 cpm/tzg of 50-kDa protein). We
have no experience in performing protein labeling using isotopes of iodine
and for this the reader is referred to an excellent treatise in this series. ~
A drawback to chemical labeling is that the modified protein may lose
one or more of its activities. Hence, the labeled protein must be tested for
activity relative to unlabeled protein. Reductive methylation is the least
invasive technique because it introduces only one or two methyl groups.
In our experience, 12 of the 13 different proteins that were labeled retained
85 to 100% activity. End-labeling with 32p introduces a larger modification
and some proteins may be expected to lose function, but the three proteins
we have 32p-end-labeled retain their activity.
Proteins that are radiolabeled metabolically (in vivo) with either
[35S]methionine o r 3H- or 14C-labeled amino acids should retain full activity.
However, the drawback to metabolic labeling is the need to purify the
radioactive protein, and during the prolonged use of centrifuges and cold
room equipment it is difficult to contain the radioactivity. The chemical
labeling methods described here also carry a health hazard, but the procedures can be performed in a fume hood in a matter of a few hours. It is
important that the operations be performed using all available precautions
including gloves, laboratory coat, and film badge, and that proper guidelines
are followed to dispose of the radioactive waste.
3H-Labeling by Reductive Methylation
The procedure described here modifies only a few lysine residues per
protein molecule and is adapted from methods used in Arthur Kornberg's
laboratory] The reaction, I'2 outlined in Eq. (1), involves the addition of
formaldehyde, which forms a Schiff base with lysine followed by reduction
using NaB3H4 and results in net replacement of a proton for a [3H]methyl
group on the primary amine of lysine. Once lysine is methylated, it is
more reactive toward a second round of methylation. 1 The charge of the
methylated lysine is conserved; the pK of monomethylated lysine is in4 B.-L. Li, J. A. Langer, B. Schwartz, and S. Pestka, Proc. Natl. Acad. Sci. USA 86, 558 (1989).
5 M. A. Blanar and W. J. Rutter, Science 256, 1014 (1992).
~' C. W. Parker, this series, Vol. 182, p. 721.
7 K.-i. Arai. S.-i. Yasuda, and A. Kornberg, J. Biol. Chem. 256, 5247 (1981).
432
POLYMERASE ACCESSORY FUNCTIONS
[32]
creased by about 0.3 pH unit and the pK of dialkylated lysine is lower
than lysine by about 0.6 pH units. 2 When only a few methyl groups are
incorporated into a protein, the monoalkylated lysine species predominates. 2
H
H
I
I
Protein-NH 2 + C ~---O ~
I
H
Protein-.N. ~ C
H20
HH
NaB3H4
I I
~. > Protein_N-C-3H
I
H
. .
I
(1)
H
The extent and velocity of the reaction increases with pH, very little
reaction occurs at pH 7.0 and maximal labeling is achieved at pH 9.5. 2
The elevated pH is needed to form the Schiff base between lysine and
formaldehyde (a readily reversible reaction), and to preserve the sodium
borohydride, which is rapidly decomposed as the pH is lowered. To prevent
extensive labeling the reaction is carried out at pH 8.5. The reaction can
be performed at pH 7.0 using cyanoborohydride, 8 although the temperature
must be elevated and the reaction time may be longer. Reductive methylation occurs only with lysine and with the amino terminus, no reaction occurs
with side chains other than lysine, and disulfide bonds are not reduced)
Most of the radioactivity is not incorporated into protein but is consumed
by side reactions such as reduction of formaldehyde to methanol and breakdown of borohydride by solvent. Buffers containing primary and secondary
amines must be avoided because they form the Schiff base and consume
the reactants. We have used sodium borate although buffers with tertiary
amines or phosphate buffer can be used. Besides formaldehyde, other
aldehydes and ketones can be used (e.g., acetone and acetaldehyde), but
the resulting alkyl group on the lysine is then bulkier (isopropyl and ethyl,
respectively). 2 Hence we use formaldehyde to produce the smallest modification possible. Note that formaldehyde itself can modify proteins by forming inter- and intraprotein cross-links; however, the destructive reactions of
formaldehyde with proteins during reductive alkylation has been examined
with none of these products being observed under conditions that are much
more extensive in time and temperature than those used here. 8
Materials for Reductive Methylation
We usually purchase 0.5 Ci of NaB3H4 of 50 to 75 Ci/mmol (Du PontNew England Nuclear, NET-023X). 0.5 Ci of 75 Ci/mmol NaB3H4 is 6.7
