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MSH6 is an obligate partner in mismatch repair mediated mutation surveillance : an in vivo study Mark, Sean Christian 2002

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M S H 6 IS A N O B L I G A T E P A R T N E R I N M I S M A T C H R E P A I R M E D I A T E D M U T A T I O N S U R V E I L L A N C E : A N IN VIVO S T U D Y B y Sean Christian Mark B . S c , The University of Victoria, 1998 A THESIS S U M B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E I N T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Genetics Graduate Program) We accept this thesis as conforming to the required standard A T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A p r i l 2002 © Sean Christian-Mark, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of C^e^S^CS> C\ aJi^ob*- ^ 0 The University of British Columbia Vancouver, Canada Date DE-6 (2/88) Abstract The D N A mismatch repair ( M M R ) system is primarily responsible for purging newly synthesized D N A of errors incurred during semi-conservative replication. Lesion recognition is initially carried out by one of two heterodimeric protein complexes, M u t S a or MutSp\ While the former, comprised of M S H 2 and M S H 6 , recognizes mispairs as well as short (1-2 nucleotide) insertions/deletions (IDLs), the latter, made up of M S H 2 and M S H 3 , is primarily responsible for recognizing 2-6 nucleotide IDLs. A s most of the functional information of these heterodimers is derived from in vitro studies, it was of interest to study the in vivo consequences of a lack of M u t S a . To this end, B i g Blue™ mice, that carry a lacf transgenic A, shuttle-phage mutational reporter, were crossed with Msh6'~ mice to evaluate the specific contribution of M u t S a to genome integrity. Consistent with the importance of M u t S a in lesion surveillance, small intestine epithelial cell D N A derived from lacf Msh6'~ mice exhibited striking increases (average of 41-fold) in spontaneous mutant frequencies. Furthermore, the lad gene mutation spectrum was dominated by G : C to A : T transitions, highlighting the critical importance of the M u t S a complex in preventing this frequently observed type of spontaneous mutation. i i Table of Contents Abstract ii Table of Contents iii List of Figures v List of Tables vi List of Abbreviations vii Acknowledgements viii Dedication ix Chapter 1 Introduction 1 1.1 D N A damage and repair pathways 1 1.2 The D N A mismatch repair system 2 1.2.1 Mismatch repair in Escherichia coli 2 1.2.2 Eukaryotic mismatch repair 4 1.3 Mismatch repair deficiency and carcinogenesis 7 1.4 MMR-deficient mouse models 9 1.5 Transgenic shuttle phage in vivo mutation detection systems 10 1.6 Thesis goals 13 1.7 Thesis summary 13 Chapter 2 Materials and methods 14 2.1 Transgenic mice 14 2.1.2 Breeding strategies 14 2.1.3 Chemicals and reagents 14 2.1.4 Preparation of tail D N A for genotype testing of mice 14 2.1.5 Determination of the Ms/26 genotype 14 2.1.6 Determination of the lad genotype 15 2.1.7 Agarose gel electrophoresis 15 2.1.8 Obtaining mouse tissues 16 2.1.9 Isolation of small intestine epithelial cells 16 2.2 Measurement of l a d mutational frequency 16 2.2.1 Isolation of genomic D N A 16 2.2.2 Maintenance o f the SCS-8 cells 18 2.2.3 Preparing the SCS-8 plating culture 18 i i i 2.2.4 Performing the packaging reaction 18 2.2.5 Plating the packaged D N A samples 19 2.2.6 Screening and analyzing assay trays 19 2.2.7 Verifying putative mutant plaques 19 2.2.8 Statistical analysis of mutant frequencies 20 2.3 Determination of lad mutation spectrum 20 2.3.1 Amplification of mutant lad genes 20 2.3.2. Purification of lad template for sequencing '. 20 2.3.3 Sequencing of the lad mutants 20 2.3.4 Sequence analysis for mutation detection 21 Chapter 3 DNA from small intestinal epithelial cells deficient in MSH6 have an increase in mutation frequency 24 3.1 Introduction 24 3.2 Results Elevated l a d mutant frequencies in MSH6~'~ D N A 26 3.3 Discussion 26 Chapter 4 Mutation spectra of MSH6 deficient small intestine epithelial cells 31 4.1 Introduction 31 4.2 Results Mutation spectra 33 4.3 Discussion 33 Chapter 5 Discussion 38 5.1 Summary of thesis 38 5.2 D N A mismatch repair-deficiency leads to tumorigenesis via subsequent mutations in oncogenes and tumor suppressors 38 5.3 Potential future directions 39 References 41 iv List of Figures Figure 1.1: M M R is shown at the replication fork of Escherichia coli 3 Figure 1.2: Simplified diagram of eukaryote mismatch repair '. 