From the Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon 97331-7301
Received for publication, March 20, 2003 , and in revised form, May 14, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In eukaryotes, mismatches are recognized by heterodimers with different specificities: base mismatches, and one or two looped-out extra nucleotides by MSH2·MSH6 and a range of extrahelical loopouts by MSH2·MSH3 in most eukaryotes (13), a subset of base-base mismatches by an additional MSH2·MSH7 in plants (4). The high similarity between MutS and its eukaryotic homologs suggests conservation of the biochemistry of MMR initiation. In contrast, although MutL-homolog MLH1·PMS2 (MLH1·PMS1 in yeast) heterodimers are required for mismatch correction, neither homologs of UvrD or MutH, nor of any ssDNA-specific exonucleases, have been implicated in eukaryotic MMR. This suggests less evolutionary conservation of post-recognition mechanisms. The absences of DNA-adenine methylation and, apparently, of MutH orthologs, indicate that different nascent-DNA features, perhaps leading-strand 3' ends and lagging-strand 3' or 5' ends, are used to strand-specifically initiate excision toward the mismatch. In mammalian cell extracts, mismatches provoke initiation of excision at pre-existing nicks or gaps in exogenous DNA substrates, with high efficiency and specificity (5, 6).
Despite the apparent divergence of mechanisms of MMR excision, numerous partial-reaction studies with purified proteins suggest general conservation of the initial steps involving MutS and MutL homologs. However, the mechanism by which mismatch-recognition is coupled to site-specific initiation of excision remains controversial. In one class of coupling models, mismatch-bound MutS/MSH dimers are postulated to bind ATP and then to move away along the DNA contour, searching for excision-initiation signals either by ATP hydrolysis-dependent translocation (7), or by ATP binding-dependent diffusional sliding, with hydrolysis occurring later (8). Or searching might be performed by more elaborate sliding complexes, to which other proteins have been recruited (25), Alternatively, MutS homolog proteins complexed with MutL homolog proteins and ATP are proposed to remain at or near mismatches, where they use DNA looping to search through space for excision initiation signals (9, 10).
Mechanisms of subsequent progressive excision are poorly understood,
especially in eukaryotes. Most simply, an exonuclease might progressively
digest one strand of dsDNA, as does the 5'-3' activity of
eukaryotic exonuclease I, which has been firmly implicated in MMR
(11,
12). No similar eukaryotic
3'-5' exonuclease has been linked to MMR, but the recent
observation that aphidicolin inhibits 3'-5' excision points to
exonucleases integral to the DNA replication polymerase and
(13). Remarkably, recent
reports show exonuclease I to be required for both 5'-3' and
3'-5' excision in MMR in cell-free extracts
(11). Other mechanisms might
involve DNA helicases that track along either strand and/or the DNA binding
activities of MutL
(14).
Thus, internal DNA structures that can block protein movement might interfere with coupling of mismatch recognition to initiation of excision, and/or progress of excision toward the mismatch. We have used two recently described techniques (13, 15) to investigate both coupling of mismatch recognition to initiation of excision and the subsequent excision process. First, we have employed our new method for production of gapped circular plasmids (16) to create multiple gaps, into which we ligated not only mismatch-creating oligomers, but also other oligomers containing DNA hairpins, which might be expected to (a) block translocation of putative searching complexes and/or (b) impede progress of excision, in either case preventing mismatch correction. We also measured excision, near the nicks and elsewhere, to distinguish between case a, where no excision would be initiated, and case b, where it would be initiated but blocked before reaching the mismatch. We find a hairpin in the shorter nick-mismatch path to severely inhibit mismatch correction, but not initiation of excision; however, excision proves unable to continue past the hairpin.