From the Graduate Center for Toxicology, the
¶ Department of Pathology and Laboratory Medicine, and the
Lucille P. Markey Cancer Center, University of Kentucky Medical
Center, Lexington, Kentucky 40536
Received for publication, October 18, 2002
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Previous work has shown that small DNA loop
heterologies are repaired not only through the mismatch repair (MMR)
pathway but also via an MMR-independent pathway in human cells.
However, how DNA loop repair is partitioned between these pathways and
how the MMR-independent repair is processed are not clear. Using a novel construct that completely and specifically inhibits MMR in HeLa
extracts, we demonstrate here that although MMR is capable of
bi-directionally processing DNA loops of 2, 4, 5, 8, 10, or 12 nucleotides in length, the repair activity decreases with the increase
of the loop size. Evidence is presented that the largest loop that the
MMR system can process is 16 nucleotides. We also show that
strand-specific MMR-independent loop repair occurs for all looped
substrates tested and rigorously demonstrate that this repair is
bi-directional. Analysis of repair intermediates generated by the
MMR-independent pathway revealed that although the processing of looped
substrates with a strand break 5' to the heterology occurred similarly
to MMR (i.e. excision is conducted by exonucleases from the
pre-existing strand break to the heterology), the processing of the
heterology in substrates with a 3' strand break is consistent with the
involvement of endonucleases.
DNA insertion/deletion heterologies (also called DNA loops) are
formed during normal DNA metabolism, particularly within simple repetitive sequences (1). These DNA loop structures are mutagenic if
they are not corrected. It is known that the DNA mismatch repair (MMR)1 system in both
prokaryotes and eukaryotes is capable of correcting small DNA loops
(for reviews, see Refs. 2-7). The Escherichia coli MMR
system can efficiently process DNA insertion/deletion heterologies up
to 7 unpaired nucleotides (8-11). Gel-shift analyses using mismatch
recognition complexes purified from both yeast and human cells have
demonstrated that MutS There is accumulating evidence suggesting that DNA loop repair can be
conducted in a manner independent of MMR (21-26). First, in
vitro functional repair assays have been used to demonstrate that
human cells have a pathway or pathways that can carry out strand-specific repair of looped heterologies using an MMR-independent mechanism. Umar et al. (21) and Littman et al.
(24) have identified a vigorous 5'-nick-directed loop repair activity
of this type using MMR-deficient extracts and DNA substrates containing
5-16 (21) or To understand the nature of DNA loop repair in human cells, we
constructed DNA loop heteroduplexes containing a strand break (either
3' or 5' to the heterology) and a loop of 2, 4, 5, 8, 10, or 12 unpaired nucleotides and characterized their repair in MMR-proficient
and MMR-deficient cell extracts. We demonstrate here that all these
substrates can be processed by both MMR-dependent and
-independent pathways in a strand-specific manner regardless of the
nick orientations and that the MMR-independent repair of the
heterologies with a strand break 3' to the loop appears to involve endonuclease(s).
Cell Lines and Nuclear Extracts--
HeLa cells were
grown in RPMI 1640 media with 5% fetal bovine serum (Hyclone) and 4 mM glutamine. HCT15 and NALM6 cells were grown in RPMI 1640 with 10% fetal bovine serum. HCT116 and HEC-1A cells were grown in
McCoy's 5A media with 10% fetal bovine serum. All cultures
contained 10 units/ml penicillin and 10 µg/ml streptomycin. All cells were grown at 37 °C with a 5% CO2 atmosphere.
Nuclear extracts were prepared from 6 liters of suspension cultures
(HeLa, NALM6) or 25-30 roller bottles of monolayer cultures (HCT15,
HCT116, HEC-1A) using a previously published procedure (27).
