From the ** Howard Hughes Medical Institute and the Departments of
Biochemistry and § Medicine-Medical Oncology,
Duke University Medical Center, Durham, North Carolina 27710 and the
School of Medical Technology, College of Medicine, National
Taiwan University, Taipei, Taiwan 100-02, Republic of China
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ABSTRACT |
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The repair of 12-, 27-, 62-, and 216-nucleotide
unpaired insertion/deletion heterologies has been demonstrated in
nuclear extracts of human cells. When present in covalently closed
circular heteroduplexes or heteroduplexes containing a single-strand
break 3' to the heterology, such structures are subject to a low level repair reaction that occurs with little strand bias. However, the
presence of a single-strand break 5' to the insertion/deletion heterology greatly increases the efficiency of rectification and directs repair to the incised DNA strand. Because nick direction of
repair is independent of the strand in which a particular heterology is
placed, the observed strand bias is not due to asymmetry imposed on the
heteroduplex by the extrahelical DNA segment. Strand-specific repair by
this system requires ATP and the four dNTPs and is inhibited by
aphidicolin. Repair is independent of the mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 and occurs by a mechanism that is distinct from that of the conventional mismatch repair system. Large heterology repair in nuclear extracts of human cells is also independent of the
XPF gene product, and extracts of Chinese hamster ovary cells deficient in the ERCC1 and ERCC4 gene
products also support the reaction.
Base pairing anomalies can occur within the DNA helix as a
consequence of DNA biosynthetic errors or as a result of
recombinational strand transfer between sequences that differ
genetically (1-4). Such pairing errors may take the form of base-base
mismatches or insertion/deletion
(I/D)1 heterologies, in which
one strand contains a segment of one or more unpaired nucleotides.
Strand-specific correction of base-base and I/D mismatches produced
during DNA biosynthesis plays an important role in mutation avoidance
(2, 5, 6), and mismatch rectification within the recombination
heteroduplex has been implicated in meiotic gene conversion in fungal
systems (3, 4, 7, 8).
Base-base mispairs are subject to strand-specific correction by the
mismatch repair systems of both prokaryotes and eukaryotes (5, 6, 9),
but action of this system on I/D mismatches is limited to fairly small
heterologies. The Escherichia coli mismatch repair pathway
will correct I/D heterologies up to about 7 unpaired nucleotides, but
larger heterologies are poorly processed by this system (10-13). A
similar specificity is characteristic of the human mismatch repair
system, which can correct I/D heterologies up to about 8 unpaired
nucleotides (14-16).
There is evidence, in some cases contradictory, that both prokaryotes
and eukaryotes can rectify I/D heterologies with larger unpaired
segments by a pathway distinct from the mismatch repair system. Using
transfection assay, Dohet et al. (10) demonstrated rectification of a bacteriophage Transformation of monkey cells with heteroduplex DNAs containing
unpaired single-stranded loops has indicated that mammalian cells can
rectify such structures (18-21). Heterology repair as scored by
transformation assay is biased about 2:1 for heterology removal (19),
and the presence of a single-strand break near the unpaired loop was
found to alter repair outcomes to a significant but limited degree
(21). In vitro experiments have also suggested that human
cells possess a system distinct from the mismatch repair pathway for
processing heteroduplexes with large I/D heterologies. For example,
Umar et al. (15) found that incised heteroduplexes containing 8- and 16-nucleotide I/D mismatches were repaired in nick-directed fashion in extracts of a mismatch repair-defective MLH1-/- cell line. Similar results have
been obtained by Genschel et al. (16), who
demonstrated that a nicked heteroduplex containing an 8-nucleotide I/D
heterology is processed in a strand-specific manner and to similar
extents by the mismatch repair system and by a second pathway that is
independent of MSH2 function. A 27-nucleotide I/D heterology was shown
to be processed only by the latter system. Although the asymmetric
rectification of the larger I/D heterologies in both of these studies
was consistent with nick direction, definitive conclusions on this
point have been precluded by the fact that individual heterologies were
tested in only one strand orientation. Furthermore, the nicks
responsible for putative strand direction in these studies were located
5' to the I/D heterology; the effect of 3' nick placement was not
tested, nor was the dependence of the efficiency of rectification on
the presence of a strand break.
In order to further define the nature of the reaction responsible for
repair of large extrahelical segments, we have constructed a set of
heteroduplexes containing unpaired I/D loops of 12-993 nucleotides,
evaluated the dependence of repair on presence and placement of a
single-strand break, and tested the possibility that strand placement
of a genetic heterology may confer asymmetry on the rectification process.