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Construction of SubstratesAfter the four plasmids described above were digested with endonuclease N.AlwI, the 30-nt oligomers between nicks were removed by heating to 85 °C and reannealing to room temperature, over roughly 60 min, in the presence of a 50-fold excess of a synthetic oligomer complementary to the displaced 30-mer. Gapped plasmids were then purified by benzoylated-naphoylated-DEAE cellulose (BND-cellulose) chromatography, as described previously (16). We ligated oligomer XHMM30a, 5'-ACGTAAGCTTCGAGGTGAATAGGATCATCG, into the N.AlwI gaps to create T/G mismatches (T) whose correction to C/G or T/A would yield a site for XhoI (CTCGAG) or HindIII endonuclease (AAGCTT), respectively, and purified the resulting supercoiled substrates, correspondingly designated s19ASPgt, s19APHgt, s19PPHgt, and s19PAgt. We used endonuclease N.Bpu10I to singly nick s19ASPgt, generating substrate A (Fig. 2). We doubly nicked s19APHgt with endonuclease N.BstNBI and, as described above, created a new N.BstNBI gap in the potential longer nick-mispair path, into which we ligated oligomer HP1, designed to form a hairpin (underlined; 20-bp stem, (CC) loop): 5'-TCTATGCGACGTTAAGGCTGCTACCCTTCTGCCGT(CC)ACGGCAGAAGGGTAGCAGCCCATGGAGAGTCGCTC. The supercoiled product, s19APHgtlhp, was then singly site-specifically nicked with endonuclease N.Bpu10I to produce substrate D (Fig. 2). We similarly inserted hairpin-forming oligomer HP1 into a N.BstNBI gap in the potential shorter nick-mispair path, forming s19PAgtshp, after which N.Bpu10I nicking yielded substrate C (Fig. 2). To produce doubly gapped molecules from substrate s19PPHgt, we digested exhaustively with endonuclease N.BstNBI, removed the 30-nt oligomers by heating and reannealing as described above, and isolated putative doubly gapped (quadruply nicked) products by BND-cellulose chromatography. To remove any DNA containing one or no gaps, we digested with endonuclease HpaI, whose only sites are in the potential gap regions and that cannot cleave ssDNA, then treated with exonuclease V to destroy any linear dsDNA. We ligated oligomer HP1 into both N.BstNBI gaps and purified the covalently closed product, designated s19PPHgtdhp. Single nicking with endonuclease N.Bpu10I yielded substrate B (Fig. 2). To prepare the homoduplex control substrate s19A-SPhm from pUC19ASP, we proceeded as described for substrate A, except that the 30-nt oligomer inserted created a G/C base pair instead of a mismatch, then singly N.Bpu10I-nicked the product, s19ASPgc. To prepare the hairpin-containing homoduplex substrate s19PAgcshp, we ligated oligomer HP1 into a single N.BstNBI gap, producing plasmid pUC19PAgcshp. Although no nicked substrate (analogous to A, B, C, and D and HMA) was derived from pUC19PAgcshp, we designate it HMC in Fig. 6 and in the associated text, for convenience.
|
Verification of Hairpin Structures by Gel ElectrophoresisWe
used endonucleases AflIII and BglII to cut at the respective
unique sites in substrates s19ASPgc (HMA), s19ASPgt (A), s19PPHgtdhp (B),
s19PAgtshp (C), s19APHgtlhp (D), and s19PAgcshp (HMC) (for convenience,
non-nicked substrates are given here the same designations as the mismatched
(A, B, C, and D) or homoduplex (HMA, HMC) substrates generated from them by
N.Bpu10I nicking, as described above under "Construction of
Substrates"). Double cleavage with endonucleases AflIII and
BglII at positions 511 and 22, respectively, generated two products:
a larger ABL fragment (1.7 kbp), identical among all substrates; smaller ABS
fragments (0.48 kbp) containing one (ABS-C, ABS-D, and ABS-HMC), two (ABS-B),
or no (ABS-A and ABS-HMA) hairpins, as shown in
Fig. 3 (left panel).
