DNA mismatch repair
Encyclopedia
DNA mismatch repair is a system for recognizing and repairing erroneous insertion, deletion and mis-incorporation of base
s that can arise during DNA replication
and recombination
, as well as repairing some forms of DNA damage.
Mismatch repair is strand-specific. During DNA synthesis the newly synthesised (daughter) strand will include errors commonly. In order to do this the mismatch repair machinery distinguishes 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. It is suspected that in eukaryotes, newly synthesized lagging-strand DNA transiently contains nicks (before being sealed by DNA ligase) and provides a signal that directs mismatch proofreading systems to the appropriate strand. This implies that these nicks must be present in the leading strand, but it is unclear how.
Any mutational event that disrupts the superhelical structure of DNA
carries with it the potential to compromise the genetic stability of a cell. 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
). Mismatches are commonly due to tautomerization of bases during synthesis. The damage is repaired by recognition of the deformity caused by the mismatch, determining the template and non-template strand, and excising the wrongly incorporated base and replacing it with the correct nucleotide
. The removal process involves more than just the mismatched nucleotide itself. A few or up to thousands of base pairs of the newly synthesized DNA strand can be removed.
s). Subsequent work on E. coli has identified a number of genes that, when mutation
ally 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 to the mismatch, which could be up to 1kb away. Upon activation by the MutS-DNA complex, MutH nicks the daughter strand near the hemimethylated site 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 ExoVII (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.
leads to the formation of tertiary structures on the surface of the molecule. The crystal structure
of MutS reveals that it is exceptionally asymmetric, and while its 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 (MutSα) and Msh2
/Msh3 (MutSβ). The MutSα pathway is involved primarily in base substitution and small loop mismatch repair. The MutSβ pathway is also involved in small loop repair, in addition to large loop (~10 nucleotide loops) repair. However, MutSβ does not repair base substitutions.
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 re-anneal 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 re-anneal, 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.
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. Its 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.
and the β-sliding clamp associate with MutSα/β and MutS, respectively. Although initial reports suggested that the PCNA-MutSα complex may enhance mismatch recognition, it has been recently demonstrated that there is no apparent change in affinity of MutSα for a mismatch in the presence or absence of PCNA. Furthermore, mutants of MutSα that are unable to interact with PCNA in vitro
exhibit the capacity to carry out mismatch recognition and mismatch excision to near wild type levels. Curiously, such mutants are defective in the repair reaction directed by a 5' strand break, suggesting for the first time MutSα function in a post-excision step of the reaction.
(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 MSH2
and MLH1
respectively, 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.
Nucleobase
Nucleobases are a group of nitrogen-based molecules that are required to form nucleotides, the basic building blocks of DNA and RNA. Nucleobases provide the molecular structure necessary for the hydrogen bonding of complementary DNA and RNA strands, and are key components in the formation of stable...
s that can arise during DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
and recombination
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
, as well as repairing some forms of DNA damage.
Mismatch repair is strand-specific. During DNA synthesis the newly synthesised (daughter) strand will include errors commonly. In order to do this the mismatch repair machinery distinguishes the newly synthesised strand from the template (parental). In gram-negative bacteria transient hemimethylation
Methylase
A methylase is an enzyme that attaches a methyl group to a molecule.These are found in prokaryotes and eukaryotes. Bacteria use methylase to differentiate between foreign genetic material and their own, thus protecting their DNA from their own immune system...
distinguishes the strands (the parental is methylated and daughter is not). In other prokaryotes and eukaryotes the exact mechanism is not clear. It is suspected that in eukaryotes, newly synthesized lagging-strand DNA transiently contains nicks (before being sealed by DNA ligase) and provides a signal that directs mismatch proofreading systems to the appropriate strand. This implies that these nicks must be present in the leading strand, but it is unclear how.
Any mutational event that disrupts the superhelical structure of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
carries with it the potential to compromise the genetic stability of a cell. 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
DNA repair
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...
). Mismatches are commonly due to tautomerization of bases during synthesis. The damage is repaired by recognition of the deformity caused by the mismatch, determining the template and non-template strand, and excising the wrongly incorporated base and replacing it with the correct nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
. The removal process involves more than just the mismatched nucleotide itself. A few or up to thousands of base pairs of the newly synthesized DNA strand can be removed.
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 geneGene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s). Subsequent work on E. coli has identified a number of genes that, when mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
ally 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 to the mismatch, which could be up to 1kb away. Upon activation by the MutS-DNA complex, MutH nicks the daughter strand near the hemimethylated site and recruits a UvrD
UvrABC endonuclease
UvrABC endonuclease is a multienzyme complex in E.coli bacteria involved in DNA repair mechanism by nucleotide excision repair, and it is, therefore, sometimes called an excinuclease...
helicase
Helicase
Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.-Function:Many cellular processes Helicases are a...