6 Figure 1.3: The N and C - terminal portions of (3-galactosidase cleave X-ga l to produce blue colonies 12 Figure 2.1: Agarose gels resolving P C R fragments from the Msh6 and the lacl P C R reactions.. 17 Figure 2.2: Sequence of the lacl gene 22 Figure 2.3 : Alignment of mutant forward sequence, mutant reverse and complement and wildtype lacl gene 23 Figure 3.1: Comparison of B i g Blue, M S H 6 and M L H 1 deficient lacl mutation frequencies in D N A from small intestine epithelial cells 29 v List of Tables Table 1.1: Eukaryote mismatch repair orthologs 5 Table 1.2: Incidence of defects in M M R genes causing H N P C C 8 Table 3.1: Spontaneous lacl mutant frequencies in D N A isolated from Msh6~'~ lacf small intestine epithelial cells 27 Table 3.2: Spontaneous lacl mutant frequencies of D N A isolated from small intestinal epithelial cells of control B i g Blue T M mice 28 Table 4.1: Mutation spectrum of the lacl gene within D N A isolated from small intestine epithelial cells oiMshS1' lacf mice 34 Table 4.2: Mutation spectrum of the APC gene within D N A isolated from small intestine epithelial cells of MshG1' APCmm +/~ -mice 36 Table 4.3: Mutation spectrum of the lacl gene within D N A isolated from small intestine epithelial cells of Mlhl'1' BC-1 mice 37 v i List of Abbreviations BER base excision repair HNPCC hereditary non-polyposis colorectal cancer IDL insertion / deletion loop MLH (1-3) M u t L h o m o l o g ( l - 3 ) MMR mismatch repair MSI microsatellite instability MSH(l-6) MutS homolog(l-6) NER nucleotide excision repair pfu plaque forming units PMS(l-2) post-meiotic segregation mutant (1-2) X-gal 5-bromo-4-chloro-3-indolyl (3-galactopyranoside Vll Acknowledgements In these studies I am grateful to Dr. L Sandercock for her valuable input on this thesis, Dr. A . Baross for her expert advice on the project. I am grateful to my supervisor, Dr. Frank Jirik. I thank Dr. A Rose for her input on the direction of this project. I thank S. Lines for maintaining the animal colony used and for help in the genotyping of the various strains used. I thank all members of the Jirik lab past and present for providing a wonderful learning environment. I am grateful to Dr. W . Edelmann for providing the M S H 6 deficient mice. This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society, and by the Alberta Heritage Foundation for Medical Research ( A H F M R ) . v i i i Dedication To friends and family for their love and support; my gratitude extends beyond the depths of the Marianas trench. A n d to my colleagues who helped me achieve a respect and understanding of the scientific process. ix Chapter 1 Introduction 1.1 DNA damage and repair pathways Conservation of genomic integrity is critical in the maintenance of cellular homeostasis and in a multicellular context, preventing individual cells from developing 'selfish' characteristics (Loeb and Loeb 2000). Cells are constantly struggling against mutational pressures which have the potential to damage the genetic material, introducing unwanted heritable changes. There is no shortage of D N A damage, arising either spontaneously, or from endogenous and/or environmental mutagens. Some common mutagens are: ultraviolet or ionizing radiation, alkylating agents, reactive oxygen species or by-products of metabolic processes (Schmutte and Fishel 1999). Mechanisms to repair D N A damage are an indispensable requirement to preserve a state of genomic stability. Included here is a brief description of some eukaryote repair mechanisms which prevent point mutations and insertion/deletion loops (DDLs), with an emphasis on mismatch repair ( M M R ) . The simplest mechanism of D N A repair involves the direct removal of a damaging lesion. A n example of this type of repair is that catalyzed by Methyl Guanine Methyl Transferase ( M G M T ) . M G M T protects the genome against alkylating events occurring at the 0 6 position of guanine and to a lesser extent the 0 4 position of thymine. 