Investigating the communication of mismatch recognition and strand incision in the DNA mismatch repair pathway

DNA Mismatch Repair (MMR) is crucial for preventing DNA replication errors that escape the proofreading mechanisms of DNA polymerases from becoming permanent mutations. MMR is initiated when MutS recognises a mismatch and recruits MutL to signal the degradation and resynthesis of the nascent DNA. The distinction between the nascent and template DNA strands is a critical aspect of Mismatch Repair in all organisms. In Escherichia coli, this problem is solved by MutH endonuclease, which incises the nascent strand specifically at hemimethylated GATC sequences. However, it remains unclear how the MutS-MutL complex communicates with MutH to activate strand incision, given that GATC sites can be located hundreds of bases away from a mismatch. To tackle this long standing question, we used single-molecule tracking of fluorescently-labeledMutS,MutL, andMutH proteins in live E. coli cells. Our quantitative measurements address how MutH is recruited to hemimethylated GATC sites. We find that it is neither dependent on MutS/L nor on the presence of DNA mismatches suggesting that MutH binds to hemimethylated GATC sites independently and becomes activated by interaction withMutL. Furthermore, we show that MutH competes with SeqA and Dam proteins when binding to GATC sites. Hence, there is a brief time window during which MutH has to be activated before the strand discrimination signal is lost. We used Fluorescence Recovery after Photobleaching (FRAP) to measure the stoichiometry and turnover of proteins during Mismatch Repair. Our results complement previous studies, leading to a refined model for the coordination of the E. coli Mismatch Repair pathway in vivo