Bacteriophages and Heteroduplex Construction--
A set of f1MR
bacteriophages for construction of heteroduplex DNAs has been
previously described (14, 23-26). Bacteriophage f1MR1 was derived from
f1h1 by elimination of the ClaI site at position
6040 by oligonucleotide mutagenesis, followed by insertion of a 27-base
pair synthetic duplex into the EcoRI site at position 5617, destroying the latter site (23, 24). The intermediate phage in this
construction, which retains the EcoRI site, is designated f1MR11 (Fig. 1). Bacteriophages f1MR9, f1MR24, and f1MR28 have been
described previously (14, 24). Phage f1MR32 was constructed by
insertion of 5'-AGCTGCAGCCAGCTCGAGCTGTGTGTGGC-3' annealed with 3'-CGTCGGTCGAGCTCGACACACACCGGATC-5' into f1MR1 (23, 24) that had been
digested with HindIII and XbaI. Phage f1MR30 was
constructed by insertion of a HindIII-NheI
restriction fragment derived from plasmid pBR322 (coordinates 29-229)
into HindIII, XbaI-cleaved f1MR1, whereas f1MR31
was constructed by insertion of a HindIII-XbaI fragment from plasmid pQE-30 (Qiagen, coordinates 187-1164) into f1MR1
digested in a similar manner.
Heteroduplex DNAs were constructed by hybridization of restriction
endonuclease-cleaved replicative form DNA to single strands isolated
from f1MR virions (25, 27). Substrates containing one or a few unpaired
nucleotides were prepared using phage DNAs described previously (14,
16, 26). Other heteroduplexes used in this study are summarized in Fig.
1, with the unpaired heterology placed either in the viral (V) or
complementary (C) DNA strand as specified. For preparation of the
heteroduplex containing a 62-nucleotide insertion in the C strand, the
oligonucleotide d(CTAGAC(TG)19CA) (14) was added to the
hybridization reaction. The presence of the oligonucleotide prevented
the formation of branched complexes in which a phage viral strand
hybridized to the 62-nucleotide unpaired segment within the
heteroduplex. As judged by resistance to NheI (Fig. 1),
isolated preparations of this heteroduplex were free of significant
levels of the oligonucleotide. A similar problem, which could not be
circumvented in this manner, was encountered in attempts to prepare
heteroduplexes with larger insertions in the C strand. For this reason,
unpaired 216- or 993-nucleotide heterologies were placed in the viral
strand only.
Heteroduplexes were prepared in covalently closed circular form or
contained a site-specific, strand-specific nick as indicated. In the
latter case, DNAs contained a strand break in the C strand at the
Sau96I or HincII site, or in the V strand at the
cleavage site for f1 gpII protein (25, 27). Nicks placed in this manner were 114 bp 5', 797 bp 5', or 170 bp 3' to the I/D mismatch,
respectively, as measured along the shorter path joining the two DNA
sites in the circular substrates (Fig. 1).
Nuclear Extracts and Repair Reactions--
Human cell lines HeLa
S3, MT1, H6, SO, LoVo, and HEC-1-A were grown as described
(14, 26-29). Chinese hamster ovary cell lines UV20 and UV41, which
were provided by Dr. Aziz Sancar (University of North Carolina, Chapel
Hill, NC), were grown as described previously (30). The human GM08437
cell line, which was obtained from the NIGMS/Coriell repository, was
cultured in a humidified 5% CO2 incubator in Eagle's
minimum essential medium containing 10% fetal bovine serum and 2×
concentrations of essential amino acids, nonessential amino acids, and
minimum essential medium vitamins.
Nuclear extracts were prepared according to Holmes et al.
(27). Unless specified otherwise, repair reactions (10 µl) contained 0.02 M Tris-HCl (pH 7.6); 0.11 M KCl; 1.5 mM ATP; 0.1 mM each dATP, dGTP, dTTP, and dCTP;
5 mM MgCl2; 24 fmol of heteroduplex DNA; and
50-75 µg of nuclear extract protein. After 15 min of incubation at
37 °C, reactions were terminated by addition of 30 µl of 25 mM EDTA, 0.07% SDS, and DNAs processed as described previously (27). Repair of heteroduplexes containing a large I/D
heterology was scored by restriction endonuclease assay in a manner
similar to that described previously for smaller heterologies (14, 26).
For example, heteroduplexes constructed using phage f1MR11 contain an
EcoRI site in the f1MR11 strand at the junction of the
heterology, with the unpaired segment containing an NheI site (Fig. 1). Repair to the deletion product confers EcoRI
sensitivity, whereas repair to the insertion product renders the DNA
sensitive to NheI cleavage.
