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. 2002 Apr;22(7):2037-46.
doi: 10.1128/MCB.22.7.2037-2046.2002.

Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A

Cecilia Ramilo  1 , Liya Gu, Shuangli Guo, Xiping Zhang, Steve M Patrick, John J Turchi, Guo-Min Li
Affiliations

Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A

Cecilia Ramilo et al. Mol Cell Biol. 2002 Apr.

Abstract

DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.

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Figures

FIG. 1.
FIG. 1.
DNA substrates. The heteroduplexes were constructed from f1MR phage series (see Materials and Methods) to contain (i) a G-T mismatch and a strand break (at the Sau96I site) in the complementary strand 125 bp 5′ to the mismatch (5′ G-T) or (ii) a TG dinucleotide insertion-deletion mismatch with a strand break 181 bp 3′ to the mispair (3′ /TG\ substrate). The mismatches were within overlapping recognition sites for two restriction endonucleases so that the DNA is resistant to hydrolysis by both enzymes. Preferential repair on the nicked strand renders the repair products sensitive to HindIII (in the case of the 5′ G-T substrate) or BglI (in the case of the 3′ /TG\ substrate). A homoduplex (5′ A-T) was also constructed in a manner identical to the construction of the 5′ G-T substrate. V and C, viral strand and cDNA strand, respectively. C1, C2, V1, and V2 represent oligonucleotide probes (solid bars) complementary to the Bsp106-flanking sequences in the complementary (C1 and C2) and viral (V1 and V2) strands.
FIG. 2.
FIG. 2.
Reconstitution of MMR in vitro. (A) Fractionation of a HeLa nuclear extract into three components required for MMR. (B) Reconstitution of MMR requires SS1, SS2, and FII. The DNA substrate (100 ng of the 5′ G-T heteroduplex) was incubated for 15 min at 37°C in the reaction buffer with fractions as indicated. Amounts of protein used were 15 μg of SS1, 1.5 μg of SS2, or 30 μg of FII. DNA was extracted, treated with HindIII and Bsp106, electrophoresed on an agarose gel, and visualized by ethidium bromide staining under UV illumination. ND, not detectable.
FIG. 3.
FIG. 3.
hRPA substitutes for SS2. (A) Reconstitution of MMR in SS1-FII by phosphocellulose P-11 fractions. MMR assays were performed on reaction mixtures containing 100 ng of G-T heteroduplex, 15 μg of SS1, 30 μg of FII, and 2 μl of a P-11 fraction as indicated. (B) SDS-PAGE of a MonoQ fraction that contains two major polypeptides with molecular sizes of 70 and 34 kDa and restores MMR to SS1-FII. Fraction SS2 was purified by Pharmacia HR 5/5 MonoQ column chromatography (see Materials and Methods), and the activity complementing SS1-FII was detected by an MMR assay (data not shown), electrophoresed on an SDS-12% polyacrylamide gel, and stained with Coomassie brilliant blue. (C) Western blot analyses of P-11 fractions. P-11 fractions (50 μl) were precipitated by an equal volume of 20% trichloroacetic acid and neutralized with Tris base prior to electrophoresis on an SDS-12% PAGE gel. Proteins were transferred to a nitrocellulose membrane and analyzed by Western blot analysis using antibodies against the 70- or the 34-kDa subunit of hRPA. (D) hRPA substitutes for SS2 in MMR. When present, hRPA was at 1.0 μg.
FIG. 4.
FIG. 4.
SS1 carries out ssDNA incision. Repair reactions were performed as described in the legend to Fig. 2B. DNA products were recovered and digested with the indicated restriction enzyme. (A) Analysis of the repair product on a native agarose gel. DNA was cleaved with Bsp106 (lanes 1 and 2) or BseRI (lanes 3 and 4). (B) Analysis of the repair product on an alkaline agarose gel. DNA was digested with Bsp106 and analyzed by Southern blotting. The membrane was hybridized with the 32P-labeled oligonucleotide 5′-AACGTCACCAATGAAACCAT-3′ (probe V1 [solid bar]), which is complementary to the 3′ flanking sequence of Bsp106 on the viral strand. Lane 4 (MR1) contained double-stranded f1MR1 DNA digested with Bsp106 and Sau96I, which served as a marker to locate the single-stranded nick of the viral strand by SS1. APD, aphidicolin; HL, HeLa nuclear extract; MR1, flMR1 dsDNA.
FIG. 5.
FIG. 5.