Treatment with DNA polymerase I Klenow fragment (3'
exo), in the presence of [-32P]dCTP only,
radiolabeled the 3' recessed ends generated by endonuclease
AflIII, presumably with equal specific activities. After phenol
extraction and ethanol precipitation, resuspended fragments were analyzed by
electrophoresis through a 5% native polyacrylamide-gel
(acrylamide:bisacrylamide at 37.5:1) and autoradiography
(Fig. 3, right
panel).
|
Mismatch-Repair Reactions in HeLa Nuclear ExtractsNuclear extracts were prepared from HeLaS3 cells (purchased from the National Cell Culture Center, Minneapolis, MN), as previously described (15). In brief, proteins were extracted with 0.15 M salt from nuclei released from cells broken by homogenization, then precipitated with ammonium sulfate. Standard MMR mixtures (15 µl) contained 100 ng of indicated substrates, 100 µg of nuclear extract, and 0.75 µg of bovine serum albumin, plus the following components at the indicated concentrations: 20 mM Tris-HCl, pH 7.6, 1.5 mM ATP, 1 mM glutathione, four dNTPs, each 0.1 mM, 5 mM MgCl2, 110 mM KCl. Mixtures were incubated at 37 °C for 12 min unless otherwise indicated. Reactions were terminated by the addition of 30 µl of stop solution (25 mM Na2EDTA, 0.67% sodium dodecyl sulfate, and 90 µg/ml proteinase K). After further incubation of mixtures at 37 °C for 15 min, DNA was extracted twice with an equal volume of phenol, and then precipitated with ethanol. DNA was resuspended in H2O and digested with 2 units of AseI endonuclease, plus 2 units of endonuclease HindIII or XhoI, as indicated, and 1 µg of RNase A, at 37 °C for 2 h in 15 µl of digestion buffer (10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol). Digested products were separated by electrophoresis in 1% agarose gels made in 1x TAE buffer (40 mM Tris acetate, 2 mM EDTA). Correction of G/T mismatches to G/C restores a site for XhoI endonuclease, which thus cuts substrates already linearized by AseI endonuclease into 0.9- and 1.3-kbp fragments. DNA bands were visualized by staining with ethidium bromide, then imaged with UVP ImageStore 7500 and analyzed using ImageQuant software. Repair yield equals the ratio of the summed intensities of the 0.9- and 1.3-kbp fragments to the total of this sum plus the intensity of the 2.2-kbp band corresponding to singly cut (uncorrected) DNA.
Analysis of Excision GapsTo freeze excision gaps generated during MMR, exogenous dNTPs were omitted from a standard reaction mixture containing nicked mismatched-DNA substrate or various control substrates. After incubation at 37 °C for 7 min, DNA was extracted and precipitated as described under "MMR Reactions in HeLa Nuclear Extracts," and digested at 37 °C for 2 h with 4 units of AhdI endonuclease plus 1 µg of RNase A in 15 µlof AhdI digestion buffer (20 nM Tris acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate, and 1 mM dithiothreitol). After addition of 0.5 pmol of a particular 32P-labeled oligomer probe (Fig. 5), the mixture was incubated at 85 °C for 5 min, then slowly cooled down to room temperature. Probes annealed to substrates were separated from free oligomers by electrophoresis in 1.2% agarose gels in 1x TAE buffer. After ethidium-stained DNA bands from agarose gels were measured quantitatively, as described under "Mismatch-Repair Reactions in HeLa Nuclear Extracts," gels were dried and the radioactivity in each sample measured by phosphorimaging. Dye-intensity measurements of bands from agarose gels were used to normalize radioactivity measurements for any variations in DNA recovery and loading.
|
Purification of hMutS and Analysis of Electrophoretic
Mobility ShiftHuman hMutS
protein heterodimers were
prepared essentially as described
(17). Briefly, the
3065% ammonium sulfate fraction from HeLaS3 nuclear extracts was passed
through a single-stranded DNA cellulose column and fractions were subsequently
eluted with 1 mM ATP, then chromatographed on a 1-ml Pharmacia HR
5/5 Mono Q column. Purified hMutS
was made 1.0 mg/ml in bovine serum
albumin and 10% (w/v) in sucrose, frozen in liquid nitrogen, and stored at
80 °C. SDS-polyacrylamide gel electrophoresis and Coomassie Blue
staining showed the purified protein to be nearly homogeneous (>98% pure).