0 6 methylguanine lesions are particularly cytotoxic, as at replication they mispair with thymine and i f not repaired, cause G:C-> A : T transitions. M G M T protects the genome from this subset of alkylation events by transferring the methyl group directly from the 0 6 position of guanine to its own reactive cysteine residue; in the process, becoming irreversibly modified and subsequently degraded (Major and Collier 1998). Nucleotide excision repair (NER) , protects the genome against U V induced photoproducts, most effectively repairing bulky or helix-distorting lesions such as pyrimidine dimers. N E R is initiated by two different mechanisms: by a complex of N E R recognition enzymes recognizing damaged D N A , or by the stalling of R N A polymerase II holoenzyme at a site of D N A damage, a repair process called transcription coupled repair (TCR). In both branches of N E R , D N A is unwound and incisions are made at sites 5' and 3' to the D N A damage and this is followed by exonuclease mediated strand degradation. The resulting gap is filled by polymerases and residual nicks are sealed by a ligase (Cleaver, Karplus et al. 2001). 1 Base excision repair (BER) catalyzes the repair of a wide variety of base damages including those resulting from alkylation, oxidation or spontaneously generated apurinic/ apyrimidinic (AP) sites. B E R is initiated by the formation o f an A P site which can occur spontaneously or due to the actions of D N A glycosylases, a family of enzymes which recognize and catalyze the removal of a large number of damaged bases. It is estimated that 10, 000 abasic sites are generated per day in each eukaryotic cell. A specialized endonuclease, A P endonuclease, recognizes A P sites and cleaves the sugar-phosphate backbone 5' o f the A P site generating a 5' terminal deoxyribose phosphate (dRP) moiety. Polymerase p\ a small D N A polymerase, removes the dRP and adds an appropriate nucleotide(s). The remaining nick in the sugar phosphate backbone is sealed by a D N A ligase. A n alternative form of this repair pathway exists (long patch repair) where a greater number of nucleotides are removed and replaced (Beard and Wilson 2000). Mismatch repair ( M M R ) , described below in greater detail, repairs D N A mismatches arising from incorporation of inappropriate nucleotides by D N A polymerases during replication or during gap resynthesis in the course of D N A repair (Kolodner and Marsischky 1999). 1.2 The DNA mismatch repair system 1.2.1 Mismatch repair in Escherichia coli E. coli M M R is primarily carried out by 3 proteins: MutS, M u t H and M u t L (Fig 1.1). Repair is initiated by a MutS homodimer that recognizes base:base mispairs with variable efficiency with the exception of C : C mismatches and IDLs up to four nucleotides in length. M M R targets repair to the nascent strand by M u t H mediated discrimination of the methylation state of D N A strands owing to the fact that D N A adenine methylase (Dam) mediated methylation of the nascent strand does not occur immediately after replication. M u t H can recognize hemimethylated G A T C sites in D N A up to 1000 bases away from the site o f a mismatch. The M u t L homodimer then interacts with MutS, coupling the mismatch damage recognition complex to M u t H , which nicks the D N A at the nearest hemimethylated G A T C sequence (Kolodner 1995; Prolla 1998). Repair proceeds 2 Figure 1.1: M M R is shown at the replication fork of Escherichia coli. 3 by the exonucleolytic degradation of the nascent strand from the nick site past the site of D N A damage. Resynthesis is accomplished by D N A polymerase III holoenzyme and the nick is sealed by D N A ligase (Marra and Schar 1999). 