DNA mismatch repair Lecture

Any mutational event that disrupts the superhelical structure of DNA carries with it the potential to compromise the genetic stability of a cell. Mismatch repair is a system for recognising and repairing the erroneous insertion, deletion and mis-incorporation of bases that can arise during DNA replication and recombination, as-well as repairing some forms of DNA damage . The fact that the damage detection and repair systems are as complex as the replication machinery itself highlights the importance evolution has attached to DNA fidelity.

 Examples of mismatched bases include a G/T or A/C pairing (see DNA repair). The damage is repaired by excising the wrongly incorporated base and replacing it with the correct nucleotide. Usually, this involves more than just the mismatched nucleotide itself, and can lead to the removal of significant tracts of DNA.
Mismatch repair
There are two types of mismatch repair; long patch and short patch. Long patch can repair all types of mismatches (although it is primarily replication associated) and can excise tracts up-to a few kilobases long. Short patch repair handles only specific mismatches caused by damage to the genome, and removes lengths of around 10 nucleotides. Successful mismatch repair requires the error-free execution of three events:
1. Detection of a single mismatch, of which there are eight kinds, in the newly synthesised DNA. 2. Determining which of the two base pairs is incorrect. 3. Correcting the error by excision repair.
Mismatch repair is strand-specific. During DNA synthesis only the newly synthesised (daughter) strand will include errors, and replacing a base in the parental strand would actually introduce an error. The mismatch repair machinery has a number of cues which distinguish the newly synthesised strand from the template (parental). In gram-negative bacteria transient hemimethylation distinguishes the strands (the parental is methylated and daughter is not). In other prokaryotes and eukaryotes the exact mechanism is not clear.
Mismatch repair proteins
Mismatch Repair is a highly conserved process from prokaryotes to eukaryotes. The first evidence for mismatch repair was obtained from S. pneumoniae (the hexA and hexB genes). Subsequent work on E. coli has identified a number of genes that, when mutationally inactivated, cause hypermutable strains. The gene products are therefore called the "Mut" proteins, and are the major active components of the mismatch repair system. Three of these proteins are essential in detecting the mismatch and directing repair machinery to it; MutS, MutH and MutL (MutS is a homologue of HexA and MutL of HexB).
MutS forms a dimer (MutS2) that recognises the mismatched base on the daughter strand and binds the mutated DNA. MutH binds at hemimethylated sites along the daughter DNA, but its action is latent, being activated only upon contact by a MutL dimer (MutL2) which binds the MutS-DNA complex and acts as a mediator between MutS2 and MutH, activating the latter. The DNA is looped out to search for the nearest d(GATC) methylation site nearest the mismatch, which could be up to 1kb away. Upon activation by the MutS-DNA complex, MutH nicks the daughter strand near the mismatch and recruits a UvrD helicase (DNA Helicase II) to separate the two strands with a specific 3' to 5' polarity. The entire MutSHL complex then slides along the DNA in the direction of the mismatch, liberating the strand to be excised as it goes. An exonuclease trails the complex and digests the ss-DNA tail. The exonuclease recruited is dependent on which side of the mismatch MutH incises the strand – 5’ or 3’. If the nick made by MutH is on the 5’ end of the mismatch, either RecJ or ExoVIII (both 5’ to 3’ exonucleases) is used. If however the nick is on the 3’ end of the mismatch, ExoI (a 3' to 5' enzyme) is used.
The entire process ends past the mismatch site - i.e. both the site itself and its surrounding nucleotides are fully excised. The single-stranded gap created by the exonuclease can then be repaired by DNA Polymerase III (assisted by single-strand binding protein), which uses the other strand as a template, and finally sealed by DNA ligase. Dam methylase then rapidly methylates the daughter strand.
MutS
When bound, the MutS2 dimer bends the DNA helix and shields approximately 20 base pairs. It has weak ATPase activity, and binding of ATP leads to the formation of tertiary structures on the surface of the molecule. The crystalline structure of MutS reveals that it is exceptionally asymmetric, and while it's active conformation is a dimer, only one of the two halves interact with the mismatch site.
In Eukaryotes, MutS homologs form two major heterodimers: Msh2/Msh6 and Msh2/Msh3. The Msh2/Msh6 pathway is involved primarily in base substitution and small loop mismatch repair. The Msh2/Msh3 pathway is also involved in small loop repair, in addtion to large loop (~10 nucleotide loops) repair. However, Msh2/Msh3 does not repair base substitutions.
MutL
MutL also has weak ATPase activity (it uses ATP for purposes of movement). It forms a complex with MutS and MutH, increasing the MutS footprint on the DNA.
However, the processivity (the distance the enzyme can move along the DNA before dissociating) of UvrD is only ~40–50bp. Because the distance between the nick created by MutH and the mismatch can average ~600 bp, if there isn't another UvrD loaded the unwound section is then free to reanneal to its complementary strand, forcing the process to start over. However, when assisted by MutL, the rate of UvrD loading is greatly increased. While the processivity (and ATP utilisation) of the individual UvrD molecules remains the same, the total effect on the DNA is boosted considerably; the DNA has no chance to reanneal, as each UvrD unwinds 40-50 bp of DNA, dissociates, and then is immediately replaced by another UvrD, repeating the process. This exposes large sections of DNA to exonuclease digestion, allowing for quick excision(and later replacement) of the incorrect DNA.
Eukaryotes have MutL homologs designated Mlh1 and Pms1. They form a heterodimer which mimics MutL in E. coli. The human homologue of prokaryotic MutL has three forms designated as MutLα, MutLβ and MutLγ. The MutLα complex is made of two subunits MLH1 and PMS2, the MutLβ heterodimer is made of MLH1 and PMS1, while MutLγ is made of MLH1 and MLH3. MutLα acts as the matchmaker or facilitator, coordinating events in mismatch repair. It has recently been shown to be a DNA endonuclease that introduces strand breaks in DNA upon activation by mismatch and other required proteins, MutSa and PCNA. These strand interruptions serve as entry points for an exonuclease activity that removes mismatched DNA. Roles played by MutLβ and MutLγ in mismatch repair are less well understood.
MutH
MutH is a very weak endonuclease that is activated once bound to MutL (which itself is bound to MutS). It nicks unmethylated DNA and the unmethylated strand of hemimethylated DNA but does not nick fully methylated DNA. It has been experimentally shown that mismatch repair is random if neither strand is methylated. These behaviours led to the proposal that MutH determines which strand contains the mismatch. MutH has no Eukaryotic homolog. It's endonuclease function is taken up by MutL homologs, which have some specialized 5'-3' exonuclease activity. The strand bias for removing mismatches from the newly synthesized daughter strand in eukaryotes may be provided by the free 3’ ends of Okazaki fragments in the new strand created during replication
Defects in mismatch repair
Mutations in the human homologues of the Mut proteins affect genomic stability, which can result in microsatellite instability (MI). MI is implicated in most human cancers. Specifically the overwhelming majority of hereditary nonpolyposis colorectal cancers (HNPCC) are attributed to mutations in the genes encoding the MutS and MutL homologues, which allows them to be classified as tumour suppressor genes. A subtype of HNPCC is known as Muir-Torre Syndrome (MTS) which is associated with skin tumors.
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