/zmol, which is sufficient to label 3.3 ml of protein. We usually label 1 ml
8 N. Jentoft and D. G. Dearborn, J. Biol. Chem. 254, 4359 (1979).
[321
RADIOLABELINGOF PROTEXNS
433
of 3 to 5 different proteins on the same occasion. Unfortunately, NaB3H4
of this specific activity is not available in smaller quantities. Lower specific
activity material can be purchased such as 100 mCi NaB3H4 at 5 to 15
Ci/mmol (NET-023H). Because most of the radioactivity is lost in competing side reactions, the higher the concentration of the protein, the more
labeled protein is produced per mole of NaB~H4 (i.e., the resulting specific
activity of the protein is unaffected whether a 1 or a 5 mg/ml solution of
protein is used). Protein can also be labeled using [14C]formaldehyde,3 but
the specific activity is 1000-fold lower than NaB3H4.
The other necessary materials include a small container for radioactive
solid waste, a triangular file, 1 to 200-/.d Pipetman with an extended tip
(explained below), ice bucket with each dialyzed protein in a separate open
tube, 10 mM NaOH on ice, 2 M formaldehyde on ice, 1 M lysine, and an
empty Eppendorf tube (for the NaB3H4 after it is dissolved in 10 mM
NaOH). To separate protein from reagents after the reaction, one fraction
collector is needed for each protein, 10 ml columns of Sephadex G-25
packed in disposable 10-ml plastic pipettes with a glass wool plug, and gel
filtration column buffer. Our typical column buffer is 20 mM Tris-HC1,
pH 7.5, 0.5 mM EDTA, 2 mM dithiothreitol (DTT), and 20% glycerol.
Procedure for Reductive Methylation
The night before the labeling reaction, dialyze each protein at a concentration of 1 to 5 mg/ml into 50 mM sodium borate, pH 8.5, 0.5 mM EDTA,
10% glycerol (DTT can be included). The next day the reagents and hardware must first be arranged in the fume hood. Then the sealed ampule
containing the dry NaB3H4 is gently tapped to place all the powder at the
bottom of the vial and the file is used to score the neck of the ampule. The
ampule will have 3Hz gas under pressure and it is important when snapping
the neck of the ampule to position it near the hood exhaust port. Dissolve
the NaB3H4 powder in 10 mM NaOH for a final concentration of 100 mM
NaB3H4 (this will be in the range of 67 to 100/~1 depending on the specific
activity of the NaB3H4). Due to the length of the ampule and the narrow
opening at the neck, the Eppendorf tip must be modified so that it reaches
to the bottom of the ampule. A simple modification is to fasten a 2-in.
length of polyethylene tubing onto the tip. The 10 mM NaOH is drawn
into the Pipetman and then transferred into the ampule to dissolve the
NaB3H4, then withdrawn and placed into the empty Eppendorf tube on
ice. At this point one must work quickly, but steadily, because the reagent
is decomposing (as evidenced by the slow appearance of bubbles).