For mobility shift analyses, 1 µg (
0.9 pmol) each of potential (not
yet nicked) substrate DNA was treated with endonucleases AflIII and
BglII and radiolabeled, essentially as described above under
"Verification of Hairpin Structures by Gel Electrophoresis" (and
again designated HMA, HMC, and A, B, C, D, for convenience), but with the
following changes. Products were immediately separated by electrophoresis in a
1% agarose gel (without ethidium bromide); respective 0.48-kbp ABS fragments
were cut out of the gels and recovered using a gel-extraction kit (Qiagen) as
recommended by the manufacturer, after a narrow slice of each sample gel was
stained with ethidium bromide to locate the fragments. Radiolabeling of
3'-recessed AflIII and BglII ends employed equal DNA
inputs, measured by PicoGreen (Molecular Probes, Inc.) fluorescence, plus PolI
Klenow (3' exo) and 0.2 µM
[
-32]dCTP and [
-32P]dTTP, plus 16.7
µM dATP and dGTP, to ensure that little ssDNA remained at the
ends; specific radioactivities were nearly identical among the five ABS
fragments. DNA samples were extracted with phenol, precipitated, and
resuspended in TE buffer. Equal amounts of ABS fragments (determined by
radioactivities) were used for band-shift analyses. Binding reactions (25
µl) contained 10 mM Hepes-KOH, pH 7.5, 110 mM KCl, 1
mM dithiothreitol, 0.4% glycerol, 0.25 pmol of purified
hMutS
, 6 fmol (2 ng) of 32P-labeled ABS fragments, and 125
ng of a 1-kbp DNA ladder (catalog number 15615-016, Invitrogen) as nonspecific
competitor. Mixtures were either incubated on ice for 15 min without ATP; or
ATP (200 µM) was added after 10 min and incubation continued on
ice for 5 min. After addition of 5 µl of 50% sucrose, mixtures were
immediately loaded onto a 5% polyacrylamide gel and electrophoresed at 6 V/cm
in TBE buffer (89 mM Tris boric acid, 2 mM EDTA) with
cooling. Protein-DNA complexes and free DNA were visualized by autoradiography
after gel drying, and quantified by phosphorimaging.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We considered it essential to verify the structure and purity of the hairpin- and mismatch-containing structures, which had been prepared by a technically demanding multistep process. To that end, we cleaved the substrates with endonuclease AflIII at position 511, 20 bp right of the right-hand hairpin site, and with endonuclease BglII at position 22, 153 bp left of the left-hand hairpin site, generating the same 1.7-kbp ABL fragment from all substrates, plus 0.48-kbp ABS fragments containing the respective mismatch and hairpin features (Fig. 3, left panel). We then 32P-labeled the 3'-recessed AflIII ends and separated the fragments by electrophoresis in native 5% acrylamide gels (1:37.5 bisacrylamide:acrylamide), as shown in Fig. 3 (right panel). The ABS-HM and ABS-A fragments from the no-hairpin homoduplex and G/T substrates migrated the fastest (lanes HMA and A). The hairpin near the right-hand end of fragment ABS-D retarded it slightly (lane 5), whereas the more interior left-hand hairpin retarded fragment ABS-C more dramatically (lane 4); fragment ABS-B, containing both hairpins, was retarded the most (lane 3). No detectable amounts of other structures were identified, and the sharpness of the ABS-B, ABS-C, and ABS-D bands suggests that the hairpins are stable, i.e. do not migrate as a mixture of partially unwound molecules or other structures, even though one hairpin is only 20 bp from the open end.
