Reflections on the Mechanism of DNA Mismatch Repair

markdownabstractLife 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 whic