The labeling reaction is initiated upon adding formaldehyde to the
protein(s) to a final concentration of 20 mM and then NaB3H4 to a final
434
POLYMERASE ACCESSORY FUNCTIONS
[321
concentration of 2 mM. After a further incubation of 15 min on ice, stop
the reaction by adding lysine to a final concentration of 50 raM, and separate
the protein from the reagents by passing the reaction mixture over a 10ml column of Sephadex G-25. The column is disposed of as radioactive
waste at the end of the procedure. The column is initially prepared in the
cold room and the buffer is kept on ice, but just before use it is brought
into the fume hood along with a fraction collector. Fractions of 400/xl are
collected and placed on ice. A small amount (3 txl) of each fraction is
counted to locate the protein peak, these are pooled, the protein concentration is determined, and then the [3H]protein is aliquoted and stored frozen
at - 7 0 °. The labeling procedure should take no more than 15 to 20 min,
the gel filtration about 30 min, and the counting and pooling of column
fractions, protein determination, and aliquoting may take about 1 hr.
Variables in this reaction include time, pH, temperature, and concentrations of formaldehyde and NaB3H4. We find that under the conditions
described above, the rate of methylation is nearly linear for 10 rain and
levels off by 15 rain. Also, lowering the formaldehyde from 20 to 10 mM
yields half the level of methylation. Finally, use of 2 mM NaB3H4 is sufficient
for the low level of labeling desired; 4 mM NaB3H4 increased the extent
of labeling by only 20%, and thus is put to better use at 2 mM to label
more protein. We have always performed these procedures on ice and have
not experimented with different temperatures. We have examined the effect
of increasing the pH to 9.0 and find the extent of methylation increases at
least 1.5-fold, but pH 8.5 is less harsh on the protein and is sufficient for
incorporation of one to two methyl groups.
32p-End-Labeling of Proteins
An efficient substrate for the cAMP-dependent protein kinase has been
identified as a heptapeptide Leu-Arg-Arg-Ala-Ser-Leu (or Val)-Gly (or
Ala). 9'1° The Km of this sequence for phosphate transfer to the Ser by the
kinase is 10 to 20/xM. 9'1° Use of only the five inner residues results in an
eightfold increase in Km 10 and substitution of any of the five inner residues
increases the Km by 10-400 fold, resulting in low to negligible rates of
phosphorylation.9 Radioactive proteins of high specific activity (->3000 Ci/
mmol) have been produced upon cloning five to seven residues of this
sequence onto the C terminus of human interferon o~4 and onto the
N terminus of segments of c-Fos)
We have engineered the kinase recognition sequence onto either the
9B. E. Kemp,D. J. Graves,E. Benjamini,and E. G. Krebs,J. Biol. Chem. 252, 4888 (1977).
~o(). Zetterqvist,U. Ragnarsson,E. Humble,L.Berglund,and L. Engstr6m,Biochem. Biophys.
Res. Commun. 70, 696 (1976).
132]
RADIOLABELING OF PROTEINS
435
A) N-terminus
IM
I.
R
R
A
wild type g e n e
V {
S
5' - CTGCGGCATATGCTTCGAAGAGCTTCTGTT-[15 matching nucleotides] - 3'
I
I I
Ndel site
I
BstBl site
B) C-terminus
wild type gene
4
]
L
R
R
A
S
V
(,
stop
3' - [15 matching nucleotides]-GAAGCTTCTCGAAGACAACCAATTCCTAGGTGGTC - 5'
I
I
BstBl site
I
I
Bamlll site
Fie. 1. Oligonucleotide sequences for PCR amplification of either N- or C-terminal PK
protein. The oligonucleotides shown are designed for use with a second oligonucleotide that
matches the gene of interest to produce a PCR product encoding either (A) an N-terminal
PK protein or (B) a C-terminal PK protein. The encoded amino acids of the kinase recognition
motif are shown above the nucleotide sequence. In each case, the 15 nucleotides at the 3'
terminus match the gene of interest. The NdeI and BamHI cloning sites and the BstBI
screening site are marked. The 5-6 nucleotides at the 5' terminus of these oligonucleotides
ensure efficient cleavage of the PCR product.
N or C terminus of three proteins, expressed them in E. coli, and purified
them to homogeneity. We have mainly studied the/3 subunit of E. coli D N A
polymerase III holoenzyme (Pol III) in which a six-residue site, Leu-ArgArg-Ala-Ser-Val (followed by Pro), was engineered onto the C terminus.
