Mut S: A Mismatch Repair Protein

 

 

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Mismatch Repair plays a critical role in maintaining the genomic sequence. The importance of mismatch repair is emphasized as malfunctions in mismatch repair system is a direct link to certain cancers. Moreover, the progression of tummors is a result of the failure of DNA repair system  Paul Goodfellow 2009.

The mismatch repair system fixes nucleotide basepairing mistakes that DNA polymerase did not catch during DNA replication Smith et al. 2001. When base pairs are incorrectly matched during DNA replication, a kink occurs. This kink produces a 60 degree angle with the mispairing located at each vertex of the kink. This kink widens the minor groove so that the distance between the phosphate backbone and mismatched base pair increases Naterjan et al. 2003. The repair system is closely related to DNA  replication, in that mismatch repair proteins are recruited due to an interaction with the repliactive sliding clamp (PCNA) Masih et al.2007.

 

The first step in mismatch repair involves the Mut S protein. Mut S is found in prokaryotes and homologues of Mut S are found in eukaryotes (MSH1, MSH2, MSH3, MSH4, MSH5 and MSH6.) All mismatch repair protiens have the same function, however specificities differ slightly Hayes et al. 2009.

 

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Mismatch repair in prokaryotes:

Mut S recognizes the kink and binds to the mismatched basepair Anil Rustgi 2007.  After the binding of Mut S, the mismatch protein Mut L joins the Mut S- DNA complex which activates Mut H. Mut H cuts the non-methylated strand. An exonuclease digests the non-methylated strand, starting a few basepairs beyond the mispairing. DNA pol III haloenzymes resynthesies the new corrected strand Stanislawska-Sachadyn et al. 2005.  

 

 

 

 

Figure 1. Representation of prokaryotic mismatch repair described above. Click here to go to illustration website.

 

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Mismatch repair in eukaryotes: 

Homologous to Mut S are MSH1, MSH2, MSH3, MSH4,MSH5, and MSH6. To gether, MSH2 and MSH6 form a heterodimer. Together the two can recognize 1 basepair instertion or deletion. MSH2 and MSH3 also form a heterodimer. Together the two recognize 2 to 4 baispair instertions or deletions Anil Rustgi 2007.

 

 

 

 

Figure 2. Mismatch repair in mammalian cell. A. Shows the MSH2 and MSH6 function. EXO1 is the exonuclease.

B. shows MSH4 and MSH5 function. The exonuclease is still to be determined Anil Rustgi 2007.

 

 

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Mut S Structure:

     Mut S is a protein composed of beta sheets and alpha helices.  It is homodimetric, meaning that it is has two identical monomers that only differ in their orientation. Mut S consists of various domains; a Helix-turn-helix domain, ATPase domain (C-termianl), connector domain, mismatch domain (N-terminal), and positively charged clamp domain Stoffregen and Marcey 2001.

 

     Mut S consist of two monomers, Monomer A and Monomer B ( in Figure 2., represented by green and blue helices, respectively). The green momoner directly contacts the mismatched basepair. The blue monomer makes a contact with the backbone of the DNA. The monomers are thought to bind non-specifically to DNA until a mismatch is encountered. Using their mismatch binding domains, together, these momoners clamp DNA in position .Hayes et al. 2009. The monomers have identical amino acid sequences. Two important ones in mismatch repair are, Phe 36 and Glu 38. Both amino acids are so important that with out either, mismatch repair can not occur  Naterjan et al. 2003. The C-terminal is been found to ben important for anti-recombination and cisplatin sensitization Calmann et al. 2005.  

 

 

Figure 3. Mut S structurally composed of two monomers, represented by blue and green helicies. The red spiral shows the DNA. The other red segment shows ADPase domain.Natrejan et al. 2003. Click here to go to illustration website.