Correction of Mismatches in Hairpin-containing SubstratesIn all substrates, mismatch correction via removal of the continuous strand (restoration of HindIII sites) was negligible (Fig. 4, lanes 1, 3, 5, and 7). This confirms the nick requirement for initiation of MMR excision and the absence of adventitious nicks. We previously observed a plateau in mismatch correction after 10 min at 37 °C when the mispair-nick separation was 0.15 kbp (15). Here we measured correction yields (restoration of XhoI sites) after 12 min (Fig. 4, lanes 2, 4, 6, and 8). The 65% yield for the non-hairpin substrate A (Fig. 4, lane 2) remained constant up to 30 min (data not shown). The yield for the longer-path hairpin substrate D was not significantly lower (Fig. 4, lane 8). However, the shorter-path hairpin reduced the yield for substrate C to only 18% (Fig. 4, lane 6), and both hairpins virtually abolished repair of substrate B. Interestingly, extension of the incubation to 25 min increased the yield for substrate C to 27%; further incubation up to 40 min had no effect (data not shown).
|
Differential Effects of Hairpins on Initiation and Progress of MMR ExcisionThe levels of excision gaps generated at various positions during 7 min incubation in nuclear extracts at 37 °C were measured by binding of specific short radiolabeled ssDNA oligonucleotide probes (Fig. 5) to the non-excised strand. We previously showed that the fractions of substrates excised in the absence of exogenous dNTPs were equal to the fractions corrected in complete reactions (15). (Here up to 60% of input substrates were corrected (Fig. 4).) Excision signals shown in Fig. 5 are relative to an internal standard: nonspecific 5'-3' excision of the homoduplex control substrate, measured at a point just beyond the pre-existing nick, in what would be the longer-path direction in a mismatched substrate (probe number 1). As observed previously (15), this nonspecific 5'-3' excision of the homoduplex substrate, although substantial near the nick, was very low at a point 0.8 kbp farther along the longer-path (probe number 2), i.e. it was not processive.
In heteroduplex substrates, 3'-5' excision along the shorter nick-mispair path efficiently generated gaps in the nicked strand just downstream (3060 bp) of the nick (probe number 3), not only in substrates A and D, which were efficiently corrected (Fig. 4), but also in poorly repaired substrate C and even negligibly-repaired substrate B. Thus, despite shorterpath hairpins in the latter two substrates, 3'-5' shorter-path excision toward the mismatch was initiated efficiently in all four cases. At the position of the mismatch, however, excision of the nicked strand (probe number 5) was high for the well repaired substrates A and D, but very low for the poorly repaired substrate C, and near background levels for substrate B; excision signals for the continuous strand at the mismatch position were at background level for all substrates (Fig. 5, probe number 4). Apparently, hairpins in the shorter nick-mispair path allowed initiation of excision, but not its progress. Interestingly, in mismatch substrates B and C, where hairpins blocked the shorter nick-mispair path, longer-path 5'-3' excision of the nicked strand measured at a site 0.8 kbp distal to the nick (probe number 1) was 56-fold higher than the homoduplex control value. Continuation of this mispair-provoked 5'-3' excision around the longer nick-mismatch contour in substrate C might account for the modest excision seen at the mismatch position (probe number 5) after 7 min, and for the significant but subnormal repair yields, 18% at 12 min (Fig. 4) and 27% at 25 min (not shown). Blockage of 5'-3' longer-path excision by the second hairpin in substrate B would account for the negligible excision (probe number 5) and correction (Fig. 4, lane 4) at the mismatch site.
The excision signals shown in Fig. 5 were all measured 7 min after initiation, roughly 1 min past the time at which excision typically reaches a plateau value in these extracts (13, 15), and thus most likely correspond to final yield values. We also assayed excision 2 min after initiation of reactions, in the linear initial phase of the time course (data not shown). For substrate D, whose shorter nick-mispair path was not blocked by a hairpin, the signal at 2 min for excision of nicked strand at the mispair position (probe number 5) was 34% of the signal measured just downstream of the nick (probe number 3). This corresponds to an excision progress rate of 4.7 nt/s, in good agreement with the 5.2 nt/s, measured previously (13). In contrast, signals at the mispair positions in substrate B and C were only 2% of the substrate D initial-excision signal (probe number 3), because of the excision progress blocking hairpins in their shorter nick-mispair paths. Notably, the initial-excision signals (probe number 3) for substrates B and C were 87 and 81% of that for substrate D, confirming that excision initiations at the nicks were at most slightly affected by hairpins between them and the mismatches. Thus, mismatch-provoked excision of the nicked strand, 3'-5' or 5'-3', appears to be blocked by hairpins in the continuous strand, suggesting that some excision components may track along this strand, but excision initiation is not affected. Comparison of probe number 5 versus number 4 signals (Fig. 4) for substrate A, after correction for background signals (substrate HMA), suggests that excision is over 100-fold specific for nicked versus continuous strands. Comparison of probe number 3 signals for substrates A versus HMA suggests that it is 80-fold specific for mismatched versus homoduplex DNA.
