This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
Every living organism is made up of a specific genome. The DNA sequencing in the genome is what makes each organism unique. Due to environmental factors and bad lifestyle, there is an increase exposure of organisms to mutagens, such as ionising radiation (Nagy and Soutoglou 2009). In order to maintain DNA integrity organisms have evolved ways such as, DNA Repair and DNA damage tolerance, to deal with the various mutations (Schneider, Schorr and Carell 2009). DNA repair is made up of two major mechanisms; damage reversal and damage removal (Eker et al. 2009). Therefore, DNA damage reversal, damage removal and damage tolerance are the three main surveillance mechanisms of the genome.
DNA damage reversal is when a spontaneous point mutation is corrected by single enzymes, without breaking the DNA backbone (Eker et al. 2009). DNA reversal is subdivided into two further categories; Photo-reactivation and Restoration of damaged bases by alkyltransferases and dioxygenases (Christmann et al. 2003). Photo-reactivation uses enzymes such as spore photoproduct lysase and photolysase to correct DNA lesions created by Ultraviolet radiation. Spore photoproduct lysase is specific for spore photoproducts which may be present in bacterial cells. Photolysases are flavoproteins which are specific for cyclobutane pyrimidine dimmers (CPDs). Photolyse in mammals has been a confliction issue. It is argued whether genome photoprotection was lost through evolution, since mammals remove photolesions in the DNA by the nuclear excision pathway, which belongs to the damage removal category (Eker et al. 2009).
DNA damage tolerance is one of the post-replicative mechanisms conserved from prokaryotes to eukaryotes. It bypasses any mismatch DNA lesions formed during replication, without the lesions actually being removed (Chang and Cimprich 2009). It is made up of two main mechanisms called DNA transletion synthesis (TLS) and template switching. In transletion synthesis the damaged DNA template is bypassed when low fidelity TLS DNA polymerases, such as Rev1, replicate over it. In contrast during template switching an undamaged DA template is used for replication in order to avoid the lesion (Friedberg 2005).
DNA damage removal is subdivided into three main mechanisms; Nucleotide excision repair (NER), base excision repair (BER) and Mismatch repair mechanism (MMR) (Christmann et al. 2003). BER is the simplest mechanism out of the three. In BER the mismatched base is removed by DNA glycosylase forming an abasic site (AP). Then DNA polymerases fill in the AP site and DNA seals the break in the strand. NER is a bit more complicated but follows the same principle (Germann, Johnson and Spring 2010). NER mechanism involves about 30 proteins which recognise DNA lesions instead of single base pair mismatches. When the protein recognises a lesion there is a repair protein complex assortment and incision on both sides of the lesion by an exonuclease of about 20 to 24 bases. Then the gap is filled by DNA polymerase and sealed with DNA ligase as in BER(Christmann et al. 2003). The last of the damage removal mechanisms is MMR.
Mismatch repair pathway (MMR) also known as "DNA spell checking", and is a highly conserved DNA repair mechanism from prokaryotes to eukaryotes. The majority of studies over the 50 years of research were performed on E.coli (Martin et al. 2010). It was the simplest model that scientists could use at the time in order to gain a better insight of the MMR pathway and understand how it works. Comparing MMR to the Base excision repair and Nucleotide repair mechanisms it is the only post-replicative mechanism which is targeted exclusively to the daughter strand. Corrections occur when bases fail to form the Watson-Crick base pairing (A-T and C-G)(Martin et al. 2010). In this literature review we will discuss the MMR pathway focusing on MSH2 and MSH3 proteins, their structure, function, deficiencies, and their use in research.
Chapter 1: Mismatch repair pathway outline; conservation from prokaryotes to eukaryotes.
MMR in E.Coli
MMR pathway is a series of cascade events which could be divided into 5 main steps; lesion recognition, strand discrimination, excision, repair and ligation (Hays, Hoffman and Wang 2005). The MMR pathway has been studied most extensively in E.coli than any other organism. E.coli provides the best prototype comparing to the homologous and more complicated eukaryotic MMR systems.
The first step is initiated when MutS homodimer recognises a mismatch, which could either be base-base misspairs or larger insertion-deletion loops (Li 2008). It is then recruited to the site with the help of Î²-clamp accessory protein, binding to the DNA mismatch (Pluciennik et al. 2009). MutS undergoes an ATP conformational change which results in the recruitment of a second dimer MutL (Acharya et al. 2003) . MutS and MutL undergo a second ATP conformational change resulting in the formation of a ternary complex around the DNA mismatch. The second ATP conformational change and interaction of MutS with MutL activates MutH (Spampinato and Modrich 2000).
MutH is a 28 Kdal, type II latent endonuclease responsible for strand discrimination. When it is recruited to the mismatch site it discriminates between parental and daughter strand by a "methylation-dependant" manner which is specific for E.coli and other gram-negative bacteria (Yang 2000) . During DNA replication Dam methylase (deoxyadenine-methytransferase), adds a methyl group to the Adenine of the daughter strand at the GATC site. MutH cleaves and nicks the un-methylated daughter strand at the hemi-methylated site, as it can be seen in figure 1 (Lahue, Au and Modrich 1989).
Using the means of electron microscopy and end-labelling methods in order to map excision tracts, it was proved that a GATC hemi-methylated sequence was enough to direct mismatch repair in gram-negative bacteria. This finding suggested that mismatch repair pathway could function in a bidirectional manner in reference to the mismatch (Grilley, Griffith and Modrich 1993). Apart from the "methyl-dependant" discrimination, strand breaks and discontinuities could also be used for strand discrimination.