In addition, we have placed the seven-residue site, Leu-Arg-Arg-Ala-SerVal-Gly, onto the C terminus of the A cro repressor and of EBNA1, the
latent origin binding protein of the Epstein-Barr virus. We have also placed
the seven-residue sequence Met-Leu-Arg-Arg-Ala-Ser-Val onto the N terminus of/3 and cro. These protein kinase (PK) proteins were engineered
by PCR using an oligonucleotide encoding the kinase motif (Fig. 1) and
expressed in the pET-3 system, it The kinase recognition motif can also be
introduced by site-directed mutagenesis. 4 Since the pET-3 system was used
for expression, the primer sequences contained an NdeI site for N-terminal
PK proteins, and a BamHI site for C-terminal PK proteins. The BstBI site
is not present in pET-3 and thus aids in screening clones.
Procedure for 32p-End-Labeling
Unlike reductive methylation, this technique can be performed with
small amounts of protein and the procedure is compatible with a variety
~ W. F. Studier, A. H. Rosenberg, J. J. Dunn, and S. W. Dubendorff, this series, Vol. 185,
p. 60.
436
POLYMERASEACCESSORYFUNCTIONS
[321
of buffers. We typically use a volume of 30 to 120 tzl containing 0.2 to 6
nmol PK protein in 20 mM Tris-HC1, pH 7.5, 1 to 60/xM [y-32p]ATP
(specific activity discussed below), 2 mM DTT, 100 mM NaC1, 12 mM
MgC12, and 10 mM NaF. Labeling is initiated upon adding cAMP-dependent protein kinase (0.008 U kinase/nmol PK protein) and shifted to 37°.
After 10 rain at 37°, the reaction is stopped on addition of 30 mM EDTA
(final concentration). The extent of 32p incorporated into the PK protein
can be determined by acid precipitation or by analysis in a SDSpolyacrylamide gel (for the latter, addition of 5 mM unlabeled ATP to
the sample buffer lowers background radiation). Free [y-32p]ATP can be
removed either by ultrafiltration, spin dialysis, or gel filtration. The catalytic
subunit of cAMP-dependent protein kinase from bovine heart can be purchased from Sigma and was used in previous s t u d i e s y however, we have
used the murine version expressed and purified from E. coli 12 (gift of Dr.
Susan S. Taylor, University of California at San Diego).
Under these conditions the stoichiometry of Pi incorporation is typically
0.5 to 0.8 tool Pi/mole protein (except N-terminal labeled/3 PK, discussed
below). Before embarking on these studies, it is important to determine
whether the wild-type protein is phosphorylated by protein kinase. Wildtype/3 was not phosphorylated to a significant extent (<0.9 mmol Pi/mol
/3PK) and sequence analysis of the radioactive chymotryptic peptide of
[32p]/3PK confirmed the label was in the kinase motif at the C terminus.
High specific activity protein can be achieved using straight [y-32p]ATP
(3000 Ci/mmol) at approximately 1/xM, or the relative proportion of unlabeled ATP and radioactive ATP can be adjusted to achieve the desired
specific activity. This can be very important in experimental designs using
both 3H-labeled protein and 3ZP-labeled protein in the same experiment
because the specific activity of 3H-labeled proteins is only 20 to 40 Ci/mmol
and therefore one must take care to prepare the 32p-labeled protein with
a comparable specific activity so as not to encounter problems of bleed
over between windows while counting both isotopes.