The substantial (non-processive) 5'-3' excision at the beginning of the longer nick-mispair path (probe number 2), seen for all mispair-containing substrates, is ascribed to enhanced initiation of 5'-3' excision at the gaps generated by concomitant mispair-provoked 3'-5' excision, as previously demonstrated (15).
Dissociation of hMutS from Hairpin-bounded Mismatched
DNA in the Presence of ATPIn the presence of ATP, both E.
coli MutS and eukaryotic MutS
proteins rapidly dissociate from
mismatch-containing linear DNA
(8,
1820),
but are retained when ends are blocked by streptavidin bound to biotinylated
nucleotides
(1921).
Furthermore, terminal DNA-cruciform or hairpin-like open-end Y structures trap
E. coli MutS protein on mismatched linear DNA in the presence of ATP
(10). To determine whether or
not putative ATP-dependent sliding of hMutS
might be similarly blocked
by a hairpin structure, we analyzed binding of hMutS
to 0.48-kbp ABS
fragments (cut out and purified from substrates with no, one, or two
hairpins), in the absence or presence of ATP. In standard assays of
electrophoretic mobility shift in the absence of ATP, hMutS
shifted
almost all G/T-containing 0.48-kbp (ABS) fragments (cleaved from substrates A,
B, C and D), as expected (Fig.
6, lanes 25); fractions of fragments shifted
ranged from 78 to 89%. Very little of the 0.48-kb fragment from hairpin-free
homoduplex substrate HMA was shifted (Fig.
6, lane 1). Complexes of hMutS
bound to mismatched
(G/T) DNA containing zero, one, or two hairpin(s) migrated at nearly identical
positions; the slight mobility differences closely corresponded to differences
seen for the respective hMutS
-free DNA fragments
(Fig. 3). Thus, the presence of
hairpins did not dramatically decrease the mobility or yield of
hMutS
-DNA complexes, or result in multiple shifted bands, as would have
been the case if hMutS
bound to hairpins as well as to mispairs. To
verify the absence of hMutS
binding to hairpins, we tested the ABS
fragment derived from a homoduplex version of substrate C (HMC), which
contains one hairpin. Binding was negligible, in the presence or absence of
ATP (Fig. 6, lanes 11
and 12). Schofield et al.
(10) similarly demonstrated
lack of binding to cruciform structures by E. coli MutS protein.
Surprisingly, binding to mismatch containing DNA fragments was severely
reduced by the presence of ATP, to
10% for all four G/T-containing
fragments, notably including the two-hairpin ABS fragment (from substrate B)
(Fig. 6, lanes
610). In contrast, E. coli MutS protein was previously
reported to remain on 90-bp mismatch-containing oligomers, blocked at both
ends by cruciforms or open-end Y structures in the presence of ATP
(10). The binding conditions
here were similar to those used previously, so the difference in results may
reflect specific hMutS
and E. coli MutS properties.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inhibition of mismatch correction by a hairpin between the nick and the
mispair, strong when the shorter-path was blocked and complete when both paths
were blocked, indicates that progression of some critical MMR complex(es)
along the DNA contour is (are) blocked. Excision at the mismatch position,
although almost as high (81%) as that just downstream of the nick in the
no-hairpin substrate, was reduced 14-fold by a shorter-path hairpin in the
continuous strand, and a further 4-fold by an additional longer-path hairpin
in the continuous strand. Thus, 3'-5' excision of the nicked
strand may require concomitant tracking of one or more proteins along
(5'-3') the continuous strand. If the proofreading exonuclease(s)
of DNA polymerases and/or
perform 3'-5' excision,
as inhibition of this process by aphidicolin suggests
(13), the polymerase(s)
themselves and/or attached cofactors might be blocked by a continuous strand
hairpin. Exonuclease I, which digests the 5'-3' strand in dsDNA
(2224)
is required for both 3'-5' and 5'-3' MMR excision and
error correction (11,
12). A hairpin in the
5'-3' strand might block exonuclease I in either case, or
interfere with tracking along the non-excised strand by a functional homolog
of E. coli UvrD helicase, or of MutL
, which binds to ssDNA
(14). These alternatives are
not mutually exclusive.