Figure Comparison between prokaryotic and eukaryotic MMR. This figure outlines the five main steps of the MMR pathway in E.coli and eukaryotes, showing which proteins are conserved and which proteins are unique for each. As it can be seen MutS and MutL are conserved in both pathways. MutH is only specific for E.coli prokaryotic pathway because it is methyl directed. PCNA, RFC and RPA are proteins only present in the eukaryotic pathway. In the eukaryotic pathway there are two homologous proteins for MutS; Mutsa and MutsÎ². They both follow the same steps but each one deals with different types and sizes of mismatch.C:\Users\Elena\Desktop\mmr PICTURE.jpg
The point where MutH nicked the daughter strand serves as a point of entry for the proteins involved in base excision, repair and ligation. Single strand-DNA- binding protein and DNA helicase II are loaded onto the daughter strand via interactions with MutL to create a single strand DNA (Mechanic, Frankel and Matson 2000). Excision of the daughter strand takes place with the aid of exonucleases of 3' or 5' direction. This depends on the location of the nick made by MutH in reference to the DNA mismatch. If the nick location is in a 3' to 5' direction to the mismatch, then the single stranded DNA will be digested either by ExoI or ExoX. If the nick is in the 5' to 3' direction compared to the mismatch then the ssDNA will be digested by ExoVII or RecJ (Burdett et al. 2001).
The final step of E.coli MMR pathway after the excision of the mismatched bases is to finally repair and re-synthesise the DNA strand. DNA repair is performed by DNA polymerase III and is followed by DNA ligase which essentially gules the parental and daughter strand back together (Lahue et al. 1989).
MMR in eukaryotes
MMR pathway in eukaryotes follows the same principal as the MMR pathway in E.coli which was described earlier, but different proteins are involved. For eukaryotes and more specifically for yeast and humans MutS proteins exist as two different heterodimeric proteins, MutSa (MSH2/MSH6) and MutSÎ² (MSH2/MSH3) (Kolodner and Marsischky 1999). MSH2/MSH6 is specific for recognising single insertion-deletion loops or base-base mismatches and MSH2/MSH3 heterodimer is specific for recognizing bigger insertion-deletion loops, about 16 nucleotides long (McCulloch, Gu and Li 2003) . MutSa could recognise larger insertion-deletion loops but with a much lower affinity than MutSÎ² (Kantelinen et al. 2010). MutsÎ² has also shown ability to repair base-base mismatches but with a much lower affinity than MutSa , and that its binding affinity is reduced significantly when ATP binds to the heterodimer (Harrington and Kolodner 2007). Therefore, depending on the type and size of the mismatch the appropriate heterodimer will be recruited to the site.
Identification and discrimination of the daughter strand in eukaryotes does not occur in a "methylation-dependant" manner as in E.coli. Older studies had suggested that strand discrimination that in eukaryotes was dependant on cytosine methylation, but further studies ruled out this possibility (Fukui 2010). Strand discontinuity which could be identified in the both template and daughter strand by gaps between the okazaki fragments serve as identification signals in eukaryotic and mammalian MMR(Modrich 2006).
When the appropriate MutS homologous heterodimer is recruited to the mismatch lesion site it undergoes an ATP conformational change and forms a clamp around the DNA. A second ATP conformational change results in the recruitment of another heterodimer MutLa, which is also a heterodimer in this case, made up of MLH and either PMS1 (in yeast) or PMS2 (in humans) and forms a ternary complex with the MSH2/MSH3 or MSH2/MSH6 (Wang and Hays 2004). Even if ATP has shown to play a critical role in the MMR pathway it still unknown of how its use by the MMR proteins is used to discriminate between homoduplex and heteroduplex. The main controversial issue regarding ATP is whether MMR is an ATP-driven pathway or an ATP-independent pathway reference. It has not been easy for scientists to discriminate between these two points because it is difficult to isolate in order to study the in-between steps of the pathway in order to study them in greater depth.
The complex described above can slide in either direction of the DNA strand and is known to initiate EXO1 activity. When it encounters a strand discontinuity, EXO1 is recruited to the site and degrades the nicked strand moving towards the mismatch lesion (Genschel, Bazemore and Modrich 2002). The discontinuity strand is usually bound to a proliferating cell nuclear antigen (PCNA). RFC is responsible for loading PCNA onto the DNA helix. PCNA interacts directly with preteins onvolved in all three steps of the MMR such as; MSH3, MSH6,MLH1 and EXO1. PCNA directs the MMR to excise the lagging strand by targeting the ends of okazaki fragments. Proteins such as PCNA,RPA and RFC have only been identified recently in the yeast and human MMR pathway, when methods such as Far Western analysis were used reference.
RPA acts as a regulator by binding to gaps and controlling EXO1 movement. It either supresses MutSa.EXO1 complex activity or restrics hydrolytic activity on excision products. The single-stranded DNA is stabilised by replication protein-A (RPA) which allows further EXO1 degradation (Lee and Alani 2006). EXO1 is degraded before it reaches the mismatch lesion. The DNA is unwound and the single-strand nick is filled by DNA polymerase III and sealed with DNA ligase (Genschel and Modrich 2006).