Important Considerations
One advantage to the 32p-end-labeling method is that the isotope is
available in small quantities and the manipulations are similar to endlabeling DNA, a common laboratory technique. PK proteins can also be
labeled using [ T - 3 5 S ] A T P . a3
How often do labeled PK proteins retain activity? The modification is
12 L. W. Slice and S. S. Tylor, J. Biol. Chem. 264, 20,940 (1989).
13 V. Naktinis and M. O'Donnell, unpublished (1993).
[32]
RADIOLABELING OF PROTEINS
437
su~tantial; not only are several amino acids added to the protein, but a
large phosphate group is also present. In our experience the activity of
both N- and C-labeled [32p]fleR is unchanged in several assays including
assembly of [32p]~PK o n t o primed D N A by Pol 1II, and ability of [32p]flPK
to confer highly processive D N A synthesis onto the polymerase.14 Likewise,
the [32p]EBNA1PK and both N- and C-labeled [32p]croPK retained their sitespecific D N A binding activities. It is important to note that if the amount
of ATP used in the labeling reaction is substoichiometric to the protein
(often the case in D N A end-labeling reactions), some types of activity assays
will not truly evaluate whether the phosphorylated species retains activity.
In theory one should be able to place the kinase motif at any exposed
region of a molecule and label it. However, placement of this kinase motif
at other positions in the/3 subunit has revealed limitations to this technique.
For example, the N-terminal/3Pr: is labeled approximately 4% at best. 13
The problem is likely due to the inability of the kinase to gain access to
the recognition site as addition of 0.5 M urea increased the labeling to 16%.~3
Further, we have placed the seven-residue kinase recognition sequence into
two different internal positions of/3, which are located on the surface
according to the crystal structure, but the kinase did not phosphorylate
these proteins/5
Examples of Applications
Interaction of Proteins with DNA
To identify a particular subunit on DNA in a complex mixture of several
proteins, the reaction is analyzed by gel filtration using beads with large
pores such that free proteins elute in the included fractions and resolve
from proteins bound to the large D N A (e.g., plasmid or M13 ssDNA),
which elute in the excluded fractions. For these studies we have used
agarose beads Bio-Gel A-5m and A-15m (Bio-Rad) as well as Sepharose
4B (Pharmacia-LKB), which exclude plasmids and ssDNA phage genomes,
yet include proteins of up to 1 MDa.
Examples of this technique abound in studies from Arthur Kornberg's
laboratory. For example, 3H-labeled proteins were used to identify primosomal proteins that remained on the ~bX174 ssDNA with the finding that the
DnaC protein, while essential to assemble the primosome on DNA, does
not remain on the D N A . 16 The E. coli Pol III contains a five-subunit
14p. T. Stukenberg, Ph.D. thesis (1993).
15j. Turner and M. O'Donnell, unpublished (1993).
in j. A. Kobori and A. Kornberg, Z Biol. Chem. 257, 13,770 (1982).
438
[321
P O L Y M E R A S E ACCESSORY FUNCTIONS
B16OO
A25o
,•
1400
1200
200
Sm~I Q
0
"~'1000
~
lSO
~
l
e
x
200
"To
20
fraction
C 100
,.,~i3~j-~.
90
-
0
30
it=
0
20
i0
30
mTx_
"
,
D
80
~
70
50
NA Kd= 17rim
SSB
70
-~
o
I g' a
6o
40
fraction
80
I
ular
400
50
Q
ear
, 600
DZ
"6 lOO
.
Sma I
800
[SH]-~
60
e~
50
40
50
lhr
40
30
20
30
10
15
25
35
2'o
1'0
2(
45
fraction
E 525, epitope .
.
.
.
.
F
ldnase
450 !
i
375
epitope
epitope-132
[3H]- ~
epitope
45Oc
~
40
~32p
0.E " ~K :OOH [y32p]ATP
~300
~
~o
fraction
0
0.~
0.5 1 1.5 2 4 8 30
O
225
37°C
~_..150
rain
8
0.~
75
•
i.
