Human mismatch repair: reconstitution of a nick-directed bidirectional reaction

Abstract

Bidirectional mismatch repair directed by a strand break located 3' or 5' to the mispair has been reconstituted using seven purified human activities: MutSalpha, MutLalpha, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase delta. In addition to DNA polymerase delta, PCNA, RFC, and RPA, 5'-directed repair depends on MutSalpha and EXOI, whereas 3'-directed mismatch correction also requires MutLalpha. The repair reaction displays specificity for DNA polymerase delta, an effect that presumably reflects interactions with other repair activities. Because previous studies have suggested potential involvement of the editing function of a replicative polymerase in mismatch-provoked excision, we have evaluated possible participation of DNA polymerase delta in the excision step of repair. RFC and PCNA dramatically activate polymerase delta-mediated hydrolysis of a primer-template. Nevertheless, the contribution of the polymerase to mismatch-provoked excision is very limited, both in the purified system and in HeLa extracts, as judged by in vitro assay using nicked circular heteroplex DNAs. Thus, excision and repair in the purified system containing polymerase delta are reduced 10-fold upon omission of EXOI or by substitution of a catalytically dead form of the exonuclease. Furthermore, aphidicolin inhibits both 3'- and 5'-directed excision in HeLa nuclear extracts by only 20-30%. Although this modest inhibition could be because of nonspecific effects, it may indicate limited dependence of bidirectional excision on an aphidicolin-sensitive DNA polymerase.

Fig. 1

Properties of recombinant human DNA polymerase δ. (A) Purified recombinant DNA polymerase δ was subjected to electrophoresis through a 10% SDS gel and stained with Coomassie blue (left panel). Positions of the four subunits of the protein (p125, p68, p50 and p12) are shown. Presence of each of these subunits in the isolated recombinant and the partially purified HeLa preparation was confirmed by Western analysis with antibodies against the individual subunits (right panel). (B) DNA synthesis on a poly(dA)•oligo(dT) template-primer was determined as described under Materials and Methods using 80 fmol of recombinant polymerase δ (, ○), or a comparable amount (as judged by quantitative Western blot) of HeLa polymerase δ (■, □). Reactions were performed in the absence (○, □) or presence (, ■) of 580 fmol of PCNA. (C) DNA synthesis by recombinant polymerase δ was determined on a circular 6440 base pair DNA containing a 220 nucleotide gap (Materials and Methods). The complete reaction contained DNA polymerase δ, RFC, PCNA, and RPA (); RPA omitted (■); PCNA omitted (□); RFC omitted (○); complete plus 90 μM aphidicolin (X). Complete gap repair would correspond to 5.3 pmol of DNA synthesis.

Fig. 2

Reconstitution of bidirectional mismatch repair with purified human proteins. (A) The assay for mismatch repair utilizes a 6,440 base pair circular heteroduplexes containing a G-T mismatch within overlapping restriction sites for HindIII and XhoI (6,23) and a site-specific nick in the complementary DNA strand 141 base pairs 3′ to the mismatch (shorter path, 3′-G-T) or 128 base pairs 5′ to the mispair (5′-G-T). Since repair occurring on the incised strand renders the DNAs sensitive to HindIII, cleavage of repaired molecules with HindIII and ClaI yields two rapidly migrating fragments (arrows in panels B and C). The heteroduplexes contain a NheI site 5 base pairs from the mismatch. Because the gaps produced by mismatch-provoked excision span this site, conversion of the heteroduplex to an NheI-resistant form provides a simple assay for mismatch-provoked excision (23,25). (B) Reactions (Materials and Methods) contained a 3′-G-T or 5′-G-T heteroduplex, MutSα, MutLα, and EXOI. DNA polymerase δ, PCNA, RFC, RPA, and aphidicolin were present as indicated. (C) Reactions as in panel B contained DNA polymerase δ, PCNA, RFC, and RPA. MutSα, MutLα, and EXOI were included as indicated. Although not shown, no detectable mismatch rectification was observed on the continuous strand of 3′- or 5′-heteroduplexes when incubated with complete, seven component system.