As it can be seen from figure 1 prokaryotic and eukaryotic MMR share homologous proteins between them, but eukaryotic MMR being a more evolved pathway has evolved several other proteins such as PCNA, RPA and RFC that associate with the main conserved proteins; MutS and MutL. Compairing between MutS and its holologous proteins in eukaryotes MSH2/MSH3 is the heterodimer which scientists are most uncertain about. Its sturcture and way of function is defferent from MutS and MSH2/MSH6. This details concerning this are expoited in the following sections.
Chapter 2: MutS homologous, details on structure and function
MMR proteins have been studied extensively over the years in order to determine details about their structure, and how the structure relates to their function. Since the MMR pathway has been conserved from prokaryotes to eukaryotes, the homologous proteins involved in both pathways are found to share a basic common structure (Obmolova et al. 2000). Studying of E.coli and Saccharomyces Servisiae has given a better insight to mammalian MMR pathway and its proteins (Kunkel and Erie 2005). C:\Users\Elena\Desktop\figure 2.jpg
Figure Adapted from (Sixma 2001) MutS homodimer. MutS is made up of two identical subunits, each comprising of 5 domains. Every domain has its own role in the regulation of mismatch repair. The five different domains are outlined in different colours showing the biochemical structure of MutS. This structure said to resemble the shape of inverted commas.MutS is a modular homodimer that regulates mismatch recognition in prokaryotes by bending and unbending of the DNA homoduplex (Wang et al. 2003). MutS subunits are presented in literature as a structure representing "inverted commas" or "praying hands" (Lamers et al. 2000). It is a protein that has an overall negative surface charge apart from the top part of the "inverted commas" where the groove is situated, which has a positive charge (Obmolova et al. 2000).Each of the five domains of the MutS protein have a distinct role in the MMR mechanism and any mutation of these domains could lead to impairment of the pathway.
MutS being a homodimer, is made up of two identical subunits (A and B), and each of the subunits is made up of five flexible domains.(Blackwell et al. 2001). Domain I, otherwise called the "mismatch binding" and domain and domain IV, "DNA clamp". Domain I is a globular domain situated at the N-terminus of the minor groove site (Sixma 2001), formed by six-stranded mixed Î²-sheets which are in turn surrounded by four a-helices. Domain IV, "DNA clamp", is formed by four stranded antiparallel Î²-sheets and is situated on the major groove side of the MutS dimer (Lamers et al. 2000) . When DNA binding takes place Domain I of the MutS A subunit and Domain IV of the MutS B subunit are the ones that interact with mismatch.
In E.coli Domain I donates Phe36 or Phe39 in order to interact with the mismatch nucleotides, which is considered to play an important role for recognition by base stacking (Lee, Surtees and Alani 2007). Domain IV of the MutS B subunit is positioned opposite domain I of the MutS subunit. It uses Î²-sheet turn to make contact with the DNA backbone. The opposite domains to the ones mentioned above ( domain IV of MutS A subunit and domain I of MutS B subunit) are not attached in DNA binding and are partially distorted (Obmolova et al. 2000). The asymmetric bindings of the domains results in a 60Ëš bent in the DNA (Wang et al. 2003).
Domains II "connector" and III "core" are responsible of the transmission of the allosteric signal coming from the bound DNA co-factor to Domain V "ATP-ase". Domain II is made up of parallel Î²-sheets which are surrounded by four a-helices, as in the "mismatch binding" domain. As with all five domains of the MutS dimmer, it is found that Domain II resembles another structure, in this case it was found to resemble the Holiday junction resolvase (ruvC) (Lamers et al. 2000). Domain III is the only out of the five domains of MutS that is completely made up of a-helices. It is made up of two sequenced regions that form an a-helical bundle. Since it is the central structure of MutS it directly connects to II, IV and V domains by the means of peptide bonding. It attaches to the DNA backbone through protein-DNA interfaces.
When DNA binds to the MutS dimer the A and B subunits rotate outwards to facilitate the DNA binding. This rotation is a result of antiparallel helicies formed between the two V domains, VaD and VaE. There two helices are responsible for bending the DNA in a helix -turn - helix motif. Domain V confronts to a structure also known as the Walker motif and resembles the structure of ABC transporters because of the ATPase binding site (Obmolova et al. 2000). This ATPase binding site is essential for controlling the MMR mechanism. There are two ATPase binding sites in each MutS homodimer, one in each subunit. They have been found to be responsible for asymmetric binding in mismatch. In reference to the biochemical basis of this activity Arg697 seems to play a key role in the asymmetric binding. It is present part of the structure in both subunits and achieves ATP binding asymmetry by promoting binding in one subunit when in the meantime preventing hydrolysis in the other subunit. Any alteration in Arg697 could cause problems to the whole pathway (Lamers, Winterwerp and Sixma 2003).
MSH2/MSH3 heterodimer : structure and function
As explained earlier on, eukaryotic MMR comprises of two homologous heterodimers in correspondence to MutS; MutSa (MSH2/MSH6) and MutSÎ² (MSH2/MSH3). These two heterodimers share MSH2 as a common part of their structure, but have different lesion specificity. MSH3 and MSH6 are the subunits responsible for binding DNA mismatches, and MSH2 is the subunit responsible for stabilisation of the conformational changed DNA (Lee et al. 2007). Since MSH2/MSH3 and MSH2/MSH6 have different binding specificities it is expected that they will have difference in structure.
MutS and MSH2/MSH6 both bind DNA in an asymmetric manner. It has been identified that MSH2/MSH6 complex is found at a 6-10 folder greater than MSH2/MSH3 in humans, meaning that MSH2/MSH6 is the most abundant heterodimer between the two (Harrington and Kolodner 2007). Studies have revealed that prokaryotic MutS is structurally homologous to MSH2/MSH6, but not to MSH2/MSH3. This is because MSH3 and MSH6 have very little sequence homology concerning their DNA binding domains, due to the fact that the they are specific for different types of lesions (Shell, Putnam and Kolodner 2007) .