 

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Mut S Function:

Mut S recognizes mispaired bases and unpaired bases, including small DNA insertion/deletion loops Stanislawska-Sachadyn et al.2005. Mut S is highly effective, it can recognize insertion/deletions up to three unpaired nucleotide base pairs long, as well as all base mispairing except for C:C  Yamamoto et al. 2000.  This even includes O6 meG mispaired with either C or T Rasmussen and Samson 1996. O6- methyl guanine is a cytotoxic and mutagenic DNA adduct. It can be formed by particular agents such as tabacco smoke, methylnitrosourea, as well as other Sn1 methylacting agents Singh et al 1996. Additionally, Mut S removes potential mutations to avoid further recombination and prevents recombination between related but not homologous DNA sequences Calmann et al 2005.

 

 

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Figure 4. A. Mut S protein. B. Mut S and DNA interaction. C. Detailed image of Mut S interaction with a a G:T mismatch. Mismatches are symbolized by purple "M."  Arrows indicate Hydrogen bonds and Van der Wals interactions. D. Image of Mut S interaction with an unpaired T.  

                                                                   Click here for larger image and to go to illustration website.

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Mut S 

How it works:

Mut S recognizes the kink produced by a mispairing. Primary recogniton occurs by the clamp domain. The clamp domain has limited contact with the DNA. However, its positive charge allows an attraction to the negativley charged DNA backbone, this alows it to hold DNA in place, with the help of the mismatch domain. Once DNA is help in place, binding to the mismatched basepair and DNA strand can occur. Mismatch repair is specific for the mispaired bases (Figure 4.)

     For example, if there is G:T mispairing, the following will occur;  

     In this particular case, Mut S attachement, involves amino acids, P36 and Glu 38. The use of the amion acids is made possibe by of the widening of the minor grove (produced by the 60 degree angle) and because of increased DNA flexibility due to mismatch or loop mutation.

MutS attaches by stacking P36 of one momoner onto the mismatch basepair. The base is moved so that the Nitrogen on it can be brought to Glu 38. Now Glu 38, can form a Hydrogen bond with the bases under Phe 36 stack (guamine and thymine). The Hydrogen bond occurs between the carbony oxygen of glutamate and Nitrogen of the base Neterjan et al. 2003 and Stroffregen and Marchy 2001.

 

 

 

 

Figure 5. Different interactions with Mut S and various nucleotide mismatches.

A. G:T mismatch, unbound to Mut S 

B. G:T mismatch, bound to Mut S 

C. C:A mismatch, unboud to Mut S

D. C:A mismatch, bound to Mut S 

E. G:G mismatch, unbound Mut S 

F. G:G mismatch, bout Mut S 

G. mismatch binding domains, superposition 

H.  A:A mismatch, bound to Mut S.

Click here for larger image and  to go to illustration website

 

 

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Inactivation of Mut S:

     

If there is a mutation in the Mut S protein where Phe 36 is unable to function, then Mut S can never bind to DNA, making repair impossible. If there is a mutation in Glu 38, mismatch repair system is destroyed. Ultimately, it will result in a mutant phenotype expresion in bacteria and can cause various cancers in humans Naterjan et al. 2003.

 

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HNPCC (Hereditary Nonployposis Colon Cancer):

 Genomic instability is the inate property of tumer cells. Instability is a result of mismatch repair system malfunctions Rodriguez-Bigas et al 1997. Hereditary nonployposis colon cancer and other sporatic cancers have been linked to defective mismatch repair function, underlying the importance of mismatch repair Hayes et al. 2009.

HNPCC is an autosomal dominant inherited genetic mutation, of any mismatch repair protein. The mutation increases the chances of developing colon cancer. Additonally, it causes extracolonic tumors, such as in the ovaries, stomach, bile duct, kindney, ureter, and bladder Anil Rustgi 2007 . 2-5% of indiviuals with colon cancer have HNPCC. Additonally, people with HNPCC have an 80% chance of developing colon cancer by the age of 44 Myers 2006.