Proc. Natl. Acad. Sci. USA
Vol. 92,pp.1950-1954, March 1995
Restoration of mismatch repair to nuclear extracts of H6
colorectal tumor cells by a heterodimer of human MutL homologs
Guo-MIN LI* AND PAUL MODRICH*tt
tHoward Hughes Medical Institute and *Department of Biochemistry, Duke University Medical Center, Durham, NC 27710
Contributed by Paul Modrich, November 16, 1994
defective in strand-specific mismatch repair and bear defects
in both alleles of the hMLHI gene. We have purified to near
homogeneity an activity from HeLa cells that complements H6
nuclear extracts to restore repair proficiency on a set of hetero-
duplex DNAs representing the eight base-base mismatches as
well as a number ofslipped-strand, insertion/deletion mispairs.
This activity behaves as a single species during fractionation and
copurifies with proteins of 85 and 110 kDa. Microsequence
analysis demonstrated both of these proteins to be homologs of
bacterial MutL, with the former corresponding to the hMLHI
product and the latter to the product of hPMS2 or a closely
related gene. The 1:1 molar stoichiometry ofthe two polypeptides
and their hydrodynamic behavior indicate formation of a het-
erodimer, which we have designated hMutLa. These observa-
tions indicate that interactions between members ofthe family of
human MutL homologs may be restricted.
Hypermutable HG colorectal tumor cells are
Frequent microsatellite mutations have been observed in a
variety of human cancers, including most tumors associated
with hereditary nonpolyposis colorectal cancer (HNPCC)
(1-3) as well as a significant fraction of sporadic colon, gastric,
pancreatic, endometrial, ovarian, and small cell lung carcino-
mas (reviewed in ref. 4). Microsatellite alterations in such
tumors have been postulated to arise through somatic muta-
tion due to a replication error (RER+) phenotype (1, 5). This
mutator hypothesis has been confirmed for a number ofRER+
colorectal tumor cell lines with the demonstration that (CA)n
microsatellite and HPRT mutabilities are elevated by as much
as two to three orders of magnitude in such cells (6-8).
Analysis of HNPCC kindreds has implicated four genes in
the disease (9-13). These loci specify protein homologs of the
bacterial mismatch repair activities MutS and MutL, with
hMSH2 encoding a MutS homolog and hMLH1, hPMS1, and
hPMS2 specifying MutL homologs. A germ-line defect in any
one of these genes is apparently sufficient to confer predis-
position to tumor development (10-13). Although somatic
cells of HNPCC-afflicted individuals are heterozygous for a
repair defect, examination of mismatch repair genes in several
RER+ tumors has revealed defects in both parental alleles,
with inactivation of the wild-type allele attributable to somatic
mutation in two cases (10, 11, 13, 14). Inactivation of a
mismatch repair function therefore appears to be the rate-
limiting step in development of a substantial fraction ofRER+
tumors. Analysis of mismatch repair in nuclear extracts of
RER- and RER+ colorectal and endometrial tumors has
yielded results consistent with these genetic conclusions and
has established a direct link between repair deficiency and the
RER+ phenotype (refs. 6 and 14; G.-M.L., M. Longley, J.
Drummond, S. Markowitz, B. Vogelstein, and P.M., unpub-
Strand-specific mismatch repair in human cells is similar to
the Escherichia coli methyl-directed pathway with respect to
mismatch specificity and an unusual bidirectional excision
capability (15-17). The mechanistic complexity of this type of
repair system is evident in the case of the bacterial reaction,
which is dependent on 10 activities (18). To further clarify the
nature of the human repair reaction, we have undertaken
isolation ofthe required activities. In this paperwe describe the
isolation of an activity that complements nuclear extracts of
the complementation group exemplified by the RER+ colo-
rectal tumor cell line H6 (6), which has been shown to be
defective in both alleles of hMLHl (11). We show that H6
complementing activity isolated from HeLa cells is a het-
erodimer comprised of the MutL homologs hMLH1 and
MATERIALS AND METHODS
Cell Lines, Nuclear Extracts, and Enzyme Assays. H6 (6)
and HeLa S3 cells (15, 17) were cultured and nuclear extracts
were prepared as described. Mismatch repair was assayed by
published methods (6, 15) except that salt concentrations of
individual reaction mixtures (15 pl containing 50 ,ug of H6
nuclear extract) were adjusted to compensate for variable
levels of salt in samples (1 p,l) scored for complementing
activity. The final concentration ofKCl was maintained at 0.11
M KCl, except in the case of fractions from hydroxylapatite
chromatography, where final KCI concentration was 0.045 M
and potassium phosphate was 0.03 M. Incubation was for 15
min at 37°C.