The reason for difference in binding specificity between the two proteins is not understood very well, but it is thought to be connected with the specific amino acid sequencing that is present in each of the two proteins. In respect to their biochemical structure that MSH6 recognises mismatches in the Phe-X-Glu motif, which is the same as in MutS, but in contrast MSH3 uses Lys-X-Lys/Arg motif (Lyer et al. 2006). Tyr42 substitutes Phe39 in MSH2 and brings out another controversial issue, of whether Tyr42 plays any role in MSH2/MSH3 activity regarding MMR pathway (Lee et al. 2007). Even if MSH3 lacks Phe, it has six other conserved residues that allow it to attach onto the DNA backbone (Alani et al. 2003). This gives a slightly better understanding that even if MutSa and MutSÎ² are able to correct the same mismatch, the mechanism used is different.
MSH2/MSH3 heterodimer was studied mostly in yeast and human MMR pathways, which conferred that the nucleotide binding activity occurs in a similar manner in both organisms. MSH2/MSH3 is known to repair IDLs but it has also shown ability to bind differently to the ATP and ADP nucleotide in comparison to MutS and MSH2/MSH6. Both MSH2 and MSH3 seem to have similar binding affinities for ATP and ADP, therefore the binding was said to happen in a stochastic, otherwise random manner (Owen, Lang and McMurray 2009). ATP and ADP bind to the walker motif on fifth domain, but do not bind to both subunits simultaneously, even if they showed not to have any apparent discrimination between MSH2 and MSH3.
In solution ADP-bound forms of the MSH2/MSH3 heterodimer were stable, and could not be replaced with ATP, even if it was found in excess concentration. ATP binding on the other hand could displace ADP from the MSH3 subunit when it bound to MSH2. Further studies performed to investigate the ADP/ATP nucleotide binding dynamics in the presence of DNA. These studies revealed that when DNA binds to MSH2/MSH3 it alters the stochastic ADP/ATP binding, decreasing ADP affinity in MSH3 to a greater extent than in MSH2, thus resulting in ADP-MSH2-MSH3- empty intermediate. When ATP binds and hydrolyses MSH3 this initiates an ADP/ATP exchange in MSH2 (Owen et al. 2009). Human MSH2/MSH3 also seems to be involved in trinucleotide DNA expansion. CAG-hairpin plays a main role in this process. If CAG-hairpin binds to MSH2/MSH3 complex this could result in inhibition of ATP hydrolysis which could lead to inhibition to normal mismatch repair (Owen et al. 2005).
When mismatch binding is initiated in the MMR pathway, DNA binding to the heterodimer induces a cycle of ATP/ADP hydrolysis in reference MSH2/MSH3. Both MSH2/MSH3 subunits have shown to participate in ATP hydrolysis but also in ADP retainment. ATP binding in the other hand could displace ADP from the MSH3 subunit when it bound to MSH2. When MSH2/MSH3 bound to DNA, this caused a decrease of ATP affinity in both subunits, affecting MSH3 to a greater extent than MSH2. This finding suggested that only ADP-MSH2-MSH3-empty complex could bind in a stable manner to the mismatch lesion. In solution ADP-bound forms of the MSH2/MSH3 heterodimer were stable, and could not be replaced with ATP, even if it was found in excess concentration. ATP binding though could displace ADP from the MSH3 subunit when it bound to MSH2 (Owen et al. 2009).
Even if MSH2/MSH3 complex has been described in literature with reference to the MutS and MSH2/MSH6 model, not everything concerning function and structure could be explained (Lee et al. 2007). There are still many unanswered questions concerning ATP regulation, MSH3 specific recognition, MSH3 structure and biochemical differences concerning domain specificity (Kantelinen et al. 2010). Generation of knockout mice and in vivo analysis of purified MSH2 ad MSH3 proteins is used to provide a better insight of how these proteins work and provide greater depth of MMR pathway regulation (Gallitaliadoros et al. 1995).
Chapter 3: MSH2/MSH3 deficiencies
Problems caused by MSH2/MSH3 deficiencies
Maintaining genome integrity and stability is important for the normal functioning of an organism. Losing any part of the MMR pathway could jeopardise maintaining genome fidelity (Edelbrock, Kaliyaperumal and Williams 2009). MSH2 and MSH3 proteins are key regulators of the initiation process of MMR and therefore crucially important for the whole pathway. Any genetic alterations that could lead to MMR deficiency in these proteins could have adverse effects on the genome (Hsieh and Yamane 2008).
Through research it was identified that MMR deficient cells show microsatellite instability. The different types and degrees of microsatellite instability (MSI) depend upon the gene affected which causes deletion of MMR proteins. MSH2 and MSH2/MSH3 deficiency leads to severe microsatellite instability embryonic stem cells. MSH2 resulted in base substitutions and frameshift mutations leading to mononucleotide and dinucleotide repeats in tumours. Recent study results have shown that MSH3 deficiency is responsible for dinucleotide MSI instability, but not mononucleotide repeat instability(Abuin, Zhang and Bradley 2000). When embryonic stem cells are deficient to these key regulator proteins MMR does not function properly. It leads to lack of error recognition and repair, which in turn leads to increase of size of these error repeats. Difference in nucleotide repeats in oncogenes and tumour suppressants could lead to pathogenic problems reference .
