Reflections on the Mechanism of DNA Mismatch Repair

Life can be separated from dead organic matter by looking at two characteristics: growth and reproduction. For both of these, cells at some point need to split into two daughter cells. However, before cell division can take place, all the genetic information, encoded in DNA, needs to be copied. This process is called replication. Failure to replicate DNA correctly leads to mutations. These mutations can cause progenitor cells to have defects and die, or can cause cancer in higher organisms. DNA mismatch repair (MMR), the subject of study in this thesis, increases the fidelity of replication by removing mismatches left by the replication machinery.

Chapter 1 describes the mechanism of MMR, and implications of mutations that arise when mismatches are left uncorrected. Some of the most prevalent forms of hereditary cancers can be traced back to dysfunction of proteins involved in MMR. In Escherichia coli, MMR is initiated by MutS upon recognition of a DNA mismatch, resulting in ATP-dependent recruitment of MutL and activation of MutH. MutH is an endonuclease that is able to nick hemi-methylated DNA at a GATC motif, which provides an entry point for MMR to remove the strand with the error. The MMR pathway is conserved in most organisms, and MutS and MutL, the initiators of MMR in E. coli, are structurally very similar to their eukaryotic equivalents MutSα and MutLα. This emphasizes the importance of MMR, and sets the stage for consecutive chapters.

Chapter 2 deals with strand discrimination and excision during MMR. In E. coli, DNA is methylated by DAM methylase at GATC sites. Transiently hemimethylated GATC sites provide the signal for distinguishing the newly synthesized DNA from the template strand. The efficiency of MMR in vivo depends on the number of GATC sites and the distance between mismatch and nearest GATC site. We quantitatively studied the rate of nicking by MutS, MutL and MutH, and subsequent strand excision by UvrD and ExoI, while varying the number of GATC sites and their distance from a GT mismatch. We find that in vitro, multiple nicks increase the efficiency of excision, while strand discrimination remains efficient over distances of 1 kb. Interestingly, we find a similar mechanism in human MMR. We propose a model where a single activated MMR complex facilitates efficient excision and repair by creating multiple daughter strand nicks.

Chapter 3 focuses on the time frame in which the individual steps are carried out. Using Monte Carlo simulations, we found that a simple diffusive model is able to predict many features of our data, which is a good indication that the incision complex uses a random walk to communicate between the mismatch and GATC sites. Combined with order-of-addition experiments, these simulations suggest that conformational changes in MutL are rate limiting for strand incision. Furthermore, the simulations show that the nicking efficiency can be increased by multiple loading of MutS sliding clamps. This multiple loading could assure that strand discrimination takes place before the signal required to discriminate between the parental and daughter strand disappears.

Chapter 4 investigates the ATPase cycle of MutL. MutL is often described as a molecular matchmaker, and is able to activate the endonuclease activity of MutH and initiate excision of the error containing strand by loading UvrD onto the nick. Nucleotide binding in MutL was shown to switch a MutL dimer from an open to a closed conformation. These conformational changes govern the interaction of MutL with DNA and other MMR proteins. In this chapter we studied the interaction of MutL and several MutL mutants, deficient in either nucleotide binding, ATP hydrolysis or DNA binding, in functional assays with MutS, MutH, UvrD and DNA. We show that nucleotide binding and hydrolysis are not needed for the interaction of MutL with MutS. However, nucleotide binding is needed for activation of MutH and UvrD, and greatly increases the affinity of MutL for DNA. In this chapter we come to a model where MutL binds MutS in an open conformation and is able to activate downstream factors independent of MutS after being loaded onto the DNA in a closed conformation.

Chapter 5 deals with the distribution of GATC sites in the genome of E. coli. This distribution is not uniform, and is characterized by clusters of GATC sites and stretches devoid GATC sites. Because we know that the number and distance between GATC sites is important for the efficiency of MMR, we investigated large chromosomal DNA fragments without GATC sites for evidence of inefficient MMR. In E. coli cells without functional MMR, conversion of A(T) to G(C) is the predominant mutation. We show that DNA fragments larger than 3000 base pairs devoid of GATC sites have an elevated GC content, which suggests there is a locally diminished MMR efficiency. Also the genomes of other gram negative bacteria have DNA fragments devoid of GATC sites, and show a similar increase in GC content. These GATC deserts often include rearrangement hotspots (rhs) and Rhs genes. The absence of GATC sites in the Rhs genes provides an explanation for the increase of rearrangements and polymorphisms found in Rhs genes, and provides interesting insight in their function in inter-cellular competition and host-pathogen interaction.