Endonuclease activity on duplex DNA, 3' -*5' and 5' -*3'
exonuclease on native and denatured DNA, and ATPase were
determined by minor modifications of previously described
Protein Microsequencing. Protein microsequencing was
performed at the Harvard Microchemistry Facility (Cam-
bridge, MA) according to Lane et al. (20). H6 complementing
activity (fraction VII) was electrophoresed through 8% poly-
acrylamide in the presence of SDS and electrotransferred in
0.025 M Tris glycine (pH 8.4) for 3 hr at 40 V at 4°C onto a
poly(vinylidene difluoride) membrane (Millipore). Protein
was visualized with Ponceau S stain, excised from the mem-
brane, and submitted to digestion in situ with trypsin. Resulting
peptides were separated by narrow-bore high-performance
liquid chromatography on a Vydac C18 (2.1 mm x 150 mm)
reverse-phase column using a Hewlett-Packard model 1090
equipped with a 1040 diode array detector, and selected
fractions were subjected to automated Edman degradation on
a model 477A protein sequencer (Applied Biosystems).
Isolation of H6-Complementing Activity. Nuclear extracts
derived from the RER+ colorectal tumor cell line H6 are
Abbreviation: HNPCC, hereditary nonpolyposis colorectal cancer.
tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
Proc. NatL Acad Sci USA 92 (1995)
deficient in strand-specific mismatch repair (6), a defect
attributed to loss of function of both hMLHJ alleles (11).
Initial experiments indicated that the in vitro repair defect
could be complemented by a partially purified fraction derived
from HeLa cells (6), and we have pursued isolation of this
putative component of the human mismatch repair system.
H6-complementing activity, which fractionated as a single
species through extensive purification, has been enriched
-5000-fold from crude HeLa nuclear extracts (Table 1).
Near-Homogeneous H6-Complementing Activity Contains
hMLHI and hPMS2 Gene Products. During the last two steps
Purification of H6-complementing activity
Nuclear extract 1530
III. S Sepharose
VI. Mono S
VII. Sucrose gradient
All purification steps were carried out at 0-4°C and all buffers
contained 1 ,g of leupeptin per ml, 1jigof pepstatin A per ml, 2mM
dithiothreitol, and 0.1% phenylmethylsulfonyl fluoride (concentration
relative to a 23°C saturated solution in isopropyl alcohol). Ammonium
sulfate-concentrated, dialyzed HeLa nuclear extract (fraction I, 80 ml,
20 mg/ml) from 180 liters of culture was prepared as described (15).
Fraction I was chromatographed on a phosphocellulose P-I1 column
(Whatman; 13.6 cm x 12.6 cm2) as described (6) to yield fraction II
(99 ml). Fraction II was dialyzed against 2-liter portions (one change,
1 hr per change) of 0.025 M Hepes-KOH, pH 7.5/0.1 mM EDTA
(buffer A) containing 0.05 M KCI until the conductivity was compa-
rable to that of buffer A containing 0.1 M KCI, and the dialysate was
applied at 20 ml/hr to an S Sepharose column (Sigma; 6.2 cm x 3.2
cm2) equilibrated with bufferAcontaining 0.1 M KCI. The columnwas
washed with 40 ml of equilibration buffer and developed with a linear
gradient of KCI (200 ml, 0.1-0.5 M) in buffer A. Fractions that
complemented H6 nuclear extracts eluted at -0.19 M KCl (fraction
III, 25 ml). Fraction III was clarified at 13,000 x g for 10 min and
loaded at 2 ml/min onto an 8-ml Pharmacia HR10/10 column packed
withHPLC grade hydroxylapatite (Calbiochem) equilibrated with 0.08
M potassium phosphate, pH 7.4/0.05 M KCl. After washing with 20
ml of equilibration buffer, the column was eluted with a 320-ml linear
gradient ofpotassium phosphate (0.08-0.5 M, pH 7.4) containing 0.05
M KCl. Fractions eluting from the column were supplemented with
EDTA to a final concentration of 0.1 mM. Complementing activity,
which eluted at a phosphate concentration of 0.25 M (fraction IV, 32
ml), was dialyzed against two 1-liter portions (1 hr per change) of0.025
M Tris HCl, pH 7.6/0.1 mM EDTA/0.1 M KCI. After clarification by
centrifugation the dialysate was applied at 1 ml/min onto a 1-ml
Pharmacia HR 5/5 Mono Q column equilibrated with dialysis buffer.