Mammalian cell lines which are deficient in MSH2 protein show increased resistance to killing by 6-thioguanine (6TG) and similar drugs. Comparing embryonic stem cells deficient for MSH3 and MSH2 it was observed that MSH2 deficient cells showed a bigger resistance to killing than MSH3. When 6TG is incorporated into the DNA upon replication, its methylation, leads to a methylated 6TG/T mismatch. The mismatch would be normally recognised by MutSa (MSH2/MSH6) but when a cell is deficient to MSH2, the MMR initiation step does not occur in the expected manner. This mismatch is then replicated leading to hypermutability and resistance to killing. Resistance to cytotoxic killing drugs could be considered of great clinical importance, such in transplant patients (Abuin et al. 2000).
Due to the fact that MSH2 is the central MMR protein, cells that are deficient for it would lead to a mutator phenotype, in contrast MSH3 deficiencies would lead to a weak mutator type. Studies in mouse embryonic stem cells showed that Apc-/-MSH2-/- deficient mice had developed intestinal adenomas, and that there were several number of Apc mutations per adenoma tested (Sohn et al. 2003). Also, germline and somatic mutations in embryonic stem cells lacking MSH2 indicated that there is a link between MSH2 and Hereditary Non-polyposis colon cancer syndrome (Vaish 2007). In reference to studies performed on embryonic stem cells derived from the 129 murine strain it was demonstrated that MSH2 plays a role in preventing homologous recombination, thus its deficiency results in the opposite effect, hyper-recombination (de Wind et al. 1995).
Further studies in mice derived from 129 x C57BL/6 strains have been performed in order to investigate MSH2 deficiency in areas such as spontaneous mutations (Burr et al. 2007), alteration of Pre-B-cells(Jenab-Wolcott et al. 2000) and the effect on immunoglobulin class switch in relation to hypermutation (Ehrenstein and Neuberger 1999). All three studies revealed that MSH2 deficiency seemed to play the key role in the problems generated. MSH2 deficient homozygous mice showed increase rates of spontaneous mutation when in contrast MSH2 deficient heterezygous mice that did not seem to have a significant increase in mutation rates (Burr et al. 2007). MSH2 deficiency is known to cause loss of function of the p53 tumour suppressor protein. This in turn leads to increase resistance to apoptosis. In relevance to the alteration of Pre-B-Cell transformation study, it is noted that when Pre-B-Cell come into contact with Abelson Virus it is transformed and undergoes apoptosis. Comparing to other cells in MSH2 deficient mice, Pre-B-Cells recovers from apoptosis due to lack of p53 factor (Jenab-Wolcott et al. 2000).
An interesting approach to this area of research was studying the effect of MSH2 deficiency concerning a different subject area, such as immunology. Due to MSH2 deficiency causing hypermutation MSH2 was suggested to have role in class-switch recombination, when B cells are stimulated and change the immunoglobulin on their surface from IgM B to IgE, IgA or IgG. Because of this effect MSH2 deficient mice produced a significant lower amount of IgG immunoglobulin even if their IgM levels were marked as normal (Ehrenstein and Neuberger 1999).
As it can be seen from the various studies over the years MSH2 and MSH3 deficiencies can cause many problems and many pathogenic problems apart from cancer, which seems to be the most obvious and extensively studied pathogenesis in relation to MSH2 deficiency (Zhang et al. 2001). When comparing between MSH2, MSH3 and MSH2/MSH3 deficiencies it could be clearly identified that embryonic stem cells deficient for both MSH2/MSH3 heterodimer showed a more significant increase in mutation rate than cells deficient for either of the proteins.
MSH2/MSH3 deficiencies used in the advantage of science
Despite the bad effects caused by deficiencies in MSH2 and MSH3, scientists have generated embryonic stem cells deficient to these to proteins for research purposes. MSH2/MSH3 deficient cells could be used to get a better insight of the MMR pathway and also to express desirable phenotypes in transgenic mice that could not be achieved when MSH2 and MSH3 proteins are present.
Transgenic mice have been used widely in research because they are an easy model to work with. They are the closest model which is easily handled in laboratories, that resembles mammalian, thus human genome. Knockout mice have been widely used in phenotyping (Baribault and Kemler 1989). Scientists knock out genes from embryonic stem cells of mouse strains and see how the mouse if affected by the deletion of a specific gene. This allowed scientists to investigate complex biological systems, by dissecting the function of individual components. Some gene deficiencies have helped us understand how various proteins and pathways work, such as genes in diabetes, obesity, alcohol tolerance, the MMR pathway and many more reference.
C57BL/6 and 129 are both non-autoimmune strains that are widely used for generating gene-targeted animals (Heidari et al. 2006). Studies going back to the 80's have shown that murine pluripotent stem cells such as these two strains could be used to pass a genetic alteration to an offspring through the germline of the transgenic mouse (Bradley et al. 1984) . It is important to establish the origin of the embryonic stem cells used in specific experiments in order to determine whether the results obtained from transgenesis could be improved by exploiting the precursor cells involved for generating the strain used (Brook and Gardner 1997).
There are various ways of how the gene targeting processes are carried out, such as, the use of a targeting construct and oligonucleotide-mediated gene targeting. A targeting construct is used to target the desired chromosomal locus. In this targeting construct a selected marker gene such as a neomycin gene is flanked with DNA sequences that are homologous to that of the locus. Therefore when the genetic material of the targeting construct is enters the cell homologous recombination will take place between the DNA sequences is the marker gene and those of the chromosomal locus. This results in incorporation of the new genetic material into the genome and disruption of the targeted gene (Capecchi 1989).