Analysis of Repair Intermediates Produced under Conditions of
Limited Repair DNA Synthesis--
Trapping of intermediates resulting
from excision of I/D heterologies was accomplished by omission of
exogenous dNTPs from reactions and thereby restricting repair DNA
synthesis (25). Excision tract end points were mapped relative to an
indicated restriction endonuclease cleavage site by indirect end
labeling (31) after electrophoresis through 1.5% alkaline agarose
(0.03 N NaOH, 2 mM EDTA) or 10% denaturing
polyacrylamide gels (50 mM Tris borate, pH 8.3, 1 mM EDTA, 8.3 M urea). After transfer and UV
cross-linking to an ICN Biotrans nylon membrane, that portion of a
heteroduplex strand of interest was visualized by probing with a 5'
32P-labeled oligonucleotide that hybridized near the strand
terminus produced by restriction cleavage (25). Oligonucleotides V2531 (d(ATGGTTTCATTGGTGACGTT), corresponding to f1MR11 viral strand nucleotides 2531-2550) and V5216 (d(ATTGTTCTGGATATTACCAG),
corresponding to viral strand nucleotides 5216-5235) were used as
probes to end label complementary strand products produced by
Bsp106 or SspI cleavage (Fig. 1). Labeled species
were visualized by exposure of membranes to Kodak XAR-5 film, and
radioactivity was quantitated by analysis of film with cooled
charge-coupled device imager (Photometrics) or by PhosphorImager analysis.
Strand-specific Repair of Large I/D Heterologies Is Directed by a
5' Strand Break--
Although the human mismatch repair system is able
to repair I/D mismatches in a nick-directed, strand-specific manner
(14, 15, 26), the activity of this system on such structures appears to
be restricted to heterologies containing less than about eight unpaired
nucleotides (15, 16). Incised heteroduplexes containing 12-, 16-, and
27-nucleotide I/D heterologies are also processed by human nuclear
extracts, but in these cases, rectification is independent of MSH2 and
MLH1 mismatch repair proteins (15, 16). Although repair of such
structures has been found to occur on the incised DNA strand, the role
of the nick in directing the reaction has been uncertain due to the
fact that the tested substrates contained the heterology in only one of
the two possible unpaired configurations. Hence, it has been unclear
whether the observed repair asymmetry is due to presence of a strand
break or to asymmetry imposed on the DNA by the unpaired heterology itself.
In order to clarify the nature of this reaction, we have constructed a
set of circular heteroduplexes containing unpaired heterologies ranging
in size from 12 to 993 nucleotides (Fig. 1), in several cases in both possible
configurations, and have evaluated the role of strand-specific
single-strand breaks in the processing of such structures. We designate
these heteroduplexes according to the DNA strand containing the
unpaired segment, the number of unpaired nucleotides in the heterology,
and placement of the strand break 5' or 3' to the heterology as viewed
along the shorter path joining the two DNA sites in the circular DNA. In this convention, 5'-C27 indicates a heteroduplex containing an
unpaired 27-nucleotide loop in the complementary DNA strand with the
strand break located 5' to the heterology, whereas 3'-V216 refers to
the heteroduplex with a 216-nucleotide unpaired segment in the viral
strand with the nick located 3' to the heterology. With all of the
substrates used, heterology rectification and any associated strand
specificity can be monitored by restriction endonuclease assay (Fig.
1).
As summarized in Table I, unpaired
heterologies of 27 or 216 nucleotides were subject to limited
processing in HeLa nuclear extracts when present in a covalently closed
circular DNA. However, heterologies in this size range were efficiently
rectified in open circular DNAs containing a single-strand break 114 or
797 bp 5' to the unpaired region in the 6.4-kilobase pair circular heteroduplexes (Fig. 2 and Table I).
Repair of these DNAs displayed a substantial bias (4-18-fold) toward
the incised DNA strand, although significant repair was detected on the
closed DNA strand in some cases (see Fig. 2, 5'-C62 heteroduplex). The
efficiency of nick-directed correction of these large heterologies was
30-70% of that observed for a G-T mismatch or T, A, or GT I/D
mispairs, all of which are processed by the conventional mismatch
repair system (14, 16, 26, 27, 32, 33). Significant repair with some
strand bias was also observed for the 5'-heteroduplex containing a
993-nucleotide unpaired region, but this substrate was processed less
well than those containing the smaller nonhomologous segments.
A single-strand break located either 3' or 5' to a mispair is
sufficient to provide strand specificity for mismatch repair MutS
It is important to note that whereas all of the 5'-heteroduplexes used
in this study contained the strand break in the complementary strand,
tested substrates included several with the unpaired nonhomology present in either the complementary or viral strand (Table I). When the
nonhomologous segment was present in the C strand, processing of the
5'-heteroduplex produced the deletion repair product, whereas presence
of the nonhomology in the V strand yielded the insertion repair
product. Consequently, the strand-specific asymmetry observed for
repair of 5'-heteroduplexes cannot be attributed to the simple presence
of an unpaired segment in a particular DNA strand. Rather, this effect
must be due to the 5' strand break. This conclusion is also consistent
with the finding that unpaired heterologies of 27-216 nucleotides are
processed only at a basal level and without evident strand bias when
present in closed circular DNAs or in heteroduplexes containing a nick
3' to the heterology (Table I and Fig. 3). The observation that the
efficiency of repair directed by a 5' strand break decreases with
increasing distance between the heterology and the nick also supports
this view. Thus, although basal processing of unpaired heterologies
does occur, the presence of a 5' strand break substantially increases
the efficiency of the reaction and confers strand specificity on the process.