Fig. 3

Dependence of excision and repair DNA synthesis on DNA polymerase δ. (A) Reactions containing 5′-G-T () or 3′-G-T (■) heteroduplexes (Fig. 2A) were performed as described under Materials and Methods except that hydrolytically defective EXOI D173A was substituted for EXOI as indicated, DNA polymerase δ concentration was varied as shown, and dNTPs were omitted from those reactions used to score excision. Mismatch repair (solid lines) was scored by cleavage with ClaI and HindIII. Mismatch-provoked excision was determined by cleavage with ClaI and NheI (dashed and hyphenated lines indicate excision occurring in the presence of wild type EXOI or hydrolytically defective EXOI D173A, respectively). (B) Reactions using a 5′-heteroduplex or an otherwise identical G•C homoduplex control (lanes 1 and 4) were performed as in panel A in the absence (lanes 1-3) or presence of dNTPs (lanes 4-8). DNA polymerase δ concentration was varied as indicated. Reaction products were digested with SspI, denatured, resolved by electrophoresis through 1.8% alkaline agarose, and probed with ³²P-labeled oligonucleotide V5891 (Materials and Methods). As illustrated schematically in the diagram on the right, this oligonucleotide hybridizes to the 5′-end of the incised strand of the SspI fragment that contains the mismatch. This probe thus permits visualization of the fate of the 3′-terminus at the strand break. (C) Reactions in the absence (lanes 1-5) or presence of dNTPs (lanes 6-10) and indicated amounts of DNA polymerase δ were performed as in panel B except that a 3′-G-T heteroduplex or the corresponding A•T homoduplex control, (lanes 1 and 6) were used. After cleavage with BstYI and denaturation, products were separated and probed with ³²P-labeled oligonucleotide V194 (Materials and Methods) to monitor the fate of the 3′-termini in the incised strand of the heteroduplex.

Fig. 4

Fine mapping of excision and re-synthesis tracts. (A) Reactions containing 200 μg HeLa nuclear extract and 96 fmol (400 ng) 3′-G-T heteroduplex, 3′-A•T homoduplex, 5′-G-T heteroduplex, or 5′-G•C homoduplex DNA as indicated, were carried out in 40 μl volume under excision or repair conditions (Materials and Methods) for 10 min at 37°C. Repair reactions (lanes 6-9 and 14-17) contained 100 μM each of dATP, dGTP, TTP, 90 μM dCTP, and 10 μM dCTP[αS]. Excision reactions (lanes 2-5 and 10-13) were performed in a similar manner except that dNTPs were omitted and reactions were supplemented with 90 μM aphidicolin to suppress DNA synthesis supported by endogenous nucleotides present in extracts (20). After digestion with SspI, DNA samples were untreated (lanes 2-9) or treated with 5 mM iodine in ethanol (lanes 10-17), products resolved on a denaturing gel and probed with 5′-³²P-labeled oligonucleotide V5891 (Materials and Methods). As illustrated schematically in the diagrams to each side of the gel, this probe hybridizes to the incised complementary strand adjacent to the SspI site and can be used to locate 3′-termini on both 3′- and 5′-DNA substrates. Numerical coordinates shown in the diagram on the right indicate fragment size in nucleotides (left axis) or distance from the mismatch (right axis). The 744 nucleotide species is the product of ligation. Lanes 1 and 18 are markers. (B) Reactions (40 μl) containing 200 ng (780 fmol) MutSα, 140 ng (780 fmol) MutLα, 8 ng (84 fmol) EXOI, 400 ng (3600 fmol) RPA, 50 ng (580 fmol homotrimer) PCNA, 130 ng (440 fmol) RFC, 40 ng (160 fmol) DNA polymerase δ and 200 ng (48 fmol) 3′-G-T heteroduplex, 3′-A•T homoduplex, 5′-G-T heteroduplex, or 5′-G•C homoduplex DNA (Materials and Methods) were incubated for 8 min at 37°C. Repair reactions (lanes 6-9 and 14-17) contained 100 μM each of dATP, dGTP, TTP, 90 μM dCTP, and 10 μM dCTP[αS]. Excision reactions (lanes 2-5 and 10-13) were performed in a similar manner except that dNTPs were omitted. Samples were processed and analyzed as in (A). Lanes 1 and 18 are markers.