~
;i
incubation time (hrs)
-
10
0
10
20
time (min)
FiG. 2. Applications of radiolabeled proteins to study the molecular dynamics and structural
details of multicomponent complexes, (A) The [3H]y complex matchmaker assembles the
[32p]/3PK dimer onto SSB coated primed M13mp18 ssDNA. Gel filtration on Bio-Gel A-5m
resolves the [32p]/~eKbound to DNA (fractions 10-15) from the [3HIT complex that dissociates
from DNA and elutes in the included fractions. The experiment was performed essentially
as described in Fig. 2.3 of Stukenberg) 4 (B) During gel filtration, [3H]~ clamps coelute with
30
[32]
RADIOLABELING OF PROTEINS
439
subassembly called the y complex, which acts in a similar fashion as DnaC
protein; 3' complex places the/3 subunit of Pol III onto a primed template
but then departs from the D N A . 17 Figure 2A shows the use of two labeled
subunits in this latter reaction. The 3H-labeled y complex was used to place
[32p]/3PK onto a primed template and the reaction was analyzed by gel
filtration. Analysis of the column fractions shows a stoichiometric amount
of [32p]/3PK dimers comigrating with the DNA, but the [3H]T complex migrates as free protein complex in the included fractions.
Topological Binding of Protein to DNA
The/3 subunit of Pol III is a ring-shaped dimer that completely encircles
DNA. ~8 Once the/3 ring is assembled onto D N A (by the y complex and
ATP) it tethers the rest of the Pol III machinery to the template,and slides
along with it for highly processive D N A synthesis. ~9 This simple solution
~7p. T. Stukenberg, P. S. Studwell-Vaughan, and M. O'Donnell, J. Biol. Chem. 266, 11,328
(1991).
is X.-P. Kong, R. Onrust, M. O'Donnell, and J. Kuriyan, Cell 69, 425 (1992).
~9M. O'Donnell, J. Kuriyan, X.-P. Kong, P. T. Stukenberg, and R. Onrust, Mol. Biol. Cell 3,
953 (1992).
(bind to) a circular plasmid with a single nick, but upon linearization of DNA the [3H]/~
clamps fall off and elute in the included fractions. (Adapted from Fig. 3 in Stukenberg et al. t7
Copyright The American Society for Biochemistry & Molecular Biology.) (C) Dissociation
of [3H]6' from the y complex was observed by incubating 14 pmol of y complex (reconstituted
with [3H]8') with 108 pmol &' in 60/.d of buffer 20 mM Tris-HCl, pH 7,5, 100 mM NaCI,
and 10% glycerol at 22°. At the indicated times, the samples were analyzed by filtration on
a 24-ml Superose-12 column. (D) Equilibrium gel filtration analysis of the interaction between
[3H]x and the SSB-DNA complex was performed on a 5-ml column of P-30 (Bio-Rad) using
500 nM [3H]x in the column buffer and injecting on the column 1.7 nmol SSB in complex
with M13mpl8 (10/xg ssDNA) in column buffer. Fractions were collected and quantitated
by scintillation counting. The Kd of 17 nM was calculated from the amount of [3H]x bound
to the SSB-DNA in the peak. (E) Protomer interchange among/3 dimers using a mixture of
2 pmol [3H]fl2 and 2 pmol hemagglutinin epitope tagged432 in 50/zl of 20 mM HEPES, pH
7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol followed by immunoprecipitation at the
indicated times using monoclonal antibody to the epitope (12CA5, BAbCO) as described by
Kolodziej and Young. 23 The [3H]fl that coimmunopreeipitated with the epitope tagged-fl
indicates the extent of subunit exchange to form the heterodimer between the [3H]/3 and
epitope tagged-/3. The experiment was repeated at the indicated temperatures. (F) In the
kinase protection assay, 180 pmol /3PK was preincubated for 30 rain at 15° either with or
without 1 nmol 88' complex, followed by treatment with protein kinase in a 30-/.d reaction
mixture as described in the text. At the indicated times 3-/zl aliquots were analyzed by SDS
polyacrylamide gel and autoradiography (inset). The kinase motif is located on the C termini,
which extrude out from the same face of the J3 dimer as indicated in the scheme.