DNA Substrates--
The DNA used to create the 5-, 8-, and
12-nucleotide looped substrates is the same as described (22) although
the derivative phage was separately constructed in our lab. The phages
used to create the 10-nucleotide looped substrate were
constructed using the oligonucleotides
5'-AGCTGCAGCCAGCTCGAGTGTGTCTGTGTGTGGC-3' and
5'-CTAGGCCACACACAGACACACTCGAGCTGGCTGC-3' as described (20). Heteroduplex DNA substrates were prepared using DNA derived from f1MR
phage series as described (27, 28). Substrates with a 5' nick were
created by digesting double-stranded phage DNA with Sau96I
(see Fig. 1), whereas the 3' nick was made by the gpII protein as
described (29). The fd:M13 hybrid and fd:fd homoduplex were constructed
in the same manner as the looped heteroduplexes, as described
previously (30).
DNA Loop Repair Assays--
Repair assays were performed as
described previously (20) in 15 µl-reactions containing 75 µg of
nuclear extract and 100 ng of heteroduplex DNA substrate at 37 °C
for 15 min. DNA samples were isolated by phenol extraction and ethanol
precipitation. The amount of repair was quantified either by the
restriction enzyme sensitivity assay (27) or by Southern blot analysis
(20). For the first assay, the DNA samples were digested with two
restriction enzymes: BspDI to linearize all DNA in
the sample and either XhoI or XcmI to score the
amount of repair. The 5, 8, 10 and 12-nucleotide looped sequences
contain the XhoI recognition sequence, and the non-looped
strand contains the XcmI sequence (see Fig. 1). If, for
example, repair of the strand containing the loop occurs such that the
loop sequence is retained in the product, then the product becomes
sensitive to XhoI digestion, and the doubly digested
repaired products can be separated from the singly digested unrepaired products on an agarose gel. Ethidium bromide stained-repair gels were
quantitated using digital photography and Kodak's Image 2.0.2 image
analysis software. Repair was defined as the percent conversion of the
starting substrate from a single 6.4-kb species (cut only by
BspDI) to the doublet of 3.1- and 3.3-kb species (cut by
BspDI and either XhoI or XcmI).
The Southern blot analysis used to score repair was similar to one
described previously (20). Isolated DNA was digested with 2 units each
of Sau96I and NheI (New England Biolabs), and the
digestion products were separated through a 10% denaturing acrylamide
gel (7 M urea, 19:1 acrylamide:bis-acrylamide,
1× TBE) and then transferred to a nylon membrane. Southern blot
analysis was performed essentially as described (31, 32) using
32P end-labeled probes V5744-5768
(5'-TTGATTAGGGTGATGGTTCACGTAG-3') and C5746-5765
(5'-CGTGAACCATCACCCTAATC-3') to probe the complementary and viral
strands, respectively. Autoradiographs were scanned into Kodak Image
2.0.2 image analysis software for quantitation. Repair was defined as
the percent conversion of a strand from the starting substrate length
to the length of the opposite strand (i.e. long to short or
short to long for loop removal or loop addition, respectively).
MMR-inhibited Loop Repair Assays--
MMR-inhibited loop repair
assays were performed as described above using 250 ng of loop
substrate and 75 ng of either fd:fd homoduplex (nonspecific inhibitor)
or fd:M13 hybrid DNA (MMR-specific inhibitor). Repair to the nicked
strand was monitored by restriction enzyme sensitivity using 2 units
each of BseRI and the appropriate scoring enzyme as
described (30). Repair of 5'GT, 5'-2V, and 3'-4C was scored by
restriction digestions as described (20). Quantitation was performed as
described for normal repair assays.
Analysis of Reaction Intermediates--
Loop repair reactions
were performed in the absence of exogenous dNTPs for intermediate
analysis as described (31, 32). All other reaction conditions were
identical to those listed for regular DNA loop repair assays. DNA
products were digested with either 2 units of SspI (Fig. 5)
or 2 units of SspI and Sau96I (Fig. 6) and
subjected to Southern blot analysis as described above using a 6%
acrylamide gel. The probes used were V5216-5235 (5'-ATTGTTCTGGATATTACCAG-3'), C5259-5235
(5'-GAAGAACTCAAACTATCGGCCTTGC-3'), and C5746-5765 (see above).