Mismatch-provoked and nick-directed excision by SS1. Reactions were performed by incubating a DNA heteroduplex (5′ G-T) or homoduplex (5′ A-T) with SS1 (30 μg) or HeLa nuclear extracts (HL, 50 μg) containing 100 nM aphidicolin (APD), as indicated. Products were cleaved with Bsp106, electrophoresed on alkaline agarose gels (1.5%), and transferred to nylon membranes as described in Materials and Methods. The membrane was hybridized with probe C1 (5′-32P-ATGGTTTCATTGGTGACGTT-3′) (lanes 1, 2, 3, 7, and 8) or probe C2 (5′-32P-GATTCTGTCGCTACTGATTAC-3′) (lanes 4, 5, 6, 9, and 10), which are complementary to the 5′ and 3′ flanking sequences of the Bsp106 site in the complementary strand, respectively. HL, HeLa nuclear extract; ITM, excision intermediates; solid bars, 32P-labeled probes.
FIG. 6.
FIG. 6.
Role of hRPA in MMR. The DNA substrate was incubated with SS1 in the presence or absence of hRPA or SSB, as indicated, and products were analyzed by Southern blot hybridization. The membrane was probed with 32P-labeled V1 (5′-AACGTCACCAATGAAACCAT-3′) (A) or 32P-labeled C1 (5′-ATGGTTTCATTGGTGACGTT-3′) (B). APD, aphidicolin; HL, HeLa nuclear extract; Blank, reaction containing no protein; solid bars, 32P-labeled probes.
FIG. 7.
FIG. 7.
FII contains gap-filling and ligase activities required for MMR. (A) FII carries out gap-filling and ligase reactions. The DNA substrate was incubated with SS1, hRPA, and FII as indicated, and products were analyzed by Southern hybridization using probe C1 (5′-32P-ATGGTTTCATTGGTGACGTT-3′). (B) Conversion of nicked circular DNA into supercoiled DNA by FII. The 5′ G-T substrate was incubated with E. coli DNA ligase (lane 4) at a final concentration of 4 U/μg of DNA or with 30 μg of FII (lane 5) at 20°C for 2 h in the presence of ethidium bromide (0.29 nM/μg of DNA). Double-stranded f1MR1 replicative-form DNA with (lane 2) or without (lane 1) Sau96I digestion was used as a reference for linear (LN), supercoiled (SC), and open circular (OC) DNA, as indicated. (C) DNA polymerase activity in FII. FII was assayed for DNA polymerase activity as described in Materials and Methods. Reaction mixtures contained 7.5 (lanes 1 and 4), 15 (lanes 2 and 5), or 30 (lanes 3 and 6) μg of protein and were incubated for 15 min at 37°C. To test for inhibition by monoclonal antibody SJK132-20 (MoAb), reaction mixtures were incubated with 1 μg of antibody for 15 min on ice prior to initiation of the polymerase reactions (lanes 4 to 6). (D) The gap-filling activity of FII requires hRPA. The DNA heteroduplex was incubated with SS1-hRPA, and the repair intermediates were purified by phenol extraction and ethanol precipitation. Intermediates were incubated with FII in the presence or absence of hRPA or SS1. Products were either analyzed by Southern blotting using the 32P-labeled probe C1 (5′-ATGGTTTCATTGGTGACGTT-3′) after cleavage by Bsp106 (lanes 1 to 5) or digested by Bsp106 and HindIII, followed by agarose gel electrophoresis as described in the legend to Fig. 2B to determine if the mispaired base is removed during the gap-filling reaction (lanes 6 to 8). APD, aphidicolin; ITM, excision intermediates; solid bar, 32P-labeled probe.
FIG. 7.
FIG. 7.
FII contains gap-filling and ligase activities required for MMR. (A) FII carries out gap-filling and ligase reactions. The DNA substrate was incubated with SS1, hRPA, and FII as indicated, and products were analyzed by Southern hybridization using probe C1 (5′-32P-ATGGTTTCATTGGTGACGTT-3′). (B) Conversion of nicked circular DNA into supercoiled DNA by FII. The 5′ G-T substrate was incubated with E. coli DNA ligase (lane 4) at a final concentration of 4 U/μg of DNA or with 30 μg of FII (lane 5) at 20°C for 2 h in the presence of ethidium bromide (0.29 nM/μg of DNA). Double-stranded f1MR1 replicative-form DNA with (lane 2) or without (lane 1) Sau96I digestion was used as a reference for linear (LN), supercoiled (SC), and open circular (OC) DNA, as indicated. (C) DNA polymerase activity in FII. FII was assayed for DNA polymerase activity as described in Materials and Methods. Reaction mixtures contained 7.5 (lanes 1 and 4), 15 (lanes 2 and 5), or 30 (lanes 3 and 6) μg of protein and were incubated for 15 min at 37°C. To test for inhibition by monoclonal antibody SJK132-20 (MoAb), reaction mixtures were incubated with 1 μg of antibody for 15 min on ice prior to initiation of the polymerase reactions (lanes 4 to 6). (D) The gap-filling activity of FII requires hRPA. The DNA heteroduplex was incubated with SS1-hRPA, and the repair intermediates were purified by phenol extraction and ethanol precipitation. Intermediates were incubated with FII in the presence or absence of hRPA or SS1. Products were either analyzed by Southern blotting using the 32P-labeled probe C1 (5′-ATGGTTTCATTGGTGACGTT-3′) after cleavage by Bsp106 (lanes 1 to 5) or digested by Bsp106 and HindIII, followed by agarose gel electrophoresis as described in the legend to Fig. 2B to determine if the mispaired base is removed during the gap-filling reaction (lanes 6 to 8). APD, aphidicolin; ITM, excision intermediates; solid bar, 32P-labeled probe.