In order to get an efficient and yet fast generation of mouse mutants oligonucleotide-mediated gene targeting is usually used. It is an effective method that uses a stretch of gene sequence information to alter the sequencing of a gene in order to provide the desired effect (Dekker, Brouwers and Riele 2003). Oligonucleotide-mediated gene targeting has been studied extensively on prokaryotic and eukaryotic organisms such as; Saccharomyces cervisiae, E.coli and mice. Oligonucleotide-mediated gene targeting, otherwise known as oligotargeting, uses three main types of oligonuclotides including single-stranded, chimeric DNA/RNA and triple-helix forming (Dekker et al. 2006) . This method is also used to target genes of a specific chromosomal locus. Oligotargeting can take place as a base insertions or base substitutions (Dekker et al. 2003). The efficiency of this method is strongly dependant of the number of nucleotides used to target a sequence, how complicated they are and if the embryonic stem cell is deficient in MSH2/MSH3 (Dekker et al. 2006).
MSH2/MSH3 is known to correct IDLs therefore when an oligonucleotide is induced in a sequence MMR proteins recognise it and initiate the MMR repair cascade events, resulting in the oligonucleotide excision. Therefore as it has been identified from various studies MMR proteins and specifically MSH2/MSH3 suppress oligotargeting by blocking the fusion of the new genetic information and homologous recombination of the induced nucleotides. Studies performed in MSH2 deficient embryonic stem cells have shown that oligotargeting efficiency increased significantly, thus proving its suppression by MSH2 (Dekker et al. 2006).
The main aim of the laboratory project is to investigate the function of Î²-defensins. These are small antimicrobial peptides which play key role in an organisms innate immunity (Cagliani et al. 2008). They have six conserved cystiene residues and are a cationic polypeptide of about 4-5 kDa in humans. Beta-defensins were firstly isolated from a patient's psoriatic lesion (Hinrichsen et al. 2008). They are present in epithelial cells of many organs such as trachea, lungs, skin, thymus, and intestine. Beta-defensins seem to play an important role in innate immunity but have also shown ability to initiate inflammatory and adaptive immune responses. Beta-defensins are not only important for humans but for plants and animals . Therefore identifying their function in vivo is important. Beta-defensin 14 in mice seems to be identified as the orthologue of human beta-defensin 3. Therefore by studying the functions of beta-defensin 14 in mice could provide a great insight towards the human orthologue, which in turn could be used in reference to future studies and clinical application (Roehrl et al. 2008).
In order to investigate the function of beta defensins MSH2 deficient mice need to be generated of the C56BL/6 and 129 cell line. This can be done by the oligotargeting method described above. When MSH2 deficient embryonic stem cells are generated oligotargeting will be used again to inactivate the beta defensin gene cluster . C56BL/6 and 129 murine strains have shown to have a predisposition to autoimmune phenotypes (Carlucci et al. 2007). Also MSH2 deficiency may cause rise of other mutations apart from beta defensin. These points should be taken into account because if undesired phenotypes accumulate along with the desired phenotype of beta-defensin knockout, it could shadow the results and not lead to a clear observation of beta-defensin function.
Abuin, A., H. J. Zhang & A. Bradley (2000) Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations. Molecular and Cellular Biology, 20, 149-157.
Acharya, S., P. L. Foster, P. Brooks & R. Fishel (2003) The coordinated functions of the E-coli MutS and MutL proteins in mismatch repair. Molecular Cell, 12, 233-246.
Alani, E., J. Y. Lee, M. J. Schofield, A. W. Kijas, P. Hsieh & W. Yang (2003) Crystal structure and biochemical analysis of the MutS center dot ADP center dot Beryllium fluoride complex suggests a conserved mechanism for ATP interactions in mismatch repair. Journal of Biological Chemistry, 278, 16088-16094.
Baribault, H. & R. Kemler (1989) EMBRYONIC STEM-CELL CULTURE AND GENE TARGETING IN TRANSGENIC MICE. Molecular Biology & Medicine, 6, 481-492.
Blackwell, L. J., K. P. Bjornson, D. J. Allen & P. Modrich (2001) Distinct MutS DNA-binding modes that are differentially modulated by ATP binding and hydrolysis. Journal of Biological Chemistry, 276, 34339-34347.
Bradley, A., M. Evans, M. H. Kaufman & E. Robertson (1984) FORMATION OF GERM-LINE CHIMERAS FROM EMBRYO-DERIVED TERATOCARCINOMA CELL-LINES. Nature, 309, 255-256.
Brook, F. A. & R. L. Gardner (1997) The origin and efficient derivation of embryonic stem cells in the mouse. Proceedings of the National Academy of Sciences of the United States of America, 94, 5709-5712.
Burdett, V., C. Baitinger, M. Viswanathan, S. T. Lovett & P. Modrich (2001) In vivo requirement for RecJ, ExoVII, ExoI, and ExoX in methyl-directed mismatch repair. Proceedings of the National Academy of Sciences of the United States of America, 98, 6765-6770.
Burr, K. L. A., A. van Duyn-Goedhart, P. Hickenbotham, K. Monger, P. P. W. van Buul & Y. E. Dubrova (2007) The effects of MSH2 deficiency on spontaneous and radiation-induced mutation rates in the mouse germline. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 617, 147-151.