The Human MMR Pathway Is Capable of Processing at Least
12-Nucleotide Loops--
To determine the upper limits of loop size
that MMR can process, MMR-proficient HeLa extracts were examined for
repair of circular heteroduplexes with different loop sizes either in
the complementary strand or in the viral strand. In addition to loops, these substrates also contain a strand break either 5' (in the C
strand) or 3' (in the V strand) to the loop (Fig.
1). This set of substrates allowed us to
test for bi-directional nick-directed repair of DNA loops. Repair of
these substrates was scored either by the commonly used restriction
enzyme assay (27) or by Southern blot analysis (20). As shown in Fig.
2, HeLa nuclear extracts were capable of
correcting all looped substrates in a strand-specific manner with
repair being targeted in each case to the nicked strand regardless of
whether the nick was 5' or 3' to the heterology. The levels of repair
were fairly constant between substrates containing from 5 to 12 unpaired nucleotides (nt), although there were a few variations (Fig.
2). The repair exhibited a ~30-fold bias toward the nicked strand, as
judged by the fact that the repair rate to the nicked strand was around
15% but only 0.5% to the continuous strand. These results suggest
that the repair of 5-12-nt loops has nick-directed, bi-directional
processing characteristics similar to that seen for mammalian base-base
MMR (31).
To ascertain whether the loop heterologies were processed by the MMR
system, an MMR-specific inhibitor (30) was used in our loop repair
assay to block all MMR activity in HeLa extracts. This inhibitor was
derived from hybridization of bacteriophages fd and M13 DNA and
contains ~200 mismatches per molecule. We have previously shown that
this hybrid can completely inhibit in vitro MMR (30). If the
loop repair observed was indeed conducted by MMR, addition of the
fd:M13 hybrid in the reaction would completely block HeLa extracts from
repairing looped heteroduplexes. These experiments are summarized in
Fig. 3 and Table I. As expected, repair
of a G/T mismatch was completely blocked by addition of fd:M13 heteroduplex DNA. This inhibition
was not due to the presence of extra DNA in the reaction since addition
of the same amount of fd:fd homoduplex DNA did not alter the
amount of repair (Fig. 3). However, the repair of loop substrates of
2-12 nucleotides, although reduced, was not completely abolished under
the same conditions regardless of whether the substrates contained a 5' or a 3' strand break (Fig. 3; Table I). These results suggest that the
repair of loops of 2-12 nucleotides in HeLa extracts contains both
MMR-dependent and -independent components. Although the
amount of MMR-independent repair remained fairly constant (at about
10%, Fig. 3 and Table I) for all 2-12-nt substrates tested, the
amount of MMR-dependent repair was gradually reduced as the
loop size increased (from ~33% for 2 nt down to ~3% for 12 nt,
Table I).
MMR-independent Repair of 2-12-nt Loops Is
Bi-directional--
A previous study has shown that
MMR-independent repair of a DNA loop that is 27 nucleotides in length
or larger in human cells can be only directed by a 5' nick (24). Our
results, however, suggest that both 5' and 3' nicks can direct loop
repair using an MMR-independent mechanism. To confirm that
MMR-independent loop repair of 2-12-nt heterologies is bi-directional,
we performed loop repair in nuclear extracts derived from several well
characterized MMR-defective cell lines; MLH1-deficient
HCT116, PMS2/MSH6-deficient HEC-1A, and
MSH6-deficient HCT15. The results (shown in Fig.
