Functions of MutLalpha, replication protein A (RPA), and HMGB1 in 5'-directed mismatch repair

Abstract

A purified system comprised of MutSalpha, MutLalpha, exonuclease 1 (Exo1), and replication protein A (RPA) (in the absence or presence of HMGB1) supports 5'-directed mismatch-provoked excision that terminates after mismatch removal. MutLalpha is not essential for this reaction but enhances excision termination, although the basis of this effect has been uncertain. One model attributes the primary termination function in this system to RPA, with MutLalpha functioning in a secondary capacity by suppressing Exo1 hydrolysis of mismatch-free DNA (Genschel, J., and Modrich, P. (2003) Mol. Cell 12, 1077-1086). A second invokes MutLalpha as the primary effector of excision termination (Zhang, Y., Yuan, F., Presnell, S. R., Tian, K., Gao, Y., Tomkinson, A. E., Gu, L., and Li, G. M. (2005) Cell 122, 693-705). In the latter model, RPA provides a secondary termination function, but together with HMGB1, also participates in earlier steps of the reaction. To distinguish between these models, we have reanalyzed the functions of MutLalpha, RPA, and HMGB1 in 5'-directed mismatch-provoked excision using purified components as well as mammalian cell extracts. Analysis of extracts derived from A2780/AD cells, which are devoid of MutLalpha but nevertheless support 5'-directed mismatch repair, has demonstrated that 5'-directed excision terminates normally in the absence of MutLalpha. Experiments using purified components confirm a primary role for RPA in terminating excision by MutSalpha-activated Exo1 but are inconsistent with direct participation of MutLalpha in this process. While HMGB1 attenuates excision by activated Exo1, this effect is distinct from that mediated by RPA. Assay of extracts derived from HMGB1(+/+) and HMGB1(-/-) mouse embryo fibroblast cells indicates that HMGB1 is not essential for mismatch repair.

FIGURE 1.

Mismatch-provoked excision in A2780/AD nuclear extracts. The upper diagram illustrates the nature of the substrates and excision assays used in this study. 6.44 kb circular heteroplexes contained a G-T mismatch (A·T base pair in homoduplex controls) and a site-specific strand break, which was located 5′ to the mismatch as viewed along the shorter path between the two sites. Although DNAs used contain 4 AseI sites, only the site proximal to the mismatch shown in the diagram was used in this work. Two methods were used to score excision products. A single NheI site, separated from the mismatch by 5 base pairs, is rendered endonuclease-resistant by mismatch-provoked excision, an effect that was monitored by digestion of reaction products with NheI and ClaI (23). In the second method, 5′-termini produced by excision were localized by digestion with AseI, resolved by electrophoresis through alkaline-agarose gels, and products probed by indirect end-labeling using a ³²P-labeled oligonucleotide that hybridizes to the processed strand adjacent to the AseI site (gray bar in right diagram). Lower, NheI resistance assay was used to score 5′-directed excision in nuclear extracts of A2780/AD cells, which are deficient in MLH1 and PMS2 (17). Excision reactions contained 5′ G-T heteroduplex (closed symbols, nick 128 bp 5′ to mismatch) or otherwise identical A·T homoduplex control DNA (open symbols), 50 μg of nuclear extract without (circles), or supplemented with 280 fmol (squares) or 1100 fmol (diamonds) MutLα. The inset shows a Western blot for MSH6 and MLH1 in 50 μg of nuclear extract derived from HeLa or A2780/AD cells, in the latter case in the absence of MutLα or supplemented with 280 or 1100 fmol of the repair protein.

FIGURE 2.

Excision termination in A2780/AD nuclear extracts. Mismatch-provoked excision reactions (“Experimental Procedures”) contained 5′ G-T heteroduplex DNA (128-bp nick-mismatch separation distance) and 50 μg of A2780/AD nuclear extract supplemented as indicated with MutLα. After digestion with AseI and Sau96I, products were subjected to electrophoresis through alkaline-agarose, transferred to a nylon membrane, and probed with ³²P-labeled oligonucleotide V4634, which hybridizes to the discontinuous heteroduplex strand adjacent to the AseI site (see diagram on left and Fig. 1). The major zone of termination products (asterisk) peaks at about 870 nucleotides (the AseI cleavage site is 1,000 bp from the mismatch). A tail of smaller material extending in size down to about 350 nucleotides is also evident, particularly at longer incubation times, and this material is potentiated by the presence of MutLα.