1.2.2 Eukaryotic mismatch repair The mechanism of eukaryotic M M R is similar to that of E.coli, with two major differences. The functions of E.coli MutS and M u t L are carried out by various heterodimeric protein complexes rather than homodimers (Table 1.1). A s well , no M u t H functional homolog has yet been found and it is, at the present time, uncertain how M M R is able to differentiate between the parental and nascent D N A strands (Kolodner 1996; Jiricny 2000). Recognition of damage in eukaryotes is carried out by orthologs of MutS. In Saccharomyces cerevisiae and mammals, six MutS orthologs have been identified, M S H 1 - M S H 6 (Jiricny, 2000). In mammalian M M R , recognition of mispairs and IDLs is mediated by two heterodimeric protein complexes with overlapping functions (Acharya, Wilson et al. 1996; Kolodner 1996). In mammalian cells, M u t S a , consisting of M S H 2 and M S H 6 , supports the repair of base mismatches, single nucleotide DDLs and to a lesser extent larger IDLs (Fig. 1.2) (Edelmann, Yang et al. 1997). MutS(3, composed of M S H 2 and M S H 3 , appears to be primarily involved in the repair of EDLs from 2-8 nts and, to a lesser degree, single base loops (Palombo, Gallinari et al. 1995; Palombo, Iaccarino et al. 1996). In most cell lines M u t S a is present in higher concentrations than MutSP and is thus thought to be the dominant partner in damage recognition ((Buermeyer, Deschenes et al. 1999; Kolodner 1996; Kolodner and Marsischky 1999; Marra and Schar 1999). M u t S a , but not MutS(3, is also required in M M R mediated stabilization of the tumor suppressor p53 and the induction of apoptotis under alkylation induced stress (Hickman and Samson 1999). In eukaryotes, the function of bacterial M u t L is carried out by several heterodimeric complexes including M u t L a and MutLp\ M u t L a is composed of M L H 1 and P M S 2 (PMS1 in yeast) and is thought to be responsible for mediating most of the M M R mediated repair activity. M u t L p is composed of M L H 1 and M L H 3 in yeast. In human cells the presence of M u t L p , composed of M L H 1 and P M S 1 , has been demonstrated but no biochemical function has yet been ascribed to 4 E. coli S. cerevisiae H. sapiens MutS Msh2,Msh6, Msh3 Msh2a, Msh6a, Msh3 Mshl _b Msh4, Msh5 Msh4,Msh5 MutL Mlhl M l h l a Pmsl Pms2a Mlh2, Mlh3 Pmsl a MutH _b _b MutU(UvrD) _b _b Table 1.1: Eukaryote mismatch repair orthologs. a Mutations found in cancer families b Not identified Table from: (Buermeyer, Deschenes et al. 1999) 5 MutSa MutLa MutSa MutSp MutLa Mutsp Insertion or deletion loops Figure 1.2: Simplified diagram of eukaryote mismatch repair. this heterodimer. A n additional mammalian M u t L ortholog, M L H 3 , has been described which interacts with M L H 1 . It is possible that in mammals, the function of MutL(3 is accomplished by M L H 1 / P M S 1 and/or M L H 1 / M L H 3 (Buermeyer, Deschenes et al. 1999; Marra and Schar 1999) (Table 1.1). The mechanism by which eukaryote M M R targets repair to the nascent strand is not known. It is unlikely that strand discrimination is dependent on D N A methylation as it is in prokaryote M M R because eukaryote D N A methylation is restricted to cytosine residues at C p G sites rather than being more global at G A T C sequences. N o eukaryote homologs of E. coli's M u t H have been identified (Buermeyer, Deschenes et al. 1999). However, there is a candidate functional homolog, M E D 1 ( M B D 4 ) . M E D 1 , an endonuclease, has a methyl-CpG D N A binding motif and has been shown to interact with h M L H l . M E D 1 also possesses a C-terminal glycosylase domain, suggesting it may have a role in base excision repair (Bellacosa, Cicchill i t t i et al. 1999). Cel l lines transfected with a dominant negative mutant of M E D 1 demonstrate instability at microsatellite sequences, a common feature of M M R deficient tumors. Thus, M E D 1 may be involved in mammalian M M R but its exact role has yet to be elucidated (Bellacosa 2001). Although all of the proteins involved in mammalian M M R have yet to be identified, much progress has been made in identifying proteins with putative roles in M M R . Exonuclease I, a 5' -> 3' exonuclease that interacts with h M S H 2 and h M L H l , is able to restore bi-directional excision activity to human nuclear extracts depleted of excision activity (Schmutte, Sadoff et al. 2001; Genschel, Bazemore et al. 2002). A second exonuclease, F E N 1 (RAD27) , that plays an important role in the processing of the 5' ends of Okazaki fragments has also been shown to interact with proliferating cell nuclear antigen ( P C N A ) , a protein that interacts with other M M R components (Kolodner and Marsischky 1999). Other proteins implicated in eukaryotic M M R include: D N A polymerase 8, the single-stranded DNA-binding protein R P A , P C N A , and R F C , a factor required to load P C N A onto D N A . More research to confirm the roles of these proteins in M M R is still needed (Kolodner and Marsischky 1999). 