Might hairpins also block mismatch-activated recognition complexes,
including other proteins as well as hMutS, that slide along DNA while
searching for excision-initiation signals? At least two classes of MMR
sliding-complex models have been proposed. In one class, ATP (perhaps its
hydrolysis) is postulated to trigger sliding of MutS/MSH dimers away from
mismatches along DNA contours
(7,
8). In other models, mismatch
containing DNA·MutS
·MutL
ternary complexes,
relatively stable in the presence of ATP, which is in fact essential for their
formation, without ATP hydrolysis being required
(25), are thought to form, and
then be released from the mismatch by proliferating cell nuclear antigen
(PCNA). This release, which is more efficient when MgCl2 is
present, might in these models generate a sliding
PCNA·MutS
·MutL
complex, or both sliding
MutS
and sliding PCNA·MutL
complexes, or these complexes
further associated with other factors needed for MMR excision
(25). In these cases, PCNA
might play two roles even before DNA resynthesis: (i) tethering MutS
to
homoduplex DNA via the known MutS
·PCNA interactions, but
releasing it when a mismatch is encountered, thus enhancing the efficiency of
mismatch recognition, as suggested by other work
(26), and (ii) allowing more
elaborate recognition complexes, perhaps involving as well MutL
,
excision factors, and PCNA itself, to slide toward excision-initiation sites
(25). We find here that
purified hMutS
alone, apparently in contrast to bacterial MutS protein
(9), is not retained on
mismatched DNA blocked at both ends with hairpins in the presence of ATP.
Thus, our experiments do not falsify any hypothesis in which ATP-dependent
sliding of MutS
alone, either directly from a mismatch or after a cycle
of MutL
recruitment and release by PCNA, searches for the
excision-initiation signal, but do demand that the mode of any such sliding be
loose enough to permit movement past the hairpin. Sliding of any more
elaborate complex past a stiff hairpin extending about 70 Å from a
20-Å diameter helix would seem unlikely, virtually impossible if the
complex included PCNA, whose central hole is 34 Å in diameter
(27). Our data clearly do not
falsify the hypothesis of Hsieh co-workers
(9,
10), that mismatch-activated
searching complexes, containing hMutS
, MutL
, and perhaps other
proteins, might remain at or near the mismatch and search for the nick through
space.
Our measurements of longer-path (5'-3') excision gaps at a
point 0.8 kbp around the contour from the nick, presumably well beyond the
range of nonspecific 5'-3' excision, provide a basis for
estimation of shorter- versus longer-path preference in
mispair-provoked excision. In mismatched-substrate A (no hairpins), the
excision signal 0.8 kbp 5' to the nick was only 9 units, after
correction for the nonspecific signal seen at this point in the homoduplex
control substrate (HMA). The similarly corrected excision value 0.3 kbp
3' to the nick (at the mismatch position), was 248 units, of which 9
units or less could have been contributed by longer-path excision. Thus,
shorter-path excision appeared 27-fold higher than longerpath; this may be a
slight overestimate, if mispair-specific 5'-3' excision of 800 nt
or so was not quite complete even after 7 min, or if some 5'-3'
excision complexes had dissociated before reaching the 0.8-kbp point. In any
event, this path specificity estimate is somewhat higher than the
1020-fold estimated previously
(15). Interestingly, the
signal corresponding to low level mismatch-provoked longer-path
(5'-3') excision at the 0.8-kbp points was increased to
2330 units when a hairpin blocked the shorter-path, as if there were
competition between excision in the two directions, and the hairpin made
shorterpath excision slightly less competitive. This signal can easily account
for the (corrected) excision signal of 15 units at the mismatch site in
substrate C. The shorter- versus longer-path preference might be
explained in several ways. Sliding MutS might traverse 0.3 kbp much
more rapidly than 1.9 kbp, with its orientation upon arrival at the nick
dictating excision back along the path just traveled. On the other hand, if
recognition complexes remaining at or near the mismatch directly contacted the
excision-initiation via DNA looping, differences in properties of the longer
versus shorter DNA contours between the two sites, intrinsic to the
respective DNA segments or caused by proteins associated with them, might
favor shorter-path excision.