Fig. 4

Fine mapping of excision and re-synthesis tracts. (A) Reactions containing 200 μg HeLa nuclear extract and 96 fmol (400 ng) 3′-G-T heteroduplex, 3′-A•T homoduplex, 5′-G-T heteroduplex, or 5′-G•C homoduplex DNA as indicated, were carried out in 40 μl volume under excision or repair conditions (Materials and Methods) for 10 min at 37°C. Repair reactions (lanes 6-9 and 14-17) contained 100 μM each of dATP, dGTP, TTP, 90 μM dCTP, and 10 μM dCTP[αS]. Excision reactions (lanes 2-5 and 10-13) were performed in a similar manner except that dNTPs were omitted and reactions were supplemented with 90 μM aphidicolin to suppress DNA synthesis supported by endogenous nucleotides present in extracts (20). After digestion with SspI, DNA samples were untreated (lanes 2-9) or treated with 5 mM iodine in ethanol (lanes 10-17), products resolved on a denaturing gel and probed with 5′-³²P-labeled oligonucleotide V5891 (Materials and Methods). As illustrated schematically in the diagrams to each side of the gel, this probe hybridizes to the incised complementary strand adjacent to the SspI site and can be used to locate 3′-termini on both 3′- and 5′-DNA substrates. Numerical coordinates shown in the diagram on the right indicate fragment size in nucleotides (left axis) or distance from the mismatch (right axis). The 744 nucleotide species is the product of ligation. Lanes 1 and 18 are markers. (B) Reactions (40 μl) containing 200 ng (780 fmol) MutSα, 140 ng (780 fmol) MutLα, 8 ng (84 fmol) EXOI, 400 ng (3600 fmol) RPA, 50 ng (580 fmol homotrimer) PCNA, 130 ng (440 fmol) RFC, 40 ng (160 fmol) DNA polymerase δ and 200 ng (48 fmol) 3′-G-T heteroduplex, 3′-A•T homoduplex, 5′-G-T heteroduplex, or 5′-G•C homoduplex DNA (Materials and Methods) were incubated for 8 min at 37°C. Repair reactions (lanes 6-9 and 14-17) contained 100 μM each of dATP, dGTP, TTP, 90 μM dCTP, and 10 μM dCTP[αS]. Excision reactions (lanes 2-5 and 10-13) were performed in a similar manner except that dNTPs were omitted. Samples were processed and analyzed as in (A). Lanes 1 and 18 are markers.

Fig. 5

T7 DNA polymerase cannot substitute for DNA polymerase δ in mismatch repair. (A) Reactions (20 μl) were carried out in two stages. Stage I reactions containing 24 fmol 5′-G-T or 3′-G-T heteroduplex DNA were performed as described (Materials and Methods) in the presence or absence of 80 fmol DNA polymerase δ as indicated. Incubation was for 8 min at 37°C. For stage II incubations, reactions corresponding to lanes 2 and 4 were deproteinized by treatment with proteinase K, phenol, and chloroform, collected by ethanol precipitation, and then incubated with RFC, PCNA, DNA polymerase δ, and RPA as in stage I except that MutSα, MutLα, and EXOI were omitted. To score repair, DNA products from all reactions were digested with HindIII and ClaI, subjected to electrophoresis through 1% agarose, and visualized with ethidium bromide. Arrows indicate repair products. (B) Stage I reactions were performed as in panel A except that T7 DNA polymerase (4 fmol) was substituted for polymerase δ, and RFC and PCNA were present only as indicated. After deproteinization, products corresponding to lanes 2, 4, 6, and 8 were incubated in stage II reactions with T7 DNA polymerase and RPA in the absence of other proteins. Repair was scored as in panel A.

Fig. 6

Effects of aphidicolin on the DNA polymerase δ exonuclease. Reactions (Materials and Methods) contained DNA polymerase δ and a 5′-³²P-labeled oligonucleotide hybridized single-stranded circular f1 MR3 DNA such that the 3′-terminus was either perfectly paired or contained an unpaired dinucleotide (Materials and Methods). RFC, PCNA, and aphidicolin were present as indicated, and reactions were sampled as a function of time. Arrows in the upper panel indicate products produced by removal of one or both unpaired nucleotides.