4) indicate that, as seen for HeLa
extracts (Fig. 3 and Table I), all MMR-deficient extracts efficiently
repaired all looped heterologies tested with no significant differences
in the repair rate between the 5' and the 3' substrates. Repair in all
cases was only detected in the nicked strand but not in the continuous
strand (data not shown), indicating that the MMR-independent processing
of the looped substrates is dependent on a nick and can occur in both 5' 5'- and 3'-Nicked Loop Substrates Are Processed by Different
Mechanisms--
To understand how DNA loops are removed by the
MMR-independent pathway, loop repair was carried out using both
MMR-proficient and MMR-deficient extracts under conditions of limited
DNA synthesis, e.g. in the absence of dNTPs. By blocking
repair DNA synthesis, repair events prior to DNA resynthesis are
trapped, and the reaction intermediates can be visualized by Southern
blot analysis (24, 31). Fig. 5 shows the
repair intermediates of four looped substrates (5'-8C, 3'-8C, 5'-12V,
3'-12V) by HeLa or HCT116 nuclear extracts in the absence of dNTPs. For
the 5'-nicked substrates, intermediates accumulated over a broad range
(from the nick to loop to several points beyond the loop) in both
MMR-proficient and -deficient extracts (Fig. 5A), a
phenomenon observed for MMR intermediates (31). This finding suggests
that the removal of the heterology in 5'-looped substrates occurs
through exonucleolytic excision starting from the pre-existing nick and
proceeding toward the loop. However, for the 3'-nicked substrates, the
patterns of repair intermediates in HeLa and HCT116 cells are
significantly different. As shown in Fig. 5B, repair
intermediates in HCT116 cells were confined to an area immediately
surrounding the loop site (lanes 6 and 8), and
this occurs regardless of whether the loop is on the nicked (lane
8) or continuous (lane 6) strand. In comparison with
the results shown in Fig. 5A, it appears that the
MMR-independent pathway uses different mechanisms to process 3'- and
5'-nicked substrates. Interestingly, the bands corresponding to repair
intermediates that immediately flank the loop site were less intense
from reactions that utilized HeLa extracts (lanes 5 and
7) as compared with reactions that utilized MMR-deficient
HCT116 extracts (lanes 6 and 8). In HeLa
extracts, MMR-dependent repair likely dominates
MMR-independent repair such that fewer MMR-independent repair
intermediates appear in the reactions carried out in these extracts. In
addition, some intermediates were recovered between the loop and the
probe in the HeLa extracts, indicative of excision proceeding beyond
the heterology, as seen previously for MMR-dependent
repair (24, 31).
It is also apparent that the HeLa extracts produced bands that flank
the loop for the substrate 3'-8C (Fig. 5B, lane
5) that are less intense as compared with those made from the
3'-12V substrate (Fig. 5B, lane 7). This
observation supports our conclusion made from the functional repair
assay that loop repair activity by the MMR pathway decreases as the
loop size increases (Table I). Thus, reduced processing of the larger
loop by the MMR pathway appears to lead to more repair of the substrate
by the MMR-independent pathway.
Removal of Heterology in 3'-Nicked Substrates Does Not Originate at
the Nick--
The pattern of repair intermediates produced by the
MMR-deficient HCT116 extracts when processing 3' substrates suggest
that removal of 3'-looped heterology by the MMR-independent pathway is
a short patch repair and may involve endonucleases. To further determine the nature of excision on the 3'-nicked substrates, repair
intermediates generated under conditions of limited DNA resynthesis
were double-digested with Sau96I and SspI and
subjected to Southern hybridization using a 32P-labeled
probe located between the nick and the loop site (Fig. 6). If excision occurs from the nick to
the loop, the probe would lose its corresponding binding sequence, and
Sau96I will not be able to digest the repair intermediates
since the region containing its recognition sequence (located between
the nick and the loop) will be single-stranded. The results, shown in
Fig. 6, indicate that this is not the case. Fragments with end points
that flank the loop site are again evident in both the 3'-8C and 3'-12V
substrates (Fig. 6), supporting the idea that incisions were made
around the loop site. These fragments were only detected on the nicked strand, regardless of which strand the loop is on. It is worth noting
that reactions utilizing HeLa extracts (Fig. 6, lanes 1 and
3) again show reduced intensity of bands as compared with those utilizing HCT116 extracts (Fig. 6, lanes 2 and
4). Clearly, the processing of these substrates by the MMR
pathway (from the strand break to the heterology) in HeLa cells left
less substrate available for repair via the MMR-independent
pathway.
In this study, we examined MMR-proficient and MMR-deficient cells
for their ability to process heteroduplexes containing a loop of 2, 4, 5, 8, 10, or 12 nucleotides, which are believed to be substrates for
both MMR-dependent and -independent pathways. We
demonstrate that both pathways can repair these substrates in a
strand-specific manner and rigorously show that DNA loop repair by
MMR-independent pathway occurs bi-directionally. Mechanistic studies
revealed that the MMR-independent pathway processes looped heteroduplexes with a 3' strand break in a way distinct from that of
MMR.