1.3 Mismatch repair deficiency and carcinogenesis In 1993 a subset of hereditary colorectal malignancies were found to be defective in components of M M R (Peltomaki 1994) (Table 1.2). These hereditary non-polyposis colorectal cancers 7 MMR gene Chromosomal location Number of mutations (%) 1996 Present MSH2 2p21 48(38%) 125 (38%) MLHl 3p21-23 75(60%) 164(49%) PMS1 2q31-q33 1(1%) 1 (0.3%) PMS2 7p22 2(2%) 5(2%) MSH6 2p2l 0(0%) 30(9%) MLH3 14q24.3 0(0%) 7(2%) Total 126(101%) 332(100.3%) Table 1.2: Incidence of defects in M M R genes causing H N P C C . The present number is compared with the year 1996 when the database was established. Table from: (Peltomaki 2001) 8 ( H N P C C ) , account for 5 % of colorectal cancers. H N P C C is an autosomal dominant disorder with high penetrance, with the average age of onset being 42 years. H N P C C patients may develop colorectal cancer alone (Lynch I type syndrome) or malignancies in extra-colonic tissues, including: endometrium, ovarian, stomach, pancreas small intestine, skin, breast and urinary tract (Lynch II type syndrome) (Prolla 1998; Toft and Arends 1998; Umar and Kunkel 1996; Wang, Lasset et al. 1999). Heterozygous germline mutations in four mammalian D N A mismatch repair genes, hMSH2 and hMLHl and to a lesser extent hPMS2 and hPMSl, have been associated with H N P C C . Germline mutations in hMSH6 have been associated with a cancer predisposition syndrome that does not meet the criteria of classical H N P C C (Kolodner 1995; Prolla 1998; Toft and Arends 1998; Wang, Lasset et al. 1999) . H N P C C patients have a germline defect in one allele of an M M R component. Somatic mutation and inactivation of the second allele results in a loss of heterozygosity and a tumor susceptibility phenotype. Homozygous germline mutations have been reported in only two families, where affected individuals developed leukemias and / or lymphomas and symptoms similar to neurofibromatosis type I at an early age (Ricciardone, Ozcelik et al. 1999; Wang, Lasset et al. 1999). Epigenetic inactivation of M M R genes, for example by hypermethylation of the hMLHl promoter, have also been found in sporadic colorectal cancers (Prolla 1998; Toft and Arends 1998). D N A from M M R deficient tissues have an increase in point mutations and may also exhibit microsatellite instability (MSI). Malignant transformation in the context of an M M R deficiency is caused by an accumulation of deleterious mutations in key growth control and tumor suppressor genes. 1.4 MMR-deficient mouse models Gene targeting experiments in mice have proven very useful in elucidating the contribution of M M R components to genomic stability and in the genesis of mouse models to study H N P C C . Mice lacking M S H 2 (de Wind, Dekker et al. 1995 ; Reitmair, Schmits et al. 1995) M S H 6 (Edelmann, Yang et al. 1997; de Wind , Dekker et al. 1999), M L H 1 (Baker, Plug et al. 1996; Edelmann, Cohen et al. 1996)or P M S 2 (Baker, Bronner et al. 1995), but not M S H 3 (de Wind, Dekker et al. 1999) are predisposed to cancer. It is noteworthy that unlike humans, mice with a single functional M M R gene are not susceptible to cancer. M L H 1 deficient mice have reduced survival due to a susceptibility to T cell lymphomas and gastrointestinal adenomas and carcinomas, a phenotype similar to M S H 2 deficient mice. Cells 9 from these mice exhibit high levels of microsatellite instability. M L H 1 deficiency also leads to infertility due to a lack of sperm production in males and a lack of mature oocytes in females (Baker, Plug et al. 1996; Edelmann, Cohen et al. 1996; Prolla, Baker et al. 1998). P M S 2 deficiency leads to a susceptibility to lymphomas and to a lesser extent sarcomas. In contrast to M L H 1 and M S H 2 deficient mice, these mice do not develop gastrointestinal tumors. Their tissues exhibit a high degree of microsatellite instability. Although P M S 2 female mice are fertile, males are sterile owing to abnormal chromosome pairing in meiosis (Baker, Bronner et al. 1995; Prolla, Baker et al. 1998). Mice deficient in M S H 2 undergo normal development and are fertile. However, they have reduced lifespans due to