The column was washed with 2 ml of starting buffer and developed
with a 20-ml linear gradient of KCl (0.1-0.5 M) in 0.025 M Tris HCl,
pH 7.6/0.1 mM EDTA, and complementing fractions, which eluted at
0.3 M KCl, were pooled (fraction V, 5.0 ml). Fraction V was dialyzed
for 1 hr against 1 liter of 0.025 M potassium phosphate buffer, pH
7.4/0.1 mM EDTA (buffer B)/0.08M KCl, and after centrifugation as
above, the dialysate was applied at 1 ml/min to a 1-ml Pharmacia HR
5/5 Mono S column equilibrated with this buffer. After washing with
2 ml of equilibration buffer, the column was eluted with a linear
gradient ofKCI (20 ml, 0.08-0.4 M) in buffer B. Activity eluted at 0.28
M KCl (fraction VI, 4 ml). The Mono S pool was diluted 2.8-fold in
small aliquotswith bufferAand loaded onto a 0.3-ml phosphocellulose
P-11 column. After washing with 0.2 ml of buffer A containing 0.1 M
KC1, complementing activity was step eluted with bufferA containing
0.5 M KCI. Material concentrated in this manner (0.3 ml) was loaded
onto two 5-ml, 10-30% linear sucrose gradients in buffer B containing
0.2 M KC1. After centrifugation at 40,000 rpm at 4°C for 30 hr in a
Beckman SW 50.1 rotor (see Fig. 1), the gradients were collected from
the bottom of the tube and active fractions were pooled (fraction VII,
2.0 ml). Fraction VII was frozen in liquid N2 in small aliquots and
stored at -80°C. Protein was determined by the method of Bradford
(21) using bovine serum albumin as a standard.
in purification (Table 1), it was evident that complementing
activity was cofractionating with 110-kDa and 85-kDa
polypeptides as judged by SDS/PAGE (Fig. 1). Furthermore,
on the basis ofthe estimated protein content ofelectrophoretic
species (Fig. 1), the molar ratio of the 110-kDa to the 85-kDa
components was essentially constant across activity profiles:
0.98 ± 0.1 and 1.06 ± 0.1 (±1 SD) for Mono. S and sucrose
gradient steps, respectively.
In view of the molar equivalence of these two polypeptides
and their tight association with complementing activity, both
were subjected to microsequence analysis. Since amino-
terminal sequence data were not obtainable from the full-
length form of either polypeptide, 110-kDa and 85-kDa com-
ponents were individually hydrolyzed with trypsin and several
peptides derived from each were subjected to automated
Edman degradation (Materials and Methods). This procedure
yielded two unique peptides from each of the two components
(Table 2). Since H6 cells are known to be defective in both
alleles ofhMLHJ (11), the four peptideswere screened against
the sequence predicted for the hMLH1 gene product (11, 12).
As shown in Table 2, peptides 85-PT78 and 85-PT111, derived
from the 85-kDa component, are identical to internal se-
quences encodedwithinhMLHI cDNA. Peptides 85-PT78 and
85-PT111 were also screened against the National Center for
Biotechnology Information (NCBI) nonredundant' protein
sequence data base using the Blast algorithm. This search
revealed perfect homology of the two peptides only to the
internal'regions of hMLH1 discussed above, although limited
homology of 85-PT111 to several a collagen sequences was
also observed. Since the molecular weight of the 85-kDa
species is also in excellent agreement with the 84,596 value
predicted for the hMLHJ gene product, we have concluded
that this component of H6-complementing activity corre-
sponds to hMLH1.
Since a perfect match to sequences corresponding to tryptic
peptides 110-PT72 and 110-PT98 could not be located within
hMLH1, these two peptides were compared to those encoded
by hPMSJ and hPMS2, two recently identified genes that, like
hMLHJ, have been implicated in HNPCC and encode MutL
homologs (13). Examination ofthe predicted hPMS1 sequence
did not yield a perfect match to either peptide, but both were
present within the predicted hPMS2 gene product (Table 2),
was sedimented through a 10-30% sucrose density gradient as de-
scribed in the legend to Table 1. One microliter of each fraction was
assayed for ability to complement H6 nuclear extracts and 10-,ul
samples were analyzed for protein composition by electrophoresis
through 8% polyacrylamide slab gels in the presence of SDS. Molec-
ular weights were determined using protein standards (Sigma; MW-
SDS-200). The protein content of individual electrophoretic species
was estimated using a cooled charge-coupled device imager (Photo-
metrics, Tucson, AZ) after stainingwith Coomassie brilliant blue (see
Sucrose gradient centrifugation of hMLHa. Fraction VI
Biochemistry:Li and Modrich
1952Biochemistry: Li and Modrich
Subunit identification of H6-complementing activity
Peptide origins were evaluated by comparison with translated
cDNA sequences for hMLHl (11, 12), hPMSI, andhPMS2 (13). An X
indicates an indeterminate amino acid: Edman degradation ofpeptide
85-PT111 yielded multiple residues during the first cycle, and cycle 15
of 110-PT72 failed to yield an identifiable amino acid. These cDNA
sequences indicate lysine as residue 311 ofhMLH1 and residues 40 and
369 of hPMS2, while hMLH1 residue 341 is predicted to be arginine.
Peptide sequences are thus consistent with a tryptic origin from
hMLH1 and hPMS2.
hMLH1 residues 312-325
hMLH1 residues 342-361
hPMS2 residues 41-57
hPMS2 residues 370-386
findings confirmed by screen of the NCBI protein sequence
data base. Peptide 110-PT98 produced a single hit correspond-
ing to hPMS2. Regions homologous to peptide 110-PT72 were
identified in bacterial, yeast, and human MutL homologs due
to the origin of this peptide from within a conserved motif, but
a perfect match was observed only in the case of hPMS2.
In addition to these two unique peptides, two additional
fractions derived from tryptic digestion of the 110-kDa species
were also sequenced. Product 110-PT65 yielded two amino
acids at each cycle while 110-PT94 yielded four, and it was
possible by inspection to arrange these amino acids into two
and four sequences, respectively (not shown). Five of these
putative peptides (three tridecamers and two octamers) were
identified in the translated hPMS2 cDNA sequence, with the
preceding amino acid in each case being Lys or Arg, as
expected for peptides of tryptic origin. The sixth putative
peptide was not found in the predicted hPMS2 polypeptide,
and none of the six sequences was identified in hPMS1 or
Thus, while clearly distinct from hMLH1 and hPMS1, the
110-kDa component ofH6-complementing activitybears strik-
ing sequence similarities to hPMS2. However, the molecular
weight ofthis species as determined by SDS gel electrophoresis
is significantly greater than the 95,808 value predicted from
the hPMS2 cDNA sequence (13). This apparent size discrep-
ancy could be due to posttranslational modification, but it is
noteworthy in this respect that the available hPMS2 cDNA
isolate may be incomplete, lacking 5' coding sequence (13).
Furthermore, hPMS2 appears to be only one member of a
closely related subfamily of genes located on chromosome 7
(13, 22). With these possibilities in mind, we surmise that the
110-kDa polypeptide identified here is the product of hPMS2
or a closely related member of the hPMS2 family.
H6-Complementing Activity Is a (hMLH1)g(hPMS2)j Het-
erodimer. Cofractionation of the 85-kDa and 110-kDa MutL
homologs with H6-complementing activity and their presence
at molar stoichiometry in active fractions strongly suggested
that the two proteins interact physically. Band sedimentation
through sucrose density gradients and gel filtration offractions
VI and VII, respectively, yielded an S20,w of 6.1 S and a Stokes
radius of 74.4 A for H6-complementing activity (Fig. 2).
Assuming a typical protein partial specific volume of 0.725
cm3/g, these hydrodynamic values indicate a native molecular
weight of 187,000 (23). Use of a partial specific volume (0.732
cm3/g) calculated from the cDNA-deduced amino acid com-
positions (24) for hMLH1 and hPMS2 (11-13) yields a native
molecular weight of 193,000 and a frictional ratio of 1.95.