440
POLYI~.ERASE ACCESSORY FUNCTIONS,
[32]
to high processivity may generalize to the gene 45 protein processivity
factor of the phage T4 replicase~ a~d the PCNA processi~,ity factor of the
yeast and human potymerase ~19 Protein surroundir~gDNA seems so basic
that it may even generalize to other D N A metabolic machineries. For
example, replicative helicases such as those encoded by phage T4 and phage
T7~ E. coli DnaB, and simian vies. 40 (SV40) T antigen are all hexamers
and may surround duplex D N A for their action (i.e., l~e'the sixfold pseudosymmetric/3 dimer).
raitial experiments using [3H]~ revealed this "topological binding"
mode of/3 to DNA and predicted its ring shape, 17 thus motivating the
crystal structure analysis. A particularly telling experiment is shown in Fig.
2B~,. Here, [3H]/3 w a s placed onto a singly nicked plasmid DNA (by -/
complex and ATP) and then gel filtered to reveal that several/3 dimers
coelute with the DNA in the excluded fractions. 17 However, linearization
o£ the DNA results in dissociation of/3, implying that it slides along DNA
and falls off the end. If/3 were to bind DNA by direct chemical interaction,
as other DNA binding proteins do, then it would have stayed associated
with the linear DNA through gel filtration as well as with circular DNA.
Hence,/3 must be bound physically to DNA by virtue of its topology. This
type of experiment is rather simple and variations on this theme may help
identify the topological binding proteins of other systems.
Subunit Exchange in Multiprotein Complexes
Radiolabeled protein can be used to measure the rate of dissociation
of a subunit from within a multiprotein complex. As an example, in Fig.
2C the five-subunit 3' complex (y66'XqJ) was labeled by reconstituting it
using [3H]6'. To this was added an eighffold molar excess of unlabeled
6' and the mixture was gel filtered at various times. Whenever the [3Hit3'
subunit dissociates from the complex, an unlabeled 6' takes its place. Hence,
over time, the column fractions containing y complex decrease in radioactivity and column fractions containing free 6' increase in radioactivity. This
technique can be extended to determine how the rate of subunit exchange
is influenced by DNA, nucleotides and other proteins.
Weak Protein-Protein Interactions
Only strong interactions among proteins can be detected by gel filtration
because it is not an equilibrium technique. However, weak interactions
can be quantitated using the equilibrium gel filtration technique? ° This
technique is normally used to determine the Ks between a protein and a
20j. p. Hummell and W. J. Dreyer, Biochim. Biophys. Acta 63, 530 (1962).
[32]
RADIOLABELING OF PROTEINS
441
small ligand molecule (e.g., a nucleoside triphosphate) where the ligand is
radiolabeled and is present throughout the column buffer. The protein
binds the radiolabeled ligand and then elutes ahead of the ligand, resulting
in a peak of radioactivity that emerges above the baseline level and is
followed by a trough where the unbound ligand would have eluted. The
Kd can be calculated from this information. This technique has been applied
in Arthur Kornberg's laboratory using [3H]/3 in the column buffer to define
the Kd of a weak protein-protein interaction between the/3 subunit and
the Pol III* assembly (Pol III lacking 13).21 Figure 2D shows an analysis of
a weak interaction between the X subunit of Pol III and an SSB-DNA
complex using [3H]x in the column buffer. 22
Exchange Rate Among Subunits of a Dimeric Protein
How rapidly do dimers of identical subunits fall apart and come back
together? For example, the protomers of the/3 dimer form a closed ring,
which must open to assemble around DNA. Do the monomer units ot a
/3 dimer rapidly come apart and then reassociate, thereby trapping DNA
inside? The rate of exchange of monomer units of a dimer cannot be
measured by gel filtration as in Fig. 2C since it is always in the dimeric
s~te. An assay to measure stability of the/3 dimer, shown m Fig. 2E, uses a
[3H]/3 dimer and a/3 dimer that is genetically tagged with the hemaggtminin
epitope (nine amino acids) at its C-terminus. 23 Upon mixiag the t'~o/3
dimers, samples of the reaction were immunoprecipitated at time imervals.