FIG. 7.
FIG. 7.
FII contains gap-filling and ligase activities required for MMR. (A) FII carries out gap-filling and ligase reactions. The DNA substrate was incubated with SS1, hRPA, and FII as indicated, and products were analyzed by Southern hybridization using probe C1 (5′-32P-ATGGTTTCATTGGTGACGTT-3′). (B) Conversion of nicked circular DNA into supercoiled DNA by FII. The 5′ G-T substrate was incubated with E. coli DNA ligase (lane 4) at a final concentration of 4 U/μg of DNA or with 30 μg of FII (lane 5) at 20°C for 2 h in the presence of ethidium bromide (0.29 nM/μg of DNA). Double-stranded f1MR1 replicative-form DNA with (lane 2) or without (lane 1) Sau96I digestion was used as a reference for linear (LN), supercoiled (SC), and open circular (OC) DNA, as indicated. (C) DNA polymerase activity in FII. FII was assayed for DNA polymerase activity as described in Materials and Methods. Reaction mixtures contained 7.5 (lanes 1 and 4), 15 (lanes 2 and 5), or 30 (lanes 3 and 6) μg of protein and were incubated for 15 min at 37°C. To test for inhibition by monoclonal antibody SJK132-20 (MoAb), reaction mixtures were incubated with 1 μg of antibody for 15 min on ice prior to initiation of the polymerase reactions (lanes 4 to 6). (D) The gap-filling activity of FII requires hRPA. The DNA heteroduplex was incubated with SS1-hRPA, and the repair intermediates were purified by phenol extraction and ethanol precipitation. Intermediates were incubated with FII in the presence or absence of hRPA or SS1. Products were either analyzed by Southern blotting using the 32P-labeled probe C1 (5′-ATGGTTTCATTGGTGACGTT-3′) after cleavage by Bsp106 (lanes 1 to 5) or digested by Bsp106 and HindIII, followed by agarose gel electrophoresis as described in the legend to Fig. 2B to determine if the mispaired base is removed during the gap-filling reaction (lanes 6 to 8). APD, aphidicolin; ITM, excision intermediates; solid bar, 32P-labeled probe.
FIG. 7.
FIG. 7.
FII contains gap-filling and ligase activities required for MMR. (A) FII carries out gap-filling and ligase reactions. The DNA substrate was incubated with SS1, hRPA, and FII as indicated, and products were analyzed by Southern hybridization using probe C1 (5′-32P-ATGGTTTCATTGGTGACGTT-3′). (B) Conversion of nicked circular DNA into supercoiled DNA by FII. The 5′ G-T substrate was incubated with E. coli DNA ligase (lane 4) at a final concentration of 4 U/μg of DNA or with 30 μg of FII (lane 5) at 20°C for 2 h in the presence of ethidium bromide (0.29 nM/μg of DNA). Double-stranded f1MR1 replicative-form DNA with (lane 2) or without (lane 1) Sau96I digestion was used as a reference for linear (LN), supercoiled (SC), and open circular (OC) DNA, as indicated. (C) DNA polymerase activity in FII. FII was assayed for DNA polymerase activity as described in Materials and Methods. Reaction mixtures contained 7.5 (lanes 1 and 4), 15 (lanes 2 and 5), or 30 (lanes 3 and 6) μg of protein and were incubated for 15 min at 37°C. To test for inhibition by monoclonal antibody SJK132-20 (MoAb), reaction mixtures were incubated with 1 μg of antibody for 15 min on ice prior to initiation of the polymerase reactions (lanes 4 to 6). (D) The gap-filling activity of FII requires hRPA. The DNA heteroduplex was incubated with SS1-hRPA, and the repair intermediates were purified by phenol extraction and ethanol precipitation. Intermediates were incubated with FII in the presence or absence of hRPA or SS1. Products were either analyzed by Southern blotting using the 32P-labeled probe C1 (5′-ATGGTTTCATTGGTGACGTT-3′) after cleavage by Bsp106 (lanes 1 to 5) or digested by Bsp106 and HindIII, followed by agarose gel electrophoresis as described in the legend to Fig. 2B to determine if the mispaired base is removed during the gap-filling reaction (lanes 6 to 8). APD, aphidicolin; ITM, excision intermediates; solid bar, 32P-labeled probe.
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