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

Abstract

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.

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

“…On substrate GT#2, we observed formation of small amounts of reaction intermediates containing a single incision at either GATC site 1 or site 2, and progressive accumulation of reaction product in which both GATC sites were incised (Figure 1B and 1E;left panel). This rapid conversion of single-nicked intermediates into double-nicked products on linearized substrate is similar to what we have observed previously on circular GT#2 35 . However, while on circular GT#2 GATC site preference was undetectable (Supplemental Figure S1), on linear GT#2 we observed a preference for nicking GATC site 2, which is close to the mismatch ( Figure 1E Figure S1).…”

“…The estimated maximal accumulation of reaction intermediates that are nicked either at GATC site 1 or GATC site 2 adds up to 30% of total substrate. This is similar to the observed amount of single-nicked intermediate obtained on circular DNA at otherwise identical conditions (see Figure 3C left panel in 35 ); indicating that also on linear DNA dual GATC site incision is processive 35 .…”

“…To explore the mechanism by which MMR proteins communicate between mismatch and multiple hemimethylated GATC sites during initiation of DNA mismatch repair, we quantitatively investigated strand incision using linear substrates containing a single G/T mismatch (or its corresponding A/T base pair) flanked by two hemimethylated GATC sites and an Alexa 647 fluorophore ( Figure 1A). Circular substrates 35 were linearized using ScaI, mixed with 50 nM MutS, 50 nM MutL and 25 nM MutH in the presence of ATP, and formation of reaction products carrying the fluorophore was analyzed using denaturing gel electrophoresis, which allowed identification of nicking events at individual GATC sites. Efficient incision of both GATC sites was observed on GT#2, carrying a G/T mismatch, while activity on homoduplex AT#2 substrate was negligible under these conditions ( Figure 1B).…”

“…We adapted our ensemble incision assay 35 such that we could quantitatively monitor incision of individual hemimethylated GATC sites that flank the mismatch on substrates GT#2 and GT#2b 35 . The estimated maximal accumulation of reaction intermediates that are nicked either at GATC site 1 or GATC site 2 adds up to 30% of total substrate.…”

“…As expected [36][37][38] , we found that dCas9 forms a specific and stable complex on different target sites and remains bound for many hours. We have previously used the D10A variant of Cas9 as in vitro endonuclease to create substrates to analyze strand excision in a reconstituted MMR system 35 . An advantage of using Cas9 and its variants in this kind of approaches is that PAM sites are ubiquitously present in DNA and thus target site selection merely requires addition of a suitable crRNA, rather than requiring cloning procedures to introduce or change the location of binding sites.…”