In their study demonstrating (a) role(s) for human exonuclease I in both
3' and 5' mismatch repair, Genschel et al.
(11) analyzed both
5'-3' and 3'-5' shorter-path excision by measuring
loss of cutting by endonuclease NheII at a site closely adjacent to a
G/T mispair, and longer-path excision by electrophoretically estimating the
length of indirectly end-labeled strands, co-linear with the nicked strands of
the substrate, that extended from a uniform end at an interior restriction
site to a variable (gap-terminated) end for as much as 3.3 kbp (to the
original nick site); shorter-path excision gaps were mapped as well. There are
qualitative parallels between their observations and those reported here.
However, the differences in specificities of excision signals for heteroduplex
versus homoduplex DNA, typically 510-fold and occasionally
higher for Genschel et al.
(11) but 7580-fold here
(Fig. 5), and the reported
relatively low ratios between G/T-provoked excision yields in MMR-proficient
versus hMuts-deficient extracts, typically 34-fold,
suggest the presence of significant background activities, making quantitative
comparisons difficult. Although their gap-mapping assay is not sensitive
enough to measure the substantial 5'-3' longer-path excision of
homoduplex substrates reported here and earlier, both studies show that this
nonspecific excision is negligible a few hundred nucleotides farther along the
longer (5'-3') path, i.e. is non-processive. In the case
of G/T substrates, both groups see substantial longer-path excision, high just
downstream of the nick, but low 0.8 kbp farther along in our case, detectable
roughly 0.1 kbp downstream in their case.
Because we previously found that in nuclear extracts 5'-3'
excision of a plasmid substrate was initiated at a pre-existing 30-nt gap
roughly 2.5 times as efficiently as at a nick, we suggested that a gap
generated by 3'-5' shorter-path excision toward a mismatch might
concomitantly stimulate longer-path 5'-3' excision
(15). Our observations here
that a small but significant fraction of longer-path excision extends all the
way around the longer-path, most likely accounting for the low but appreciable
mismatch correction when the shorter-path (only) is blocked by a hairpin,
suggest that some longer-path excision is in fact specifically
mismatch-provoked. The relationship between this mismatch-provoked excision in
nuclear extracts and the low but highly extended 5'-3' longer-path
mismatch-dependent excision seen by Genschel et al.
(11) using only purified human
MutS and exonuclease I is not clear. In 5' heteroduplex
substrates both groups observed very little 3'-5' longer-path
excision (11,
15). Whether any longer-path
3'-5' excision might extend far enough to accomplish significant
mismatch correction when the shorter- (5'-3') path is blocked
remains to be determined.
The experiments described here thus implicate a translocating complex in MMR excision, but do not rule out some searching through space or sliding/translocating models for mismatch-nick coupling. This work points toward further experiments, more stringent blocks, testing for putative multiprotein complexes that might be trapped, for example, and illustrates new techniques for constructing and testing complex mismatch-repair substrates.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed. Tel.: 541-737-1777; Fax:
541-737-0497; E-mail:
haysj{at}bcc.orst.edu.
1 The abbreviations used are: MMR, mismatch repair; hMuts, human
MSH2·MSH6 protein heterodimers; hMutL
, human MLH1·PMS2
protein heterodimer; dsDNA, double-strand DNA; ssDNA, single-strand DNA; nt,
nucleotides; kbp, one thousand DNA base pairs; PCNA, proliferating cell
nuclear antigen.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|