The low level of strand-independent repair that we observe with
covalently closed circular heteroduplexes or 3'-substrates (Table I)
could be the consequence of events directed by a 5' strand breaks
produced by endonucleases present in the extract (27). However, it is
also possible that I/D heterologies are directly recognized and
processed in a nick-independent fashion by other activities present in
the nuclear extract. Some evidence supporting the latter view is
described below.
Requirements for Large Heterology Repair in Human Nuclear
Extracts--
The only exogenous cofactors required for in
vitro repair of large I/D heterologies by HeLa nuclear extracts
are ATP, Mg2+, and the four dNTPs. Omission of any of these
components resulted in substantial decrease in repair of the several
5'-heteroduplexes tested (Table II). The
reaction was also inhibited by 90 µM aphidicolin but not
by 0.5 mM ddTTP. Because aphidicolin is a specific
inhibitor of DNA polymerases Repair of Large Insertion/Deletion Heterologies Yields a Covalently
Continuous DNA Product--
Repair by the bacterial mismatch
correction pathway has been shown to culminate with ligation of the
repaired DNA strand (38). As shown in Fig.
4, we have used an indirect end labeling
method (25, 31) to determine whether large heterology repair is
associated with ligation of the repaired DNA strand. As judged by
production of the full-length (~6400 nucleotides) linear form after
cleavage with Bsp106, the repaired C strand was recovered in
covalently continuous form in 80-95% of the molecules isolated after
incubation with HeLa nuclear extracts (Fig. 4, odd lanes
1-9). The small amounts of incised C strand evident in the
Bsp106 hydrolysates is most likely derived from unligated
heteroduplex. This point is most easily seen with the end-labeled C
strand product produced by Bsp106 cleavage of the V216(H)
heteroduplex that contained a single-strand break in the
HincII site. As shown in Fig. 1, the distance between the
Bsp106 site, which is the position of indirect end labeling,
and the HincII site is 3884 nucleotides, a value that
corresponds well to the 3900-nucleotide species observed after
Bsp106 cleavage of DNA products obtained after incubation with HeLa nuclear extract.
When the products of heteroduplex incubation with HeLa nuclear extract
were hydrolyzed with Bsp106 and the appropriate restriction enzyme diagnostic for strand-specific rectification, a fraction of the
full-length 6400-nucleotide C strand was converted to a smaller species
with a mobility similar to that of the 3100-nucleotide marker (Fig. 4,
even lanes 2-10), which is the expected size of repair
products (Fig. 1). However, with some 5'-heteroduplexes containing a C
strand nick at the Sau96I site, this repair product was
evident only as an increase in signal over a background DNA species of
similar size that is evident in samples treated with Bsp106
alone (e.g. see Fig. 4, lanes 3 and 4 or lanes 9 and 10). As discussed above for the
V216(H) heteroduplex with a HincII nick, we think it likely
that this background signal is due to persistence of some molecules
with a discontinuity in the C strand at or near the Sau96I
site. Due to the fact that the Sau96I strand break in these
heteroduplexes is separated from the heterology by only 114 bp, this
species is poorly resolved from the C strand fragment that is
diagnostic for repair.
In view of this background problem, production of the covalently
continuous repair product was confirmed by physical isolation of
covalently closed, circular duplex DNA produced upon incubation of C27
and C62 heteroduplexes with HeLa nuclear extract. As shown in Fig. 4
(lanes 11-14), repaired molecules were present in the closed circular population produced with both heteroduplexes.
Repair of Large I/D Heterologies Is Independent of MSH2, MSH6,
MLH1, PMS2, and XPF Gene Products--
In human cells, the
conventional, nick-directed mismatch repair pathway is capable of
processing the eight base-base mismatches as well as I/D heterologies
of 1 to about 8 unpaired nucleotides (14-16, 25-27, 32). Previous
experiments have shown that a 5'-heteroduplex containing a
16-nucleotide I/D heterology is subject to repair in
MLH1-/- HCT116 cells (15) and that 12- and
27-nucleotide heterologies are repaired in
MSH2-/- LoVo cells (16). Fig.
5 confirms and extends these
observations. As expected, a 5'-heteroduplex containing a CA
dinucleotide insertion in the complementary DNA strand is not
significantly repaired in MSH2-/- LoVo cells
(28), MLH1-/- H6 cells (39), or
PMS2-/- MSH6-/-
HEC-1-A cells (40) and is subject to limited correction in MSH6-/- MT1 cells (26, 41), with repair in
extracts of the latter cell line due to presence of the MSH2·MSH3
MutS
Kirkpatrick and Petes (42) have found that rectification of an unpaired
26-nucleotide heterology during meiotic segregation in S. cerevisiae depends on the MSH2 and RAD1 gene
products. As noted above, we have found the repair of large
heterologies in human nuclear extracts to be independent of human MSH2.