Previous studies in human cells have suggested that both
MMR-dependent and MMR-independent pathways are capable of
repairing DNA loop structures with small insertion/deletion DNA loops
being processed by MMR and large loops being processed by an
MMR-independent system (20-22, 24). However, how large a DNA loop can
be processed by MMR was not clear. In E. coli, it has been
documented that MMR can correct unpaired heteroduplexes containing no
more than 7 nucleotides (8, 10, 11, 34, 35). In yeast, the upper limit
of the loop size increases to 13 nucleotides (18, 19). Given the
overlapping activities of DNA loop repair in human cells, it is
difficult to distinguish loop repair activity conducted by MMR from
that conducted by an MMR-independent pathway. Using fd:M13 hybrid as an
MMR inhibitor, we successfully separated DNA loop repair activities in
human cells. Interestingly, when the loop size increases, the ability
of MMR to process looped heteroduplexes decreases proportionally (Table
I and Fig. 7). Our data show that the MMR
pathway in human cells can process DNA loops containing at least 12 unpaired nucleotides. Although we did not test loop substrates
containing more than 12 nucleotides in this study, the data in
Table I suggest that the MMR system is capable of processing DNA loops
more than 12 nucleotides long. When the percent of repair inhibited by
fd:M13 (i.e. the percent of repair contributed by the
MMR-dependent pathway, Table I) was plotted
versus loop size, we can extrapolate that the loop repair
activity by MMR will drop to zero when the loop size reaches 17 nucleotides (Fig. 7). Therefore, the largest loop size that MMR can
process is likely to be 16 nucleotides with larger loops being
processed by entirely MMR-independent pathways. Our data are consistent
with a previous observation by Wilson et al. (17) that
although human MutS
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(a heterodimer of MSH2 and MSH6) can bind to
small DNA loops with 1-4 nucleotides, whereas MutS
(a heterodimer
of MSH2 and MSH3) can interact with DNA loops up to 24 nucleotides
(12-17). Genetic studies in yeast suggest that the yeast MMR system
may be capable of correcting DNA loops up to 13 unpaired nucleotides
(18, 19). Functional in vitro MMR assays have shown that the
human MMR can repair DNA loops with at least 8 unpaired nucleotides
(20-22). However, the upper limits of loop size that can be processed
by the human MMR system have not been adequately defined.
27 (24) nucleotides in the loop. Although the first study found that a 3' nick could direct repair of
2-5-nucleotide loops via an MMR-independent mechanism (21), the later
study observed no significant repair of this type for 27- or
216-nucleotide loops (24). Functional assays have also identified a
vigorous MMR-independent, 5'-nick-directed loop repair in yeast cells. This activity was identified using yeast lysates to affect
repair on a 27-nucleotide looped substrate but, unlike what has been observed for human extracts, a very weak 3'-nick-directed activity (at
least 2-fold less than the 5'-nick-directed repair) was also identified
(25, 26). Thus, the limits and requirements of MMR-independent loop
repair in eukaryotes still need to be clarified.
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Fig. 1.
DNA loop substrate
configurations. The location of unpaired DNA is indicated by a
lollipop. An example of the base sequence at the site of the
heterology for the 5'- and 3'-8C substrates is shown. The
XcmI recognition sequence is underlined. The
XhoI recognition sequence is in bold. The nick in
the complementary (C) strand, created by Sau96I,
is located 130 bp 5' to the loop site. The nick in the viral
(V) strand, created by gpII, is 186 bp 3' to the loop site.
All loop sequences contain the XhoI recognition sequence,
and non-looped strands contain the XcmI recognition
sequence. Substrates are named by the convention: (nick location)-(loop
size)(looped strand); e.g. 5'-8C has a 5' nick and an
8-nucleotide loop in the C strand.
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Fig. 2.
Strand-specific, nick-directed loop repair in
HeLa nuclear extracts. Loop repair assays were performed using 75 µg of HeLa cell nuclear extract and 100 ng of substrate DNA. Repair
to either strand was scored using the restriction enzyme sensitivity
assay for all substrates except 3'-8V and 3'-12V, for which the
Southern blot assay was used (see "Experimental Procedures").