FIGURE 3.

Effects of MutLα on 5′-directed excision in a purified system. Excision reactions (“Experimental Procedures”) contained per time sample 24 fmol of 5′ G-T heteroduplex DNA, 400 fmol of MutSα, 900 fmol of RPA, 21 fmol of Exo1, and MutLα as indicated. Excision was visualized by hybridization assay (probe V4634) after AseI digestion and alkaline electrophoresis through 2% agarose (“Experimental Procedures”) as illustrated in the diagrams on the left. Separation distances between the nick and the mismatch were 128 (A), 494 (B), or 808 bp (C). Two major termination zones were observed with each DNA (asterisks). As discussed in the text, stability of these zones was modulated by the presence of MutLα.

FIGURE 4.

Excision on homoduplex DNA in the purified system. Excision reactions were performed as described in the legend to Fig. 3C except that the substrate contained an A·T base pair instead of a G-T mismatch.

FIGURE 5.

HMGB1 effects on excision tracts in the purified system. Mismatch-provoked excision reactions, performed and analyzed as in Fig. 3, contained per time sample 400 fmol of MutSα, 21 fmol of Exo1, 280 fmol of MutLα, and as indicated, 900 fmol of RPA, 3.3 pmol of HMGB1, or neither RPA or HMGB1. G-T heteroduplexes contained a strand break located 128 (A), 494 (B), or 808 bp (C) 5′ to the mispair.

FIGURE 6.

Effects of RPA, MutLα, and HMGB1 on the processive action of MutSα-activated Exo1. Protein challenge was used to evaluate effects of repair activities on processive hydrolysis by activated Exo1. A, reactions contained (per time sample) 24 fmol of gapped homoduplex DNA substrate (gap size 314 nucleotides) and 400 fmol of MutSα. After 5 min at 37 °C, hydrolysis was initiated at time 0 by addition of 21 fmol of Exo1 in the absence (buffer, left) or presence of either 280 fmol of MutLα (middle) or 3.3 pmol of HMGB1 (right). Reaction conditions were the same as those used for mismatch-provoked excision (“Experimental Procedures”) except that the volume per time sample was reduced to 18.4 μl to accommodate the further addition at 1 min of 1.6 μl buffer (16 mm HEPES-KOH pH 7.5, 100 mm KCl, 0.3 mm dithiothreitol, 1 mg/ml BSA, 5% (v/v) glycerol). Reactions were sampled (20 μl), hydrolysis quenched, and products visualized after cleavage with ClaI, alkaline-agarose gel electrophoresis, Southern transfer, and hybridization with probe V2532 (“Experimental Procedures”). B, reaction conditions were as in panel A, except that hydrolysis was initiated by addition of Exo1 only. At 1 min, reactions were supplemented per time sample with 900 fmol of RPA (left), 280 fmol of MutLα (middle), or 3.3 pmol of HMGB1 (right) in 1.6 μl of buffer.

FIGURE 7.

Mismatch repair in HMGB1-deficient MEF cell extracts. Mismatch repair of 5′ (gray and black bars) or 3′ (white and stippled bars) G-T heteroduplexes in HMGB1-proficient or -deficient MEF cell extracts was determined in the absence (gray and white bars) or presence (black and stippled bars) of 3.3 pmol of recombinant HMGB1 as described under “Experimental Procedures.” Heteroduplex orientation refers to the 3′ or 5′ location of the strand break relative to the mispair as viewed along the shorter path linking the two sites in the circular substrates used. As indicated in the upper diagram, repair was scored by restoration of HindIII sensitivity (26) as judged by cleavage with HindIII and ClaI. Reactions were done in triplicate, and error bars indicate 1 S.D. Insets show the results of HMGB1 Western blots obtained with 100 μg of HMGB1-proficient (left) or -deficient (right) extract unsupplemented (left lanes of each gel) or supplemented (right lanes) with 100 ng of recombinant human HMGB1. Because of the presence of a His₆ tag, the recombinant protein runs slower than endogenous HMGB1. Ponceau S staining of the Western membrane was used as a loading control.