Cagliani, R., M. Fumagalli, S. Riva, U. Pozzoli, G. P. Comi, G. Menozzi, N. Bresolin & M. Sironi (2008) The signature of long-standing balancing selection at the human defensin beta-1 promoter. Genome Biology, 9.
Capecchi, M. R. (1989) ALTERING THE GENOME BY HOMOLOGOUS RECOMBINATION. Science, 244, 1288-1292.
Carlucci, F., J. Cortes-Hernandez, L. Fossati-Jimack, A. E. Bygrave, M. J. Walport, T. J. Vyse, H. T. Cook & M. Botto (2007) Genetic dissection of spontaneous autoimmunity driven by 129-derived chromosome 1 loci when expressed on C57BL/6 mice. Journal of Immunology, 178, 2352-2360.
Chang, D. J. & K. A. Cimprich. 2009. DNA damage tolerance: when it's OK to make mistakes. In Nat Chem Biol, 82-90. United States.
Christmann, M., M. T. Tomicic, W. P. Roos & B. Kaina (2003) Mechanisms of human DNA repair: an update. Toxicology, 193, 3-34.
de Wind, N., M. Dekker, A. Berns, M. Radman & H. te Riele (1995) Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer. Cell, 82, 321-330.
Dekker, M., C. Brouwers, M. Aarts, J. van der Torre, S. de Vries, H. V. de Vrugt & H. te Riele (2006) Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3. Gene Therapy, 13, 686-694.
Dekker, M., C. Brouwers & H. T. Riele (2003) Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides. Nucleic Acids Research, 31.
Edelbrock, M. A., S. Kaliyaperumal & K. J. Williams (2009) DNA mismatch repair efficiency and fidelity are elevated during DNA synthesis in human cells. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, 662, 59-66.
Ehrenstein, M. R. & M. S. Neuberger (1999) Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation. Embo Journal, 18, 3484-3490.
Eker, A. P., C. Quayle, I. Chaves & G. T. van der Horst (2009) DNA repair in mammalian cells: Direct DNA damage reversal: elegant solutions for nasty problems. Cell Mol Life Sci, 66, 968-80.
Friedberg, E. C. 2005. Suffering in silence: the tolerance of DNA damage. In Nat Rev Mol Cell Biol, 943-53. England.
Fukui, K. (2010) DNA mismatch repair in eukaryotes and bacteria. J Nucleic Acids, 2010.
Gallitaliadoros, L. A., J. D. Sedgwick, S. A. Wood & H. Korner (1995) GENE KNOCK-OUT TECHNOLOGY - A METHODOLOGICAL OVERVIEW FOR THE INTERESTED NOVICE. Journal of Immunological Methods, 181, 1-15.
Genschel, J., L. R. Bazemore & P. Modrich (2002) Human exonuclease I is required for 5 ' and 3 ' mismatch repair. Journal of Biological Chemistry, 277, 13302-13311.
Genschel, J. & P. Modrich. 2006. Analysis of the Excision Step in Human DNA Mismatch Repair. In Methods in Enzymology, ed. a. P. M. Judith Campbell, 273-284. Academic Press.
Germann, M. W., C. N. Johnson & A. M. Spring (2010) Recognition of damaged DNA: structure and dynamic markers. Med Res Rev.
Grilley, M., J. Griffith & P. Modrich (1993) BIDIRECTIONAL EXCISION IN METHYL-DIRECTED MISMATCH REPAIR. Journal of Biological Chemistry, 268, 11830-11837.
Harrington, J. A. & R. D. Kolodner (2007) Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs. Molecular and Cellular Biology, 27, 6546-6554.
Hays, J. B., P. D. Hoffman & H. X. Wang (2005) Discrimination and versatility in mismatch repair. DNA Repair, 4, 1463-1474.
Heidari, Y., A. E. Bygrave, R. J. Rigby, K. L. Rose, M. J. Walport, H. T. Cook, T. J. Vyse & M. Botto (2006) Identification of chromosome intervals from 129 and C57BL/6 mouse strains linked to the development of systemic lupus erythematosus. Genes and Immunity, 7, 592-599.
Hinrichsen, K., R. Podschun, S. Schubert, J. M. Schroder, J. Harder & E. Proksch (2008) Mouse beta-defensin-14, an antimicrobial ortholog of human beta-defensin-3. Antimicrobial Agents and Chemotherapy, 52, 1876-1879.
Hsieh, P. & K. Yamane (2008) DNA mismatch repair: molecular mechanism, cancer, and ageing. Mechanisms of ageing and development, 129, 391-407.
Jenab-Wolcott, J., D. Rodriguez-Correa, A. H. Reitmair, T. Mak & N. Rosenberg (2000) The absence of Msh2 alters abelson virus Pre-B-Cell transformation by influencing p53 mutation. Molecular and Cellular Biology, 20, 8373-8381.
Kantelinen, J., M. Kansikas, M. K. Korhonen, S. Ollila, K. Heinimann, R. Kariola & M. Nystrom (2010) MutS beta exceeds MutS alpha in dinucleotide loop repair. British Journal of Cancer, 102, 1068-1073.
Kolodner, R. D. & G. T. Marsischky (1999) Eukaryotic DNA mismatch repair. Current Opinion in Genetics & Development, 9, 89-96.
Kunkel, T. A. & D. A. Erie (2005) DNA mismatch repair. Annual Review of Biochemistry, 74, 681-710.
Lahue, R. S., K. G. Au & P. Modrich (1989) DNA MISMATCH CORRECTION IN A DEFINED SYSTEM. Science, 245, 160-164.