Values are the average determined from at least three separate assays,
and error bars represent the standard error of the mean. A
G/T mismatch is repaired at ~50% efficiency (data not shown).
Stippled bars represent repair of the nicked strand, and
black bars represent repair of the continuous strand.
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Fig. 3.
Analysis of the MMR-dependent and
-independent contributions to loop repair. Loop repair assays were
performed in reactions containing 250 ng of substrate DNA and 75 µg
of HeLa nuclear extract in the presence of no additional DNA
(black bars), 75 µg of fd homoduplex DNA (white
bars), or 75 µg of fd:M13 heteroduplex DNA (cross-hatched
bars). The extent of nick-directed repair was monitored using the
restriction enzyme sensitivity assay. Values are determined from at
least three separate assays as described previously.
Relative loop repair by MMR-dependent and -independent
pathways
3' and 3'
5' orientations. A subset of these substrates were
also tested for repair by extracts derived from an
MSH2-deficient lymphoblastoid cell line, NALM-6 (33). Again,
nick-directed and bi-directional loop repair was observed in this
MSH2 mutant cell line (data not shown). It is worth
mentioning that the repair rate in these MMR-deficient cells is
comparable with that observed in HeLa extracts supplemented with fd:M13
(compare Fig. 4 with Fig. 3). In sum, we demonstrate using either
MMR-deficient or MMR-inhibited nuclear extracts that 5' and 3' nicks
direct nearly equivalent amounts of repair for several of our loop
substrates. These results strongly suggest that MMR-independent repair
of 2-12-nt looped substrates occurs bi-directionally.
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Fig. 4.
Bi-directional loop repair in
MMR-deficient extracts. Loop repair was performed as described in
the legend for Fig. 2 using 100 ng of looped substrates and 75 µg of
nuclear extracts. Each bar represents the average repair
from two substrates with the same strand break orientation and loop
size but with the loop located on opposing stands (e.g.
group 5'-5 combines results from both 5'-5C and
5'-5V substrates). Black bars represent repair for HeLa,
white bars represent repair for HCT15, cross-hatched
bars represent repair for HCT116, and gray bars
represent repair for HEC-1A extracts. Values are determined from at
least three separate assays for each substrate as described
previously.
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Fig. 5.
Southern blot analysis of loop repair
intermediates. Nuclear extracts (75 µg) from either HeLa or
HCT116 cells were used in the repair assay in the absence of exogenous
dNTPs to block DNA resynthesis. DNA products were purified, digested
with SspI, and separated through a 6% denaturing acrylamide
gel followed by electrotransfer to a nylon membrane. The membrane was
hybridized with a 32P end-labeled oligonucleotide to
visualize the repair intermediates. A diagrammatic representation of
the region visualized is shown on either side of the autoradiographs:
gray boxes, probe binding site; dashed lines,
non-probed strand; black lines, probed strand. As shown in
A, 5'-nicked substrates with the loop in either the nicked
(5'-8C) or the continuous (5'-12V) strand were visualized with probe
V5216-5235. As shown in B, 3'-nicked substrates with the
loop structure in either the nicked (3'-12V) or the continuous (3'-8C)
strand were visualized with probe C5259-5235. Control reactions that
contained dNTPs showed only the full-length and nicked molecules,
corresponding to repaired/unrepaired ligated substrates and unligated
substrates, respectively (data not shown).
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Fig. 6.
Processing of 3'-nicked substrates does not
occur by excision from the pre-existing nick. DNA
resynthesis-inhibited loop repair reactions were performed as described
under "Experimental Procedures." Symbols in the
schematic are defined as described in the legend for Fig. 5. DNA
products were purified and digested with SspI and
Sau96I, separated through a 10% denaturing acrylamide gel,
and then electrotransferred to nylon membranes. The probe used was
C5746-5765, which binds in between the nick and loop site.
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can recognize DNA loops up to 24 nt, only those
with
16 nt can activate the ATPase activity of MutS
.