Based on these hydrodynamic measurements and the molar
stoichiometry of the two polypeptides alluded to above, we
surmise that H6-complementing activity isolated from HeLa
cells is a heterodimer of hMLH1 and hPMS2 subunits, similar
to the highly asymmetric shape of the bacterial (MutL)2
homodimer (19). We have designated this activity hMutLa.
hMutLa Restores Bidirectional, Strand-Specific Mismatch
Repair to H6 Nuclear Extracts. (CA), microsatellite and
HPRT mutabilities are elevated >100-fold in H6 tumor cells
(6-8), and nuclear extracts derived from this cell line are
defective in repair of base-base and slipped-strand insertion/
deletion mispairs (6). The human mismatch repair system has
an unusual bidirectional capability, and the strand break that
directs correction may reside either 3' or 5' to the mispair (17).
The nature of the H6 defect is such that mismatch-provoked
excision is blocked from the 3' and the 5' sides of the mispair
(6). As shown in Table 3, purified hMutLa restored strand-
specific repair proficiency to H6 nuclear extractswhen assayed
on a set of heteroduplexes representing each of the eight
base-base mismatches as well as several slipped-strand mis-
pairs. Moreover, repair in hMutLa-complemented extracts
was efficiently directed by a strand break located either 3' or
5' to the mispair, as expected for bidirectional mismatch cor-
rection. H6 nuclear extracts were highly responsive to addition
of the purified protein (Fig. 3). The maximal level of repair
achieved at about 25 ng of added hMutLa is comparable to
that observed in extracts of repair-proficient RER- colorectal
tumor cell lines (6).
Screen of fraction VII for simple activities revealed the
absence of detectable mismatch binding as judged by gel shift
assay. This fraction was also free of endonuclease (<5 pmol/
FIG.2.Sedimentation coefficient and Stokes radius of H6-
complementing activity. (Upper) Fraction VI (2 jig in 0.1 ml) was
subjected to band sedimentation on sucrose density gradients as
described in the legend to Table 1. The sedimentation profile of
hMutLa was determined by mismatch repair assay, and the sedimen-
tation coefficient of the protein was estimated by comparison to
migration of marker proteins (catalase, 11.3 S; -y-globulin, 7.1 S;
ovalbumin, 3.55 S) run in a parallel gradient. (Lower) The Stokes
radius ofhMutLa was determined by filtration of the protein (fraction
VII, 2 ,ug in 0.1 ml) at 1 ml/hr through a Sephacryl S-300 column (26.5
x 0.38 cm2) equilibrated with 0.025 M potassium phosphate, pH
7.4/0.1 mM EDTA/2.0 mM dithiothreitol/10% sucrose/0.2 M KCl/1
,ug of leupeptin per ml/1 jigof pepstatin A per ml/0.1% phenyl-
methylsulfonyl fluoride. The column was calibratedwithapoferritin (67.3
A), catalase (52.2 A), ovalbumin (30.5 A), and RNase A (16.4 A).
Proc. Natl. Acad. Sci. USA 92(1995)
Proc. Natl. Acad. Sci. USA 92 (1995)1953
hMutLa restores broad specificity mismatch repair to H6
Mismatch 5' Nick
Repair assays (see text) contained 50 ,tg of H6 nuclear extract, 10
ng of hMutLa, and 24 fmol of heteroduplex DNA containing a nick
in the complementary strand at the Sau96I site or in the viral strand
at the gpII site. These incisions are located 125 bp 5' or 181 bp 3' to
mismatch, respectively, as viewed along the shorter path in the circular
heteroduplexes (6, 17). Repair on the continuous heteroduplex strand
was typically <10% of that on the incised strand (not shown). Values
in parentheses indicate repair in the absence of added hMutLa. ND,
mg per hr) and 3' -> 5' exonuclease on double- or single-
stranded DNA (< 1 pmol/mg per hr) but displayed trace levels
3' exonuclease on duplex and single-stranded DNA
(11-14 pmol/mg per hr) and ATPase in the presence of G-T
heteroduplex DNA (12 pmol/mg per hr).
H6 tumor cells are hypermutable and are deficient in mismatch
repair as judged by in vitro assay (6-8). Our finding that a
hMSHlhPMS2 heterodimer restores repair proficiency to H6
nuclear extracts is in accord with the genetic observation that
this cell line is defective in both alleles of hMLH1 (11).