the development of T-cell lymphomas at 4-6 months. M S H 2 deficiency leads to adenomas and carcinomas of the gastrointestine and i f mice live beyond 6 months, they are prone to skin neoplasms and other tumors. Cells from M S H 2 deficient mice also demonstrate instability at microsatellite sequences (de Wind, Dekker et al. 1995 ; Reitmair, Schmits et al. 1995). Mice deficient in M S H 6 are fertile and have a longer lifespan than M S H 2 deficient mice, developing neoplasms around one year of age. These mice are susceptible to lymphomas and, depending on the genetic background of mice may have an increase in gastrointestinal tumors. Unlike the other M M R deficient mice, their tissues have a low level of microsatellite instability ((Edelmann, Yang et al . 1997;de Wind , Dekker et al . 1999;Heyer, Yang et al. 1999). A more comprehensive discussion of the M S H 6 phenotype is included in the introduction of chapter 3. 1.5 Transgenic shuttle phage in vivo mutation detection systems Transgenic animals carrying mutational target genes offer an invaluable tool for investigating mutagenesis in the whole animal. Systems available to facilitate this type of research often differ with respect to the mutational target and the strain of host mouse, but commonly use lambda phage as a means of shuttling target genes from mammalian hosts into bacteria. Care should be taken when designing a study using an in vivo mutational assay as the choice o f mutational target may have an impact on the outcome of the study. Below is a brief description of some of the main assay systems used to study mutagenesis in an in vivo setting. 10 The SupF gene, isolated from E.coli, encodes a tyrosine amber suppressor t R N A molecule which has been adapted for the study of mutagenesis in shuttle vector plasmids (Leach, Gunther et al. 1996). The supF gene has an advantage over other mutational reporters as it has two repetitive sequences, C7 and G8 that make it a good target for polymerase slippage events. Frameshift mutations, a sub-set of mutations which are under-represented in mutational targets that have a shortage of repetitive sequences, can be detected quite readily by this target gene (Andrew, X u et al. 2000). The lacZ gene, encoding E. coli (3-Galactosidase, is used as a mutational reporter in the Muta Mouse. This gene is suitable for use in mutagenesis studies as the gene is sensitive to mutational inactivation (Gossen, de Leeuw et al. 1989; Gossen and V i j g 1993 Gossen, 1991 #368; Gossen, de Leeuw et al. 1994). The lacl gene, isolated from the E. coli lac operon, is widely used as a mutational target in in vivo mutational assays. The lacl protein forms a homotetramer which binds to the lac operon sequence and represses transcription of the lacZ gene. When a mutation occurs which prevents the formation of a functional tetramer, transcription of lacZ occurs. The lacl gene product is very sensitive to mutational inactivation. Inactivating mutations at over 600 sites have been reported from transgenic animals, with new sites still being recovered (The B i g Blue Website; deBoer and Glickman, 1998). Databases of lacl mutations have been generated which can be used to help interpret mutational spectra (Big Blue Website; (de Boer, 1998). For our mutational analysis we have chosen the B i g Blue™ mouse (Stratagene). The B i g Blue™ (Stratagene) strain contains the lacl gene and the a portion of the LacZ reporter in a retrievable bacteriopage vector. Efficient phage packaging protocols enable the recovery of the mutational target from the genomic D N A of any tissue. If an inactivating mutation is present in the lacl gene, the a portion of the lacZ gene is expressed from the incoming phage, complementing the carboxy-terminal fragment provided by an appropriate host cell; this results in p-galactosidase activity, hydrolysis of X-ga l , an analog of lactose, and the formation of a blue plaque (Kohler, Provost et al. 1991) (Fig 1.8). The mutation frequency in the retrievable reporter gene is calculated by dividing the number of blue plaques by the total number of plaques. Mutation spectra of transgenic mice can be obtained by isolation of mutant/blue plaques, P C R amplification of the lacl gene and sequencing of the amplification product. 11 p-Galactