(Received for publication, December 20, 1996, and in revised form, February 12, 1997)
From the Department of Biochemistry and § Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710
HeLa nuclear extract was resolved into a depleted
fraction incapable of supporting mismatch repair in vitro,
and repair activity was restored upon the addition of a purified
fraction isolated from HeLa cells by in vitro
complementation assay. The highly enriched complementing activity
copurified with a DNA polymerase, and the most pure fraction contained
DNA polymerase but was free of detectable DNA polymerases
and
. Calf thymus DNA polymerase
also fully restored mismatch repair
to the depleted extract, indicating DNA polymerase
is required for
mismatch repair in human cells. However, due to the presence of DNA
polymerases
and
in the depleted extract, potential involvement
of one or both of these activities in the reaction cannot be
excluded.
Mismatch repair ensures genetic stability by correcting DNA
biosynthetic errors and by blocking recombination between diverged DNA
sequences (reviewed in Refs. 1-3). Defects in human MutS or MutL
result in genetic destabilization (4-8), are the genetic basis of
tumor predisposition in hereditary nonpolyposis colorectal cancer
kindreds (9-13), and have been implicated in a subset of sporadic
cancers (5, 14-17). In addition to its ability to recognize mismatched
base pairs (16, 18), MutS
also recognizes certain classes of DNA
damage (19, 20), and MutS
or MutL
defects are associated with
resistance to certain DNA-damaging agents, including several that are
used in anticancer chemotherapy (4, 21-26).
The Escherichia coli mismatch repair reaction has been
reconstituted in vitro (27, 28). Briefly, MutS recognizes a
mismatch and MutL binds to this complex. This activates a latent,
MutH-associated endonuclease that incises the unmethylated strand of a
hemimethylated d(GATC) sequence, which may be located either 5 or 3
to the mispair. The nick serves as the primary signal that directs
repair to the unmethylated strand, and in the presence of DNA helicase II, exonucleolytic excision initiates at the nick and proceeds toward
the mismatch through the action of either RecJ or exonuclease VII when
incision is 5
to the mismatch, or exonuclease I when the nick is 3
to
the mispair (28, 29). The resulting single-strand gap is then filled by
DNA polymerase III holoenzyme, and covalent continuity is restored to
the repaired strand by DNA ligase. Single-stranded DNA-binding protein
is also a required component of the reaction.
Four activities have been implicated in the human mismatch repair
reaction. MutS (a heterodimer of MSH2 and MSH6) and MutL
(a
heterodimer of MLH1 and PMS2) were isolated by virtue of their ability
to restore strand-specific mismatch correction to nuclear extracts of
repair-deficient tumor cell lines (16, 18, 30). In addition to its
subunit function in MutS
, MSH2 forms a molecular complex with the
MutS homolog MSH3 (31, 32). This MutS
complex binds to
insertion/deletion mismatches and presumably plays a role in their
processing. Proliferating cell nuclear antigen
(PCNA)1 has also been implicated in the
human mismatch repair reaction (33). However, the activities involved
in the excision stage of the reaction and the DNA biosynthetic activity
responsible for repair synthesis remain to be identified. To this end
we have used biochemical methods to deplete nuclear extracts of a
required repair activity and have used depleted extracts as the basis
for an in vitro complementation assay to isolate additional
components of the system. We describe here experiments implicating DNA
polymerase
in the human mismatch repair reaction.
Human HeLa S3 cells were cultured and
nuclear extracts prepared as described previously (16, 34). All
fractionation steps were performed at 0-4 °C. Samples of
repair-proficient HeLa nuclear extract were resolved independently on
heparin-Sepharose 4B (35) and the immobilized dye resin Reactive Brown
10 (Sigma) to generate mismatch repair deficient fractions. HeLa
nuclear extract (93 mg) was diluted with 0.025 M
HEPES·KOH (pH 7.6), 0.1 mM EDTA, 2 mM
dithiothreitol, 0.1% phenylmethylsulfonyl fluoride (PMSF, concentration relative to a 23 °C saturated solution in
isopropanol), and 1 µg/ml leupeptin (buffer A) to make the
conductivity equivalent to buffer A containing 0.1 M KCl,
centrifuged briefly to remove insoluble material, and applied to a 5.5 cm × 1.5-cm2 column of heparin-Sepharose 4B
equilibrated in buffer A containing 0.1 M KCl. The material
that flowed through the column, representing 80% of the starting
material, was briefly dialyzed against buffer A containing 0.1 M KCl, frozen in small aliquots with liquid nitrogen, and
stored at 80 °C (Fraction H1). Nuclear extract (70 mg) was resolved in a similar manner on a 5.1 cm × 0.8-cm2
column of Reactive Brown 10 equilibrated in 0.025 M
Tris·HCl (pH 7.6), 0.1 mM EDTA, 2 mM
dithiothreitol, 0.1% (saturated) PMSF, 1 µg/ml leupeptin, and 0.05 M KCl. The material that did not bind the resin,
representing 60% of the starting material, was treated as above
(Fraction R1).
In vitro mismatch repair assays with nuclear extracts (50 µg) and 100 ng (24 fmol) of heteroduplex f1MR DNA were performed as described previously (5, 16, 34). Briefly, this method uses a 6.4-kilobase pair circular heteroduplex that contains a site-specific, strand-specific incision to provide strand direction to the reaction. The presence of the mismatch within overlapping sites for two restriction endonucleases serves to block cleavage by either activity, with repair conferring sensitivity to one endonuclease or the other depending on which strand is corrected. Assay for complementation of depleted nuclear extract was performed in a similar manner, except reactions (20 µl) contained 35 µg of Fraction H1 and 27 µg of Fraction R1 (referred to below as H1/R1) in place of nuclear extract, and KCl was maintained at a final concentration of 105-110 mM taking into account salt contributed by depleted extract and column fractions. Reactions were terminated by the addition of 5 µl of 0.5 M EDTA and repair scored as described previously (5, 34).
DNA polymerase activity was determined in reactions (50 µl) containing 0.02 M Tris·HCl (pH 8.0), 2 mM 2-mercaptoethanol, 10 µg of acetylated bovine serum albumin, 0.01 M MgCl2, 50 µM each dATP, dGTP, and dTTP, 50 µM [32P]dCTP (600-1900 cpm/pmol), and 40 µg of sonicated, DNase I-activated salmon sperm DNA. Trichloroacetic acid-insoluble radioactivity was determined after incubation at 37 °C for 60 min. When indicated, DNA polymerase activity was also determined in reactions (50 µl) containing 0.04 M Tris·HCl (pH 6.8), 2 mM 2-mercaptoethanol, 10 µg of acetylated bovine serum albumin, 1.5 mM MgCl2, 50 µM dATP, 50 µM [32P]dTTP (600-800 cpm/pmol), and 1 µg of either poly(dA-dT) or poly(dA)·oligo(dT)12-18, (20:1 nucleotide ratio). Acid-insoluble radioactivity was determined following incubation at 37 °C for 30 min.
The polymerase inhibitor butylphenyl-dGTP was a gift from George Wright (University of Massachusetts, Worcester, MA), and aphidicolin was from Sigma.
Isolation of Activity That Complements Depleted HeLa Nuclear ExtractHeLa nuclear extract (34) derived from 200 liters of
culture (Fraction I, 100 ml, 23 mg/ml) was thawed on wet ice, diluted in buffer A to yield a conductivity equivalent to buffer A containing 0.02 M KCl, centrifuged at 12,000 × g to
remove insoluble material, and applied at 100 ml/h to a
phosphocellulose column (Whatman P-11, 18 cm × 12.6 cm2) equilibrated with buffer A containing 0.02 M KCl. The column was washed with 450 ml of equilibration
buffer and developed with a linear gradient of KCl (2900 ml, 0.02-0.9
M) in buffer A. Fractions that restored repair of a
5-G·T heteroduplex to depleted H1/R1 nuclear extract eluted at
~0.39 M KCl (Fraction II, 250 ml). Fraction II was
dialyzed against buffer A containing 0.05 M KCl until the conductivity was equivalent to buffer A containing 0.085 M
KCl, clarified as above, and applied at 25 ml/h to a heparin-Sepharose 4B column (14.6 cm × 1.75 cm2) equilibrated in buffer
A containing 0.085 M KCl. The column was washed with 50 ml
of equilibration buffer and developed with a 260-ml linear gradient of
KCl (0.085-0.5 M) in buffer A. Fractions complementing
depleted H1/R1 extracts eluted as a broad peak centered at ~0.2
M KCl (Fraction III, 84 ml). After freezing in liquid nitrogen and storage at
80 °C, Fraction III was later thawed on
wet ice and dialyzed against 0.025 M Tris·HCl (pH 7.6),
0.1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml pepstatin A
(buffer B) containing 0.085 M KCl until the conductivity
was equivalent to buffer B containing 0.1 M KCl. After
clarification by centrifugation, the dialysate was applied at 0.7 ml/min onto a 1.0-ml HR 5/5 Mono Q (Pharmacia) FPLC column equilibrated
in buffer B containing 0.1 M KCl. The column was washed
with 2 ml of equilibration buffer and eluted with a 20-ml linear
gradient of KCl (0.1-0.5 M) in buffer B. Fractions were
supplemented with dithiothreitol to 1 mM and PMSF to 0.1%
by the addition of concentrated stock solutions. Fractions
complementing depleted H1/R1 extracts eluted at ~0.31 M
KCl (Fraction IV, 3.5 ml). Fraction IV was dialyzed against 0.025 M potassium phosphate (pH 7.0), 0.1 mM EDTA,
10% glycerol, 0.01% Nonidet P-40, 1 µg/ml leupeptin, 1 µg/ml
pepstatin A (buffer C) also containing 0.085 M KCl until
the conductivity was approximately equivalent to buffer C containing
0.085 M KCl. The dialysate was applied at 0.7 ml/min onto a
1.0-ml HR 5/5 Mono S (Pharmacia) FPLC column equilibrated in buffer C
containing 0.085 M KCl. The column was washed with 3 ml of
equilibration buffer, eluted with a 15-ml linear gradient of KCl
(0.085-0.5 M) in buffer C, and fractions adjusted to 1 mM dithiothreitol and 0.1% PMSF as before. Fractions
complementing depleted H1/R1 extracts eluted at ~0.3 M
KCl, and individual fractions were either used directly or frozen in
small aliquots with liquid nitrogen and stored at
80 °C. Fraction V in Table I represents the column fractions (17-19, 2.4 ml) containing the peak of repair complementing activity.
|
Polyclonal antibody ID, raised in
chicken against the recombinant p180 subunit of human DNA polymerase
, was kindly provided by Teresa Wang (Stanford University, Stanford,
CA) (36). Antiserum to DNA polymerase
was raised in a New Zealand
White rabbit against the synthetic multiple antigenic peptide
LYQKEVSHLSALEERFSRLW, corresponding to residues 1036-1055 of the
catalytic subunit of calf thymus DNA polymerase
(37). Antibodies to
human DNA polymerase
were generously provided by Stuart Linn
(University of California, Berkeley). Monoclonal antibodies raised
against recombinant rat PCNA were obtained from Boehringer Mannheim.
Mouse monoclonal antibodies against human single-stranded DNA-binding
protein (RPA) and rabbit antibodies raised against the DNA polymerase
accessory factor RF-C were gifts from Jerard Hurwitz (Sloan-Kettering
Cancer Center, New York). Rabbit anti-chicken IgG cross-linked to
horseradish peroxidase (HRP), rabbit anti-mouse IgG cross-linked to
HRP, and goat anti-rabbit IgG cross-linked to HRP were from Sigma.
Sheep anti-mouse IgG cross-linked to HRP was purchased from Amersham, and 125I-labeled goat anti-rabbit IgG was from ICN.
For immunological blot analysis, proteins were loaded onto 8% SDS-polyacrylamide gels and electrophoresed at 200 V in a mini-gel apparatus (Bio-Rad) until bromphenol blue present in the loading dye had run off the gel. Proteins were transferred to a nylon membrane (Biotrans, ICN), which was probed with primary antibodies as indicated, and immune complexes visualized using an ECL chemiluminescence kit (Amersham) in the case of peroxidase linked secondary antibodies, or by autoradiography in the case of 125I-labeled secondary antibodies.
DNA Polymerases and PCNAImmunopurified human KB cell DNA
polymerase was a gift of William Copeland (NIEHS, Research Triangle
Park, NC). One unit of DNA polymerase
activity represents the
amount of enzyme required to incorporate 1 nmol of labeled dNMP into
activated salmon sperm DNA in 1 h at 37 °C (38). Purified calf
thymus DNA polymerase
was a generous gift of Antero So (University
of Miami, Miami, FL). One unit of DNA polymerase
activity is
defined as the incorporation of 1 nmol of dNMP into
poly(dA)·oligo(dT) in 1 h at 37 °C in the presence of 2-4
µg/ml PCNA (39). The plasmid pT7hPCNA, encoding human PCNA under the
control of a T7 promoter, was a gift of Bruce Stillman (Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY). Human PCNA was expressed in
E. coli BL21(DE3) and purified to apparent homogeneity by a
modification of the method of Fein and Stillman (40).
In an effort to isolate unidentified activities involved in human mismatch repair, including those for which mutations might render cell lines inviable, we sought to generate biochemically deficient nuclear subfractions that were defective in mismatch repair in vitro. HeLa nuclear extract was resolved into crude bound and unbound fractions on both heparin-Sepharose 4B and Reactive Brown 10 resins (see "Experimental Procedures"). None of the individual subfractions obtained in this manner were able support mismatch repair in vitro, but reactions containing both unbound fractions (this mixture was designated H1/R1) converted DNA heteroduplexes into a new species with an eletrophoretic mobility consistent with that expected for a gapped DNA molecule (data not shown). This suggested that H1/R1 was lacking one or more components required for repair DNA synthesis. This depleted fraction proved to be deficient in a single required activity, a DNA polymerase required for the repair synthesis reaction.
HeLa nuclear extract was resolved by chromatography on phosphocellulose, and column fractions were assayed for their ability to restore mismatch repair activity to depleted H1/R1 extract. This column yielded a single, symmetrical peak of complementing activity, which was further purified as described under "Experimental Procedures" and summarized in Table I. The most highly purified fraction (Fraction V) was enriched about 2,800-fold relative to nuclear extract.
H1/R1 complementing activity was purified by virtue its ability to
restore G·T heteroduplex repair. In addition to its inability to
support G·T heteroduplex repair, H1/R1 extract was also defective in
repair of a two-nucleotide insertion/deletion mismatch. As shown in
Fig. 1, repair of both types of mismatch was fully
restored by addition of Fraction V. Since strand directionality of the human mismatch repair reaction can be provided by a nick located either
3 or 5
to the mismatch (41), the ability of Fraction V to restore
repair with both heteroduplex orientations was also tested. Fig. 1
demonstrates that the activity of Fraction V is independent of mismatch
nick orientation.
As noted above, although mismatch rectification did not occur in H1/R1
extract, heteroduplex processing was observed, with repair blocked at a
late stage in the reaction. This suggested that the depleted extract
might be deficient in a DNA polymerase required for repair synthesis,
and polymerase activity was found to co-purify with complementing
activity (Fig. 2), prompting further test of this
possibility by immunological blot analyses. Whereas DNA polymerases
,
, and
were all present in HeLa nuclear extract, depleted
H1/R1 extract contained DNA polymerase
and reduced levels of pol
(~ 5%), but pol
was undetectable in this fraction (Fig.
3). On the other hand, pol
was the only DNA
polymerase detected in Fraction V of complementing activity, and levels
of this polymerase estimated by immunological blot analysis correlated well with H1/R1 complementing activity during purification, strongly suggesting that DNA polymerase
was the required activity lacking in
the depleted extract. Although PCNA and the human single-stranded DNA-binding protein RPA were resolved from complementing activity during phosphocellulose chromatography, both were present in depleted extract, and this fraction also contained the polymerase accessory factor RF-C (Fig. 3 and data not shown).
The participation of DNA polymerase in mismatch repair is also
supported by chemical inhibition of repair and DNA polymerase activities. Mismatch repair in HeLa nuclear extracts is blocked by
aphidicolin, indicating involvement of DNA polymerases
,
, and/or
in the reaction (34). We have confirmed this observation with the
finding that 100 µM aphidicolin completely inhibited repair when depleted H1/R1 extract was complemented with Fraction IV of
the purified activity, and DNA synthesis on activated salmon sperm DNA
supported by Fraction V was reduced approximately 2-fold by 100 µM aphidicolin. Furthermore, neither Fraction IV DNA
polymerase activity nor repair activity in complemented H1/R1 extract
was inhibited by 10 µM butylphenyl-dGTP (data not shown),
suggesting that DNA polymerase
does not have a critical role in
mismatch repair (42).
While the foregoing experiments suggested that depletion of pol accounted for the repair deficiency of H1/R1 extract, H1/R1 complementing activity was only partially purified, with Fraction V
containing about 50 distinct protein species as judged by
silver-stained SDS-polyacrylamide gels. To test the sufficiency of pol
in restoration of repair to H1/R1 extract, the complementing
activity of bona fide pol
was tested. As shown in Fig.
4, mismatch repair in the depleted extract was restored
to a similar degree by either calf thymus pol
or Fraction V of the
complementing activity isolated here. By contrast, human pol
did
not complement the depleted extract. Furthermore, repair activity
restored by either pol
or Fraction V was further enhanced by about
40% upon supplementation of H1/R1 extract with additional exogenous
PCNA. This finding is consistent with previous experiments implicating
PCNA in repair (33), although in our case the presence of PCNA in the
depleted extract (Fig. 3D) prevents a more quantitative
assessment of the role of this activity in the reaction. It is
noteworthy that PCNA (150-200 ng) was found to stimulate DNA
polymerase activity of isolated complementing activity about 2-fold on
activated salmon sperm DNA or poly(dA-dT) template-primers, but by more
than 10-fold with poly(dA)·oligo(dT) (data not shown). Stimulation of
DNA polymerase activity by PCNA on this template-primer is a hallmark
of pol
(43, 44) and provides further evidence that this activity is
the major DNA biosynthetic activity present in the fractions isolated
here.
This work presents direct evidence for involvement of DNA
polymerase in human mismatch repair. We have shown that pol
is
required for repair of base-base mismatches and small
insertion/deletion heterologies, and that the requirement for this
activity is evident when the single strand break that directs the
reaction is located either 5
or 3
to the mispair. These findings
indicate that pol
plays a general role in the reaction. While we
cannot exclude participation of DNA polymerases
or
in mismatch
repair due to the presence of significant levels of both activities in
depleted H1/R1 extract, the observed insensitivity of repair to
butylphenyl-dGTP renders pol
an unlikely candidate. Furthermore,
while the residual level of pol
in repair-deficient, depleted
extract (~ 5% of that present in fully active HeLa extracts, Fig. 3)
was insufficient to support repair, isolated complementing activity was
devoid of detectable levels of this polymerase. Thus, either the human mismatch repair reaction requires both pol
and pol
, or pol
is sufficient to meet the polymerase requirement in the reaction. We
tend to favor the latter possibility. Results obtained with Saccharomyces cerevisiae DNA polymerase mutants may also
bear on this point. Strand et al. (45) demonstrated that
inactivation of the proofreading function of yeast pol
results in a
5-10-fold increase in mutation rate of d(CA)n repeat
sequences, but proofreading mutations in pol
do not. It is
difficult to reconcile the different phenotypes based solely on the
functional roles of the two polymerases in chromosome replication (46), but differential participation of the two enzymes in mismatch repair
might account for this difference since d(CA)n mutability
correlates strongly with mismatch repair defects (45).
Umar et al. (33) have recently found that dinucleotide
repeats are unstable in yeast PCNA mutants and, as judged by two hybrid
analysis, that PCNA interacts with yeast MutL homologs PMS1 and MLH1
(human PMS2 is a homolog of yeast PMS1). They also demonstrated that
mismatch repair in human cell extracts is blocked prior to the excision
step by p21WAF1 or by a p21 peptide, which binds to PCNA and
inhibits PCNA-dependent DNA replication (47). While PCNA
involvement would be consistent with a repair role for pol since
the two proteins are known to interact (44, 48), a definitive argument
cannot be based on this observation because PCNA probably also
interacts with pol
(49).
Available information on the nature of the human mismatch repair
reaction has indicated striking similarities to the bacterial pathway,
including conservation of specificity and mechanism, and involvement of
homologs of MutS and MutL at early steps of the reaction (reviewed in
Ref. 2). The work described here extends these similarities to the DNA
polymerase requirement. Repair DNA synthesis in E. coli
methyl-directed mismatch correction is catalyzed by DNA polymerase III
holoenzyme, the enzyme that is also responsible for chromosome
replication. Like DNA polymerase III, pol plays a major role in the
synthesis of new DNA strands at the replication fork, a function it
probably shares with pol
(46).