As the "heterodimer" of one [3H]/3 and one epitope .tagged-/3 ~as formed,
the amount of [3H]/3 in the coimmunoprecipita~ increased. The results
indicate that monorneric units of the/3 dimer reqaire :a ~ong ~ e *o ~ssociate even at 37°. This assay can be exploited to study the m~laence of other
proteins and of DNA and ATP on the stability of the/3 dimer.
Kinase Protection Assay
We originally end-labeled proteins with [32p] to develop a "protein
footprinting" assay for mapping the interactive surface of one protein with
another (or with DNA), but this work is still in progress. However, we
have developed a "kinase protection" assay. An example of this assay is
shown in Fig. 2F in which/3eK is incubated with or without the ~ ' complex,
a subassembly of the y complex that binds/3. 24 Then kinase is added and
21 R. S. Lasken and A. Kornberg, .L Biol. Chem. 262, 1720 (1987).
22 Z. K e l m a n and M. O'Donnell, unpublished (1993).
23 p. A. Kolodziej and R. A. Young, this series, Vol. 194, p. 508.
24 R. Onrust, Ph.D. thesis (1993).
~2
POLYMERASE
ACCESSORY FUNCTIONS
[331
the time course of phosphorylation is monitored by SDS-PAGE followed
by autoradiography. The result shows that phosphorylation of/3Pi~ is almost
completely btocked by the ~fi' complex. The two C termini of the/3 dimer,
where the' ki,'nase motif is located, extrude from the same face of the/3
dimer (see scheme in Fig. 2F). Hence, the ability of 8fi' complex to block
these sites from phosphoryl~tion suggests that &~' may interact with the
"C-termiwai" face of the/3 ~dimer.
Acknowledgments
We are grateful to Dr. Susan S. Taylor for the catalytic subunit of cAMP-dependent
protein kinase. Supported by Public Health Service grant GM38839.
[33] C y c l i n g o f Escherichia coli D N A P o l y m e r a s e III f r o m
One Sliding Clamp to Another: Model for Lagging Strand
By
JENNIFER TURNER a n d MIKE O ' D O N N E L L
Introduction
The multiprotein replicase of the Escherichia coli chromosome, DNA
polymerase III holoenzyme (Pol III), achieves a tight ATP-activated grip
on DNA through its ring-shaped clamp protein, the/3 dimer ( / 3 2 ) . 1-4 The
/32 ring encircles DNA, thereby acting as a sliding clamp and continuously
holds Pol III to the template for remarkably high speed and processivity. 5
The model of a circular protein clamp riding along in back of the polymerase
fits nicely with the continuous synthesis of the leading strand, but conceptually it would hinder the discontinuous mode on the lagging strand where
Pol III must rapidly dissociate from the end of one Okazaki fragment to
start another fragment at the next RNA primer (i.e., Fig. 1C).
Studies of Pol III showed that it was indeed very slow to replicate more
than a stoichiometric number of primed templates. 6 After Pol III completely
replicated a circular single-stranded DNA (ssDNA) template, several
1 A. Kornberg and T. A. Baker, " D N A Replication." Freeman, New York, 1991.
2 X.-P. Kong, R. Onrust, M. O'Donnell, and J. Kuriyan, Cell 69, 425 (1992).
3 M. O'Donnell, J. Kuriyan, X.-P. Kong, P. T. Stukenberg, and R. Onrust, Mot. Biol. Cell 3,
953 (1992).
4 j. Kuriyan and M. O'Donnell, J. Mot. Biol. 234, 915 (1993).
5 p. T. Stukenberg, P. S. Studwell-Vaughan, and M. O'Donnell, Z Biol. Chem. 266, 11,328
(1991).
6 p. M. J. Burgers and A. Kornberg, J. Biol. Chem. 257, 11,474 (1982).
METHODS IN ENZYMOLOGY, VOL. 262
Copyright © 1995 by Academic Press, Inc.
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