(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 ExoVII (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 homologs
When bound, the MutS2 dimer bends the DNA helix and shields approximately 20 base pairs. It has weak ATPase activity, and binding of ATPAdenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
leads to the formation of tertiary structures on the surface of the molecule. The crystal structure
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
of MutS reveals that it is exceptionally asymmetric, and while its 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
MSH2
MSH2 is a gene commonly associated with Hereditary nonpolyposis colorectal cancer.-Interactions:MSH2 has been shown to interact with Exonuclease 1, MSH3, MSH6, CHEK2, MAX, Ataxia telangiectasia and Rad3 related and BRCA1.-Further reading:...
/Msh6 (MutSα) and Msh2
MSH2
MSH2 is a gene commonly associated with Hereditary nonpolyposis colorectal cancer.-Interactions:MSH2 has been shown to interact with Exonuclease 1, MSH3, MSH6, CHEK2, MAX, Ataxia telangiectasia and Rad3 related and BRCA1.-Further reading:...
/Msh3 (MutSβ). The MutSα pathway is involved primarily in base substitution and small loop mismatch repair. The MutSβ pathway is also involved in small loop repair, in addition to large loop (~10 nucleotide loops) repair. However, MutSβ does not repair base substitutions.
MutL homologs
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 re-anneal 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 re-anneal, 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
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
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: an endonuclease present in E. coli and Salmonella
MutH is a very weak endonucleaseEndonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
that is activated once bound to MutL (which itself is bound to MutS). It nicks unmethylated
Methylation
In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...
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. Its 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.
β-sliding clamp/PCNA
PCNAPCNA
Proliferating Cell Nuclear Antigen, commonly known as PCNA, is a protein that acts as a processivity factor for DNA polymerase δ in eukaryotic cells. It achieves this processivity by encircling the DNA, thus creating a topological link to the genome...
and the β-sliding clamp associate with MutSα/β and MutS, respectively. Although initial reports suggested that the PCNA-MutSα complex may enhance mismatch recognition, it has been recently demonstrated that there is no apparent change in affinity of MutSα for a mismatch in the presence or absence of PCNA. Furthermore, mutants of MutSα that are unable to interact with PCNA in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
exhibit the capacity to carry out mismatch recognition and mismatch excision to near wild type levels. Curiously, such mutants are defective in the repair reaction directed by a 5' strand break, suggesting for the first time MutSα function in a post-excision step of the reaction.
Defects in mismatch repair
Mutations in the human homologues of the Mut proteins affect genomic stability, which can result in microsatellite instabilityMicrosatellite instability
Microsatellites are repeated sequences of DNA. Although the length of these microsatellites is highly variable from person to person, each individual has microsatellites of a set length. These repeated sequences are common, and normal...
(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 MSH2
MSH2
MSH2 is a gene commonly associated with Hereditary nonpolyposis colorectal cancer.-Interactions:MSH2 has been shown to interact with Exonuclease 1, MSH3, MSH6, CHEK2, MAX, Ataxia telangiectasia and Rad3 related and BRCA1.-Further reading:...
and MLH1
MLH1
MutL homolog 1, colon cancer, nonpolyposis type 2 , also known as MLH1, is a human gene located on Chromosome 3. It is a gene commonly associated with hereditary nonpolyposis colorectal cancer.It can also be associated with Turcot syndrome....
respectively, which allows them to be classified as tumour suppressor genes.
A subtype of HNPCC is known as Muir-Torre Syndrome
Muir-Torre syndrome
Muir–Torre syndrome is an inherited cancer syndrome that is thought to be a subtype of HNPCC. Individuals are prone to develop cancers of the colon, breast and genitourinary tract, and skin lesions, such as keratoacanthomas and sebaceous tumours. The genes affected are MLH1 & MSH2, and involved...
(MTS) which is associated with skin tumors.
Further reading
- Griffith, Wessler, Lewontin, Gelbart, Suzuki, Miller, Introduction to Genetic Analysis, 8th Edition, W.H. Freeman and Company, ISBN 0-7167-4939-4
- Thomas A.Kunkel and Dorothy A. Erie,(2005), DNA Mismatch Repair, Annu Rev.Biochem,74:681-710
- Errol C.Friedberg, Graham C. Walker, Wolfram Siede, Richard D. WoodRichard D. WoodRichard D. Wood is an American molecular biologist specializing in research on DNA repair and mutation. He is known for pioneering studies on nucleotide excision repair , particularly for reconstituting the minimum set of proteins involved in this process, identifying proliferating cell nuclear...
, Roger A. Schultz, Tom Ellenberger, DNA repair and Mutagenesis, 2nd edition, ASM press, ISBN 1-55581-319-4