Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair

Abstract

The hemimethylated d(GATC) sequence that directs Escherichia coli mismatch repair can reside on either side of a mismatch at a separation distance of 1,000 bp or more. Initiation of repair involves the mismatch-, MutS-, and MutL-dependent activation of MutH endonuclease, which incises the unmethylated strand at the d(GATC) sequence, with the ensuing strand break serving as the loading site for the appropriate 3'-to-5' or 5'-to-3' excision system. However, the mechanism responsible for the coordinated recognition of the mismatch and a hemimodified d(GATC) site is uncertain. We show that a protein roadblock (EcoRI(E111Q), a hydrolytically defective form of EcoRI endonuclease) placed on the helix between the two DNA sites inhibits MutH activation by 70-80% and that events that escape inhibition are attributable, at least in part, to diffusion of EcoRI(E111Q) away from its recognition site. We also demonstrate that a double-strand break located within the shorter path linking the mismatch and a d(GATC) site in a circular heteroduplex abolishes MutH activation, whereas a double-strand break within the longer path is without effect. These findings support the idea that initiation of mismatch repair involves signaling along the helix contour.

Fig. 1.

Experimental system. (A) Heteroduplex DNAs contained a G·T mismatch, a single hemimodified d(GATC) site 1,024 bp from the mismatch, and EcoRI recognition sites at either, both, or neither of the positions indicated (see Fig. 3). One EcoRI site was located 309 bp from the mismatch, between the mispair and d(GATC) site (shorter path), and the second was located 145 bp to the other side of the mispair. Heteroduplexes also contained an NheI site 5 bp from the mismatch, which is rendered resistant to cleavage when MutS is bound to the mispair (12). Because the efficiency of MutH activation by MutS and MutL can be affected by superhelical density (9), circular substrates were linearized with ClaI before use to avoid experimental variability caused by possible differences in this parameter with different heteroduplex preparations. (B) ClaI linearized G·T heteroduplex (2.4 nM; two EcoRI sites) prebound with EcoRI_E111Q (12 nM) was challenged with 120 nM WT EcoRI endonuclease, and the reaction was sampled to score the remaining intact DNA (circles) (see Materials and Methods). Error bars are ±1 SD (three determinations). The curve shown was determined by nonlinear least-squares fit to a single exponential (amplitude, 2.36 nM; k = 0.0311 min⁻¹; R² = 0.991). Because the heteroduplex contained two EcoRI sites, the rate of cleavage per site is half of that shown. (No preferential cleavage of either site was observed.) The two-EcoRI-site heteroduplex also was challenged with 120 nM EcoRI in the absence of EcoRI_E111Q; the intact heteroduplex was not detectable after 30 s of reaction, but low levels of molecules that cleaved at just one site or the other were observed (squares). (C) Reactions containing 2.4 nM G·T heteroduplex with two EcoRI sites (prebound as indicated with 24 nM EcoRI_E111Q) were supplemented with 0, 12.5, 25, 50, or 100 nM MutS as the monomer. After 15 min at 37°C, samples of the reactions were challenged with NheI for 1 min. The challenge of samples from the same reaction with WT EcoRI demonstrated that >95% of EcoRI sites were occupied by EcoRI_E111Q (data not shown).

Fig. 2.

EcoRI_E111Q inhibits MutH activation on a linear G·T substrate. (A) Covalently closed circular 5′ G·T heteroduplex or A·T homoduplex DNAs containing two EcoRI sites were linearized with ClaI (Fig. 1A), prebound with EcoRI_E111Q as indicated, and then incubated with MutH, MuL, MutS, and ATP (see Materials and Methods). Reactions were sampled as a function of time and were quenched, and products were resolved by electrophoresis through alkaline agarose and transferred to a nitrocellulose membrane. The substrate and product were visualized by hybridization with an excess of the ³²P-5′-end-labeled oligonucleotide V2505 (see schematic at right), which hybridizes to the unmethylated strand. The 2,313-nucleotide segment resulting from d(GATC) incision was quantitated by using a phosphorimager. Filled circles, G·T heteroduplex in the absence of EcoRI_E111Q (mock prebound); open circles, G·T heteroduplex prebound with EcoRI_E111Q; squares, A·T homoduplex prebound with EcoRI_E111Q. Error bars are ±1 SD (three determinations). (B) Experimental procedure and symbols are as in A, but DNA substrates were 3′ G·T heteroduplex or A·T homoduplex DNAs. Incision at the d(GATC) site was scored by hybridization with ³²P-5′-end-labeled oligonucleotide C2527, which hybridizes to the unmethylated strand.

Fig. 3.

EcoRI_E111Q inhibition of MutH activation depends on EcoRI site location. (A) MutHLS reactions on 3′ G·T heteroduplex DNAs. The descriptions of the symbols are the same as for those in Fig. 2B, except that DNAs contained one or no EcoRI site, as indicated. The individual EcoRI sites present in the molecules shown in A and B correspond to the two sites present in the DNAs shown in Fig. 2. The error bars indicate the SD for three independent experiments (A) or the range of values observed in two independent experiments (B and C).

Fig. 4.

Effects of a helix discontinuity on MutH activation. (A) Closed circular 3′ G·T heteroduplexes were linearized with individual endonucleases as indicated and then subjected to treatment with MutH, MutL, and MutS as described in Fig. 2. d(GATC) incision was scored after digestion with ClaI by hybridization with ³²P-5′-end-labeled oligonucleotide C2527 (oligonucleotide C2552 for the BspHI-linearized heteroduplex). The region encompassing the shorter path between the mismatch and the d(GATC) site in the circular heteroduplex is shown in bold. (B) The analysis of d(GATC) incision after restriction enzyme linearization was as in A, except that 5′ heteroduplexes were used.