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Fig. 7.
Estimation of the maximum loop size that can
be processed by human MMR. The amount of relative repair by the
MMR-dependent pathway versus the number of
nucleotides in the unpaired heterology is plotted based on the repair
data (% of repair inhibited by fd:M13) presented in Table I. The
line represents the best fit of the data to a polynomial
expression (order 2), carried out using KaleidaGraph 3.0. (Synergy
Software, Reading, PA). Loop size 0 represents the G/T
mismatch.
Another important finding in this study is the identification and characterization of 3'-nick-directed loop repair by an MMR-independent pathway. Previous studies have shown that MMR-independent loop repair in human cells can be certainly directed by a 5' strand break (21, 22, 24), but whether or not a 3' nick can be used for loop repair was less clear. Whereas repair of heteroduplexes containing up to 5 unpaired nucleotides can be directed by a 3' nick (21), large looped heteroduplexes with a strand break 3' to the heterology are processed poorly with little strand bias (24). In this study, we clearly show that both MMR-depleted HeLa extracts and MMR-deficient extracts can efficiently process looped heteroduplexes containing 5-12 unpaired nucleotides regardless of whether the strand break is 5' or 3' to the heterology (Figs. 3 and 4, Table I). It is clear that there are at least two types of MMR-independent loop repair pathways in human cells: one that is specific for large loops (possibly 17 nucleotides or more) and occurs only through a 5' to 3' orientation and the other that is responsible for repairing small loops (16 nucleotides or less) and can work bi-directionally.
Our analysis of repair intermediates under the condition in which DNA
synthesis is blocked revealed that the MMR-independent repair pathway
processes 3'- and 5'-nicked substrates differently. In MMR,
strand-specific excision of the mispaired base is oriented from the
strand break toward the mismatch, regardless of whether the strand
break is 3' or 5' to the mismatch (31, 36). Thus, intermediates
recovered from DNA synthesis-blocked reactions span a broad range,
normally from the strand break to a point ~200 nucleotides beyond the
heterology (31, 36). The intermediates detected in 5'-looped substrates
processed by either MMR or the MMR-independent pathway are similar to
those observed in mismatched substrates (Fig. 5A). However,
in the case of 3'-nicked substrates, repair intermediates were
recovered in an area immediately flanking either side of the loop (Fig.
5B), suggesting that the removal of the heterology in this
type of substrate involves endonucleases. This hypothesis is supported
by the fact that excision from the nick to the loop did not occur since
the intermediates produced in DNA synthesis-inhibited reactions were
sensitive to Sau96I (~50 nucleotides 5' to the nick site,
Fig. 1) and since probe C5746-5765 (located between the nick and the
loop) could hybridize to the nicked strand and detect repair
intermediates (Fig. 6). These results imply that some portion of DNA
loop repair may be conducted by nucleotide excision repair or a pathway
similar to nucleotide excision repair in which damaged DNA is removed
through double endonucleolytic cleavage (37). However, our preliminary experiments using nuclear extracts from cell lines mutated in XPA, XPB, or XPG ruled out the involvement of nucleotide excision repair in loop repair.2
Therefore, the repair of DNA loops with a 3' nick is likely conducted by an activity independent of not only MMR, but also nucleotide excision repair. However, how the heterology in the 3' substrate is
removed remains to be elucidated.
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ACKNOWLEDGEMENTS |
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We thank Jianxin Wu, Steve Presnell, and Isabel Mellon for helpful discussions and comments on this work, Lu Qiu for nuclear extract preparations, and Huixian Wang for help to construct the phages used to create the 5, 8, and 12-nucleotide looped substrates.
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FOOTNOTES |
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* This work was supported by Grants CA85377 and CA72956 from the National Cancer Institute (to G.-M. L.).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 in part by a training grant (ES-07266) from the National Institute of Environmental Health Sciences. Present address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
** To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536. Tel.: 859-257-7053; Fax: 859-323-2094; E-mail: gmli@uky.edu.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M210687200
2 S. D. McCulloch and G.-M. Li, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: MMR, mismatch repair; nt, nucleotide(s).
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