Lamers, M. H., A. Perrakis, J. H. Enzlin, H. H. K. Winterwerp, N. de Wind & T. K. Sixma (2000) The crystal structure of DNA mismatch repair protein MutS binding to a G[middot]T mismatch. Nature, 407, 711-717.
Lamers, M. H., H. H. K. Winterwerp & T. K. Sixma (2003) The alternating ATPase domains of MutS control DNA mismatch repair. Embo Journal, 22, 746-756.
Lee, S. D. & E. Alani (2006) Analysis of Interactions Between Mismatch Repair Initiation Factors and the Replication Processivity Factor PCNA. Journal of Molecular Biology, 355, 175-184.
Lee, S. D., J. A. Surtees & E. Alani (2007) Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition. Journal of Molecular Biology, 366, 53-66.
Lyer, R. R., A. Pluciennik, V. Burdett & P. L. Modrich (2006) DNA mismatch repair: Functions and mechanisms. Chemical Reviews, 106, 302-323.
Martin, L. M., B. Marples, M. Coffey, M. Lawler, T. H. Lynch, D. Hollywood & L. Marignol. 2010. DNA mismatch repair and the DNA damage response to ionizing radiation: making sense of apparently conflicting data. In Cancer Treat Rev, 518-27. Netherlands: 2010 Elsevier Ltd.
McCulloch, S. D., L. Y. Gu & G. M. Li (2003) Nick-dependent and -independent processing of large DNA loops in human cells. Journal of Biological Chemistry, 278, 50803-50809.
Mechanic, L. E., B. A. Frankel & S. W. Matson (2000) Escherichia coli MutL loads DNA helicase II onto DNA. Journal of Biological Chemistry, 275, 38337-38346.
Modrich, P. (2006) Mechanisms in eukaryotic mismatch repair. Journal of Biological Chemistry, 281, 30305-30309.
Nagy, Z. & E. Soutoglou. 2009. DNA repair: easy to visualize, difficult to elucidate. In Trends Cell Biol, 617-29. England.
Obmolova, G., C. Ban, P. Hsieh & W. Yang (2000) Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature, 407, 703-710.
Owen, B. A. L., W. H. Lang & C. T. McMurray (2009) The nucleotide binding dynamics of human MSH2-MSH3 are lesion dependent. Nature Structural & Molecular Biology, 16, 550-557.
Owen, B. A. L., Z. Y. Yang, M. Y. Lai, M. Gajek, J. D. Badger, J. J. Hayes, W. Edelmann, R. Kucherlapati, T. M. Wilson & C. T. McMurray (2005) (CAG)(n)-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition. Nature Structural & Molecular Biology, 12, 663-670.
Pluciennik, A., V. Burdett, O. Lukianova, M. O'Donnell & P. Modrich (2009) Involvement of the beta Clamp in Methyl-directed Mismatch Repair in Vitro. Journal of Biological Chemistry, 284, 32782-32791.
Roehrl, J., D. Yang, J. J. Oppenheim & T. Hehlgans (2008) Identification and biological characterization of mouse beta-defensin 14, the orthologue of human beta-defensin 3. Journal of Biological Chemistry, 283, 5414-5419.
Schneider, S., S. Schorr & T. Carell (2009) Crystal structure analysis of DNA lesion repair and tolerance mechanisms. Current Opinion in Structural Biology, 19, 87-95.
Shell, S. S., C. D. Putnam & R. D. Kolodner (2007) Chimeric Saccharomyces cerevisiae Msh6 protein with an Msh3 mispair-binding domain combines properties of both proteins. Proceedings of the National Academy of Sciences of the United States of America, 104, 10956-10961.
Sixma, T. K. (2001) DNA mismatch repair: MutS structures bound to mismatches. Current Opinion in Structural Biology, 11, 47-52.
Sohn, K. J., M. Choi, J. Song, S. F. Chan, A. Medline, S. Gallinger & Y. I. Kim (2003) Msh2 deficiency enhances somatic Apc and p53 mutations in Apc+/-Msh2-/- mice. Carcinogenesis, 24, 217-224.
Spampinato, C. & P. Modrich (2000) The MutL ATPase is required for mismatch repair. Journal of Biological Chemistry, 275, 9863-9869.
Vaish, M. (2007) Mismatch repair deficiencies transforming stem cells into cancer stem cells and therapeutic implications. Molecular Cancer, 6.
Wang, H., Y. Yang, M. J. Schofield, C. W. Du, Y. Fridman, S. D. Lee, E. D. Larson, J. T. Drummond, E. Alani, P. Hsieh & D. A. Erie (2003) DNA bending and unbending by MutS govern mismatch recognition and specificity. Proceedings of the National Academy of Sciences of the United States of America, 100, 14822-14827.
Wang, H. X. & J. B. Hays (2004) Signaling from DNA mispairs to mismatch-repair excision sites despite intervening blockades. Embo Journal, 23, 2126-2133.
Yang, W. (2000) Structure and function of mismatch repair proteins. Mutation Research-DNA Repair, 460, 245-256.
Zhang, S. L., R. Lloyd, G. Bowden, B. W. Glickman & J. G. de Boer (2001) Msh2 DNA mismatch repair gene deficiency and the food-borne mutagen 2-amino-1-methyl-6-phenolimidazo 4,5-b pyridine (PhIP) synergistically affect mutagenesis in mouse colon. Oncogene, 20, 6066-6072.