Although bacterial and human mismatch repair systems are
strikingly similar with respect to specificity and mechanism
(17), the number of human genes encoding MutL homologs
(11-13, 22) and the nature of one such activity described here
suggest that the two repair systems may differ in significant
ways. In contrast to human cells, E. coli possesses a single mutL
gene, the product of which forms a stable homodimer (19).
H6 nuclear extract. Complementation of H6 nuclear extract by
hMutLa (fraction VII) was assayed as described in the text using a
heteroduplex containing a viral strand incision at the gpII site and a
/CA\ slipped-strand mispair (see Table 3).
Human MutL homolog restores mismatch repair activity to
On the other hand, the nature ofhuman MutL genes and the
hMutLa preparations characterized here is reminiscent of
yeast. Saccharomyces cerevisiae has at least two genes that
encode MutL homologs, yPMSJ (25) and yMLHI (26), and
mutations in these two genes behave in an epistatic fashion
with respect to increased mutability, suggesting participation
of the encoded products in a common mutation avoidance
system (26). Indeed, interaction between yPMS1 and yMLH1
has been demonstrated by protein affinity chromatography,
but homospecific interactions involving either protein were
not detectable by this method (27). The aggregation state of
the yeast hetero-oligomer has not been established. In view of
these heterospecific interactions in the yeast system, it is
appropriate to note that hPMS2, identified here as a subunit
of hMutLa, displays greater homology to yPMS1 than does
While the hMutLa preparations described here are biolog-
ically active, we have been unable to identify a simple activity
associated with this component of the repair system. Other
than a demonstrable nonspecific mode of DNA binding (28),
attempts to assign a simple activity to bacterial MutL have also
yielded negative results (19). In fact, the specific interactions
of bacterial MutL with heteroduplex DNA that have been
described to date all depend on the presence of additional
components ofthe methyl-directed pathway. Thus, MutLbinds
specifically to MutS-heteroduplexcomplexes in the presence of
ATP and, together with MutS and ATP, is required for
activation of MutH and for initiation of the excision stage of
repair (29). These observations have led to the suggestion that
MutL may act as an interface between MutS and other
components of the repair system (29, 30). Analysis of the yeast
PMS1 MLH1 complex has also failed to reveal specific inter-
action with heteroduplex DNA, although specific binding does
occur in the presence of the MutS homolog yMSH2 (27). In
view of these findings in other systems, our failure to identify
a simple activity associated with hMutLa is not surprising.
Evidence available to date indicates that a defect in any four
genes can confer predisposition to HNPCC, presumably as a
consequence ofmismatch repair deficiency, and three of these,
hMLHI, hPMS1, and hPMS2, specify MutL homologs (9-13).
In addition to the chromosome 7-derived hPMS2 cDNA
originally characterized (13), this chromosome harbors a fam-
ily of related genes (13, 22). Our finding that hMLH1 isolated
from cervical tumor cells is complexed with a unique hPMS2
isoform raises regulatory questions concerning production of
the set of human MutL homologs and the manner in which
their interactions are modulated as well as related issues
regarding functional roles of hPMS1 and the various members
of the hPMS2 family. Inasmuch as bacterial MutS and MutL
have been implicated in several distinct mutation avoidance
pathways (29), the multiplicity of human MutL homologs
might reflect differentiation of these functions in higher cells.
It is also possible that expression of the various human MutL
genes may be regulated according to developmental or tissue-
specific programs. This is an intriguing possibility given that
HNPCC families have been subclassified depending on the
absence or presence of extracolonic tumors (31). However, the
nature of the genetic lesions responsible for the tissue distri-
bution of tumors associated with the HNPCC subclasses has
not been defined.
We express our appreciation to Bert Vogelstein for communication
of results prior to publication; to William S. Lane and the Harvard
Microchemistry Facility for their assistance with peptide isolation,
mass spectrometry, and microsequence analysis; and to Sherry Larson
and Alison Hayes of the Duke University Cancer Center Tissue
Culture Facility for their expert help in growth of the cell lines used
in this work. This research was supported by Grant GM45190 from the
National Institute of General Medical Sciences.
Biochemistry:Li and Modrich
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