In addition, we have tested this reaction in nuclear extracts derived
from Chinese hamster ovary UV41 and UV20 cells deficient in
ERCC4 or ERCC1 gene products respectively, rodent
homologs of S. cerevisiae RAD1 and RAD10 proteins. Both
extracts displayed repaired a 5'-C62 I/D insertion/deletion heterology
normally, and similar results were obtained with the GM08437 human cell
line deficient in the XPF gene product, the human homolog of
RAD1 (not shown). Therefore, large heterology repair in extracts of
mitotic mammalian cells apparently occurs by a pathway distinct from
that responsible for heterology rectification in meiotic yeast cells.
Analysis of Repair Intermediates Implies That Repair of Large I/D
Heterologies Occurs by a Mechanism Different from Mismatch
Repair--
Restriction of repair DNA synthesis by omission of dNTPs
or inclusion of aphidicolin has permitted visualization of excision intermediates that are produced during mismatch correction (25). We
have used a similar approach in an attempt to trap excision intermediates produced during nick-directed repair of large
heterologies. Fig. 6 shows the results of
such an analysis with the V216 and C27 heteroduplexes containing a C
strand nick at the Sau96I site 114 bp 5' to the heterology
junction (Fig. 1), as well as a single nucleotide T I/D heteroduplex
with a nick at the same position. The complementary strand of each of
these substrates was largely converted to a covalently closed species
(6.4-kilobase pair band) under complete repair conditions. As observed
previously for excision tracts generated by the mismatch repair system
(25), excision intermediates were evident with the T I/D heteroduplex
when dNTPs were omitted from the reaction. Under these conditions,
about half of the molecules retained a discontinuity in the C strand, with the shortened 5'-termini mapping from the nick to about 200 nucleotides beyond the original location of the mispair. Different results were obtained with C27 and V216 heteroduplexes. Under complete
reaction conditions, about 10% of the recovered molecules contained
discrete discontinuities in the vicinity of the original location of
nick and the heterology, and these species increased dramatically upon
omission of dNTPs (Fig. 6).
In view of the proximity of the heterology and the strand break in
these heteroduplexes (separation distance of only 114 bp), these
putative excision intermediates were examined at higher resolution by
mapping relative to the 3'-end produced in the C strand by hydrolysis
with SspI, which cleaves 400 nucleotides from the position
of the I/D heterology (Fig. 1). As shown in Fig.
7, omission of dNTPs resulted in a
substantial reduction in production of repair products, an effect most
easily seen with the V216 heteroduplex, where the 933-nucleotide repair
fragment is well resolved from the 717-nucleotide fragment in the
unrepaired DNA (lanes 2 and 4). Omission of dNTPs
also resulted in accumulation of major C strand species with termini
mapping to a region just 5' of the position of the heterologies for
both C27 and V216 heteroduplexes (Fig. 7, lanes 3 and
4). A second species of about 400 nucleotides corresponding
to a discontinuity in the C strand at the location of the heterology
was also evident with both heteroduplexes, but as can be seen, this
species was also produced from the covalently closed circular form of
the C27 substrate and was observed with the incised C27 heteroduplex in
the presence of dNTPs. Hence, the latter species may be unrelated to
the strand-specific repair reaction described here, although it could
be involved in the low level of strand-independent repair that we have
observed (Table I). Because neither of these species was observed with
the incised A·T homoduplex control (Fig. 7, lane 7), their
production is clearly dependent on presence of an I/D heterology.
Discontinuities corresponding to the location of the Sau96I
strand break were observed in a small fraction of V216 and C27
heteroduplexes after incubation with HeLa extract in the presence of
dNTPs (Fig. 7, lanes 2 and 3, 515- and
542-nucleotide species). Similar discontinuities were evident in C
strand products obtained in the absence of dNTPs, but in this case, the
yield was elevated, and species of reduced chain length were present
(Fig. 7, lanes 4 and 5), indicative of excision
from the strand break. Similar degradation products were not observed
with the A·T homoduplex control DNA (Fig. 7, lane 7).
Although these observations do not suffice to establish the mechanism
for nick-directed repair of large I/D heterologies, they clearly show
that this reaction occurs by a mechanism that is distinct from that of
mismatch repair as deduced using similar methods (25).
As judged by dependence of repair on MSH2 and
MLH1 products, the processing of I/D mismatches of 1-4
nucleotides in human cell extracts occurs almost exclusively by
mismatch repair (14, 15, 26, 33). Based on the rates of repair observed
in nuclear extracts of MSH2-/- or
MLH1-/- cells as compared with
those observed when such extracts are supplemented with the deficient
activities, we have found that a 5-nucleotide I/D mismatch is rectified
primarily by the mismatch repair system, although limited MSH2 and
MLH1-independent repair does occur (Ref. 16 and this study). Analogous
experiments with 8- and 12-nucleotide I/D heteroduplexes have indicated
that the former substrate is processed to a similar degree by the MSH2- and MLH1-dependent and -independent pathways, with
rectification of the latter DNA being largely independent of the
mismatch repair system. We show here that 27-, 62-, and 216-nucleotide
I/D heterologies are efficiently processed in human nuclear extracts by
a reaction that is independent of MSH2, MSH6, MLH1, and PMS2,
clearly distinct from the conventional mismatch repair system.
Although large heterologies were subject to limited processing when
present in covalently closed circular heteroduplexes or heteroduplexes
containing a single-strand break 3' to the heterology, repair was
enhanced substantially by a single-strand break placed 5' to the I/D
heterology, and in this case, rectification was highly biased to the
incised strand. This dependence on nick heterology orientation also
distinguishes the reaction from mismatch repair, which can be directed
to a particular strand by an incision located either 3' or 5' to the
mispair (25). The two pathways are also dissimilar, as judged by the
nature of excision intermediates that accumulate upon restriction of
repair DNA synthesis (Fig. 7 and Ref. 25).
Transfection of monkey cells with SV40 heteroduplexes by Weiss and
Wilson (19, 21) has previously indicated that large I/D heterologies
are efficiently rectified in mammalian cells. Although presence of a
strand break 71 or 125 nucleotides from the unpaired loop was found to
bias rectification to a significant degree in this system, the primary
determinant of repair was found to be the heterology itself (21). In
contrast, we have observed only a limited degree of nick-independent
processing of large I/D heteroduplexes in nuclear extracts of human
cells, although we have detected a possible product of endonuclease
action at the site of the heterology in a covalently closed circular
substrate (Fig. 7, lane 6). On the other hand, we have
demonstrated efficient, strand-specific rectification events directed
by a nick located 5' to the heterology. There are several potential
explanations for the different results obtained with these two systems.
For example, our method for preparation of nuclear extracts may
selectively extract components of the nick-directed system at the
expense of those involved in a distinct pathway that acts primarily via heterology recognition. An alternate possibility is that the results of
Weiss and Wilson (19, 21) are complicated by strand loss effects like
those that have been observed during transformation of E. coli with heteroduplex DNAs (43). It is also important to note
that in contrast to the DNAs used here, the nicked heteroduplexes used
by Weiss and Wilson (21) were prepared by denaturation and
hybridization of two duplex DNAs and thus contained a mixture of the
two combinations of the parental strands. As they note (21), use of
such a mixture would artificially depress the effect of a strand break
on repair in their transfection assay if the nick-directed reaction
were to depend on a particular chemical polarity as we have found to be
the case in extracts of human cells.
Genetic analysis has indicated that large I/D heterologies are repaired
during meiosis in S. cerevisiae in a reaction that depends
on the yeast MSH2 and RAD1 gene products (42).
The nick-directed reaction that occurs in extracts of mitotic human
cells described here apparently occurs by a distinct pathway because it
is independent of MSH2 and the mammalian homologs of yeast RAD1 and
RAD10. A study by Lahue and
co-workers2 describes a
system for study of large heterology repair in extracts of mitotic
S. cerevisiae. As in the case of the cell-free human system
described here, repair in yeast extracts is independent of yeast MSH2.
Although a 60% reduction in activity was observed in extracts of
rad1 mutant cells, the addition of purified RAD1 protein or
the RAD1/RAD10 complex failed to restore normal levels of repair to
rad1 cell-free extracts.2
The demonstration of strand-specific repair of large I/D heterologies
in human cell extracts raises questions concerning the functions of
this reaction. Although such a system may function in the processing of
recombination heteroduplexes, the strand specificity of the reaction
suggests a role in correction of I/D heterologies that arise by DNA
misalignment events during chromosome replication (22). If a potential
role in replication fidelity is assumed, the obligate polarity imposed
by the requirement for a 5' strand terminus would presumably restrict
action of the system to the lagging DNA strand, where 5' terminal
discontinuities occur. Isolation of the activities involved in the
pathway and the identification of the corresponding structural genes
should serve to clarify the roles of this system.
INTRODUCTION
Top
Abstract
Introduction
References
heteroduplex containing an 800-nucleotide unpaired IS1 heterology by a pathway that was
independent of mutH, mutL, and mutS gene
function. In contrast, Carraway and Marinus (12) failed to detect
repair of large heterologies upon transformation of covalently closed
circular plasmid heteroduplexes into wild type E. coli.
Although strand breaks are known to be required for efficient
correction by conventional mismatch repair systems (6, 17), a potential
activating role for strand discontinuities in large heterology repair
in E. coli has not been reported.
EXPERIMENTAL PROCEDURES
RESULTS
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Fig. 1.
Heteroduplex structures. Heteroduplexes
with an I/D heterology were constructed by hybridization of f1MR9,
f1MR28, f1MR30, or f1MR31 with f1MR11 DNA, or by hybridization of
f1MR32 and f1MR24 DNAs (see under "Experimental Procedures"). For
purposes of comparison, 6453-bp f1MR24 (14) is identical to 6413-bp
f1MR11 except for a 40-bp insertion into the EcoRI site of
the latter phage. The f1MR11 map shown in the bottom of the figure
illustrates DNA sites used in this study. Heteroduplexes contained an
unpaired heterology within the V or C DNA strand as indicated. The
location and strand placement of single-strand breaks are indicated by
lines that contact only one DNA strand. Substrates with a
strand break in the C strand are referred to as 5' substrates because
the incision lies 5' to the heterology junction as viewed along the
shorter path between the two sites in the circular molecule.
Heteroduplexes with a discontinuity in the V strand at the gpII site
are referred to as 3' substrates for a similar reason. Sites of
cleavage by restriction endonucleases used in the analysis of repair
intermediates are designated by lines passing through both
strands of the heteroduplex. SspI cleaves the heteroduplex
at six sites, but only those sites employed in this study are shown.
Shaded regions correspond to oligonucleotide probes used for
indirect end labeling analysis.
Substrate specificity of large heterology repair
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Fig. 2.
Strand-specific repair of large I/D
heterologies. Repair in HeLa nuclear extracts (75 µg) was scored
as described under "Experimental Procedures." Heteroduplexes
contained a single-strand break in the complementary DNA strand at the
Sau96I site (114 bp 5' to the heterology as viewed along the
shorter path linking two sites in the circular DNA) and 27, 62, 216, or
993 unpaired heterologies in V or C DNA strands as indicated (Fig. 1).
DNAs were digested with Bsp106 and the appropriate
restriction endonuclease to score heterology repair (Fig. 1), and
products were separated by agarose gel electrophoresis. Repair
occurring on the continuous V strand and the open C strand is
indicated. S and P indicate unrepaired substrate
and products, respectively.
-,
MutS
-, and MutL
-dependent human mismatch repair
system (Ref. 25 and the last two entries of Table I). As described above, a single-strand break located 114 or 797 bp 5' to the heterology supports strand-specific repair of 27-, 62-, and 216-nucleotide heterologies. However, a nick located 170 bp 3' to the heterology was
ineffective in this respect. As can be seen in Table I and Fig.
3, repair of 3'-C27, 3'-V27, and 3'-V216
heteroduplexes was not significantly different from that observed with
the corresponding covalently closed, circular substrates, and no strand
specificity was evident in the low level repair values obtained with
any of these 3'-heteroduplexes.
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Fig. 3.
Dependence of large heterology repair on
placement of the strand break. Repair assays using HeLa nuclear
extract (5 mg/ml) were performed as described under "Experimental
Procedures" except that reactions were scaled up to 50 µl, and
10-µl samples were removed as indicated. V216 heteroduplexes
contained a single-strand break in the C DNA strand at the
Sau96I site 114 bp 5' to the heterology ( ), in the C
strand at the HincII site 797 bp 5' to the heterology (
),
or in the V strand at the gpII cleavage site 170 bp 3' to the
heterology (
). A covalently closed circular heteroduplex (
) was
also tested.
,
, and
(34), whereas
dideoxynucleotides are potent inhibitors of the
DNA polymerase (35,
36), these observations indicate involvement of polymerase
,
,
and/or
in repair of large I/D mismatches. DNA polymerase
has
been previously implicated in the conventional mismatch repair system
(37).
Reaction requirements
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Fig. 4.
5'-Heteroduplexes containing large I/D
heterologies are repaired to covalently closed-circular products.
Repair reactions (see under "Experimental Procedures") were scaled
up to 20 µl (lanes 1-10) or 50 µl (lanes
11-14) and contained 7.5 mg/ml HeLa nuclear extract and 10 µg/ml of the indicated 5'-heteroduplex. With the exception of the
V216(H) heteroduplex, which contained a nick in the C strand at the
HincII site, all DNAs contained a nick in the C strand at
the Sau96I cleavage site (Fig. 1). For lanes
1-10, DNA isolated from reactions was divided into two parts.
Half was linearized with Bsp106 (-), and the other half was
digested with Bsp106 and the restriction endonuclease
(EcoRI for C27 and C62; NheI for V27, V216, and
V216(H)) diagnostic for repair (+). After electrophoresis through
alkaline agarose and transfer to nylon membranes, the complementary DNA
strand was visualized by indirect end labeling by hybridization to 5'
32P-labeled d(ATGGTTTCATTGGTGACGTT) (see under
"Experimental Procedures" and Ref. 25). This synthetic
oligonucleotide, which corresponds to V strand nucleotides 2531-2550,
hybridizes to the 3'-end of the linear form of the C strand produced by
cleavage with Bsp106 (Fig. 1). For the samples shown in
lanes 11-14, DNA isolated from repair reactions was
subjected to native agarose gel electrophoresis in the presence of 0.5 µg/ml ethidium bromide (see under "Experimental Procedures") and
covalently closed relaxed DNA recovered from the gel. Relaxed DNA
samples were then analyzed as described above for lanes
1-10. DNA standards were run on each gel (not shown).
heterodimer (16, 26). Addition of purified MutS
restored a
normal level of repair on the CA I/D heteroduplex to LoVo and MT1
extracts, and MutL
restored repair to the H6 extract. However, 12-, 27-, and 62-nucleotide heterologies were repaired in all extracts, and
the addition of MutS
or MutL
had no significant effect on the
degree to which they were processed. Repair of these larger
heterologies can therefore occur by a pathway that is independent of
MSH2, MSH6, MLH1, and PMS2.
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Fig. 5.
Large heterology repair is independent of
MSH2, MSH6, MLH1, and PMS2. Nuclear extracts from LoVo
(MSH2-/- (28)), MT1
(MSH6-/- (26, 41)), H6
(MLH1-/- (39)), and HEC-1-A
(PMS2-/-,
MSH6-/-(40)) cells were assayed for repair of
I/D heterologies containing 2, 12, 27, or 62 unpaired nucleotides in
reactions containing 50 µg of extract (see under "Experimental
Procedures"). In all cases the unpaired nucleotides were within the C
DNA strand, which also contained a strand break at the
Sau96I site 114 bp 5' to the heterology. Upper
panel, LoVo extract in the absence (black bar) or
presence (cross-hatched bar) of 200 ng of MutS and MT1
extract in the absence (white bar) or presence
(stippled bar) of 200 ng of MutS
. Lower panel,
H6 extract in the absence (black bar) or presence
(cross-hatched bar) of 50 ng of MutL
and HEC-1-A extract
in the absence (white bar) or presence (stippled
bar) of 50 ng of MutL
.
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Fig. 6.
Nature of excision intermediates under
conditions of restricted DNA synthesis as deduced by indirect end
labeling. C26, V216, or single nucleotide T I/D heteroduplexes
contained a C strand nick at the Sau96I site 114 bp 5' to
the heterology. DNAs were incubated with HeLa nuclear extract as
described under "Experimental Procedures" in the presence of
exogenous dNTPs (+dNTPs) or in their absence
(-dNTPs). After linearization with Bsp106,
electrophoresis through alkaline agarose, and transfer to a nylon
membrane, C strand DNA products were probed with 5'
32P-labeled oligonucleotide V2531 (see under
"Experimental Procedures"). This oligonucleotide, which hybridizes
to the 3'-end of the C strand Bsp106 cleavage product, is
shown as a shaded bar in the figure and serves to end-label this
strand. The Sau96I strand break in the heteroduplexes used
is approximately 3.2 kilobase pairs from the probed terminus.
/T\, insertion of T.
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Fig. 7.
High resolution analysis of repair
intermediates under conditions of restricted DNA synthesis. Repair
in HeLa nuclear extracts in the presence of (+dNTPs) or
absence of exogenous dNTPs (-dNTPs) was performed as
described under "Experimental Procedures." C27 and V216
heteroduplexes contained a C strand nick at the Sau96I site
(Fig. 1). An A·T homoduplex containing a Sau96I C strand
nick and a covalently closed form of the C27 (ccc-C27)
heteroduplex were also tested. Reaction products were isolated and
digested with SspI endonuclease, and restriction products
were separated on a 10% denaturing polyacrylamide gel and transferred
to a nylon membrane (see under "Experimental Procedures"). The
membrane was probed with 5' 32P-labeled oligonucleotide
V5216 (viral strand residues 5216-5235), which hybridizes to the C
strand adjacent to the SspI cleavage site at coordinate 5216 (shaded bar on maps at top, also see Fig. 1).
Chain lengths were measured relative to size markers of 250, 416, 542, and 744 nucleotides. The maps at top indicate positions of
interest in the DNAs tested. The strand break in the V216 heteroduplex
is 515 nucleotides from the probed SspI terminus, whereas
this site is 542 nucleotides from the SspI end in the C27
heteroduplex and the A·T homoduplex. Heterologies are shown by
dashed lines; for the C27 heteroduplex, the 27-nucleotide
heterology is processed out in the reaction directed by the C strand
nick, whereas in the V216 substrate, the 216-nucleotide heterology is
repaired into the product. Thus, the SspI segment derived
from unrepaired C27 heteroduplex has a length of 744 nucleotides,
whereas that derived from the repair product has a length of 717 nucleotides. The corresponding SspI fragment chain lengths
for unrepaired and repaired forms of the V216 heteroduplex are 717 and
933 nucleotides, respectively.
DISCUSSION
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ACKNOWLEDGEMENT |
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We thank Elisabeth Penland for culturing the cells used in this work.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by Physician Scientist Award K08 CA71554 from the National Institutes of Health.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Dept. of Biochemistry, Howard Hughes Medical Institute, Rm. 150, Duke University Medical Center, P. O. Box 3711, Durham, NC 27710. Tel.: 919-684-2775; Fax:
919-681-7874; E-mail: modrich{at}biochem.duke.edu.
2 S. E. Corrette-Bennett, B. O. Parker, N. L. Mohlman, and R. S. Lahue, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: I/D, insertion/deletion; V, viral; C, complementary; bp, base pair(s).
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REFERENCES |
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