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Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair

DNA mismatch repair (MMR) is an evolutionarily-conserved process responsible for the repair of replication errors. In Escherichia coli, MMR is initiated by MutS and MutL, which activate MutH to incise transiently-hemimethylated GATC sites. MMR efficiency depends on the distribution of these GATC sites. To understand which molecular events determine repair efficiency, we quantitatively studied the effect of strand incision on unwinding and excision activity. The distance between mismatch and GATC site did not influence the strand incision rate, and an increase in the number of sites enhanced incision only to a minor extent. Two GATC sites were incised by the same activated MMR complex in a processive manner, with MutS, the closed form of MutL and MutH displaying different roles. Unwinding and strand excision were more efficient on a substrate with two nicks flanking the mismatch, as compared to substrates containing a single nick or two nicks on the same side of the mismatch. Introduction of multiple nicks by the human MutLα endonuclease also contributed to increased repair efficiency. Our data support a general model of prokaryotic and eukaryotic MMR in which, despite mechanistic differences, mismatch-activated complexes facilitate efficient repair by creating multiple daughter strand nicks.


Description of the mismatch-specific, quantitative assay to study the effect of GATC site distribution on daughter strand incision efficiency
To analyze daughter strand incision and excision efficiency in vitro, we adapted existing protocols to generate relaxed circular hemi-methylated DNA substrates (1), incorporating a fluorophore (either Alexa 488 or Alexa 647 ), a single G/T mismatch or the corresponding A/T Watson-Crick base pair, and 1, 2 or 15 GATC sites (GT#1, GT#2 and GT#15 as shown in Figure S1A; AT#1, AT#2 and AT#15). Incision of GATC sites by E. coli MMR proteins was analyzed using agarose gel electrophoresis in the presence of ethidium bromide, which allows the separation of the remaining covalently-closed substrate from the nicked product. Efficient nicking of substrate GT#1 was dependent on the presence of MutS, MutL, MutH, ATP and the G/T mismatch ( Figure S1B and C). The fluorophore did not influence the rate of the incision reaction ( Figure S1G and H) and enabled convenient and reliable quantification, circumventing the use of radioactively labeled substrates.
Homoduplex AT#1 was nicked inefficiently, especially at physiological ionic strength (150 mM KCl) and, dependent on protein concentrations, 15-30 times slower than nicking of GT#1 ( Figure S1D and E, Figure S2). Activity on homoduplex substrates has been described previously (2,3) and might be linked to recognition of aberrant nucleotides incorporated by the T4 DNA polymerase during the primer extension reaction, to base modifications occurring during the purification of the substrates, to secondary structures, or to an intrinsic activity of the MMR proteins on substrates without a mismatch.
The efficiency of mismatch repair in vivo and in vitro depends on the number of hemi-methylated GATC sites, as well as on the distance between the mismatch and the nearest GATC site (4)(5)(6). To investigate at which sub-step of the repair reaction this GATC site distribution exerts its effect, we compared incision activities of MutSLH on defined substrates with different numbers of GATC sites and different distances between mismatch and GATC site. Intuitively, one expects that finding 1 GATC site out of 15 on a 3.2-kb substrate will be faster than finding a single GATC site. Indeed we observe that at relatively low ionic strength (50 mM KCl) incision of GT#15 occured faster than incision of GT#2, which in turns was nicked faster than GT#1 (main Figure 1 left panels), indicating that the search for the GATC site, rather than the nucleolytic activity of MutH, was rate limiting for incision. In the presence of MutSL, incision at 50 mM KCl was more efficient than with MutH alone, but still dependent on the number of sites, and possibly reflected the combined activities of MutH by itself and MutSLH-induced incision. At physiological ionic strength (150 mM KCl), MutH by itself was extremely inefficient, indicating that it depends on MutSL to be loaded onto mismatched DNA; but MutSLH incises GT#1, GT#2 and GT#15 in a mismatch-dependent manner ( Figure S2 for homoduplex controls). The effect of the number of sites is largely absent because we observed a reproducible but not significant difference between incision rate constants for GT#1 (0.36 ± 0.1 min -1 ), GT#2 (0.45 ± 0.2 min -1 ) and GT#15 (0.54 ± 0.2 min -1 ; p> 0.05 upon comparison of global fit with separate fits for each substrate). This indicates that at these condition, a reaction step before the search for the GATC site also becomes rate-limiting for incision.
To investigate the influence of the distance between the mismatch and the nearest GATC site on the efficiency of daughter strand incision, we created two substrates in which we varied the position of the G/T mismatch relative to a unique GATC site (substrates GT#1 and GT#1b; main Figure 1C). We labeled these substrates with different fluorophores (Alexa 647 and Alexa 488 ), to be able to measure the incision kinetics of the two substrates within a single experiment. In this way, each substrate served as an internal control for the other, which resulted in a lower experimental error. At protein concentrations approaching saturation (200 nM MutS, 200 nM MutL, 50 nM MutH; Figure S1E), the duration of protein binding steps is minimized, and thus a putative delay caused by the communication between the mismatch and the GATC site can be studied ( Figure 1D).
To investigate the effect of number and location of GATC sites on unwinding and excision efficiency, we created GT#2b, GT#15b, and versions of GT#1b with additional nicks introduced using the D10A nicking variant of Cas9 loaded with appropriate CRISPR and tracr-RNA ( Figure S1 and S10).     Table S1.      S1: Rate constants (min -1 ) and observed rates (nM DNA min -1 ) obtained from fitting with incision model. Table lists the concentrations of MutS, MutL, MutH and DNA, the ionic strength at which the assay was performed, the rate constants (min -1 ) for the first and the second nicking event, the observed rates (nM DNA min -1 ) for the first and the second nicking event at t=0, and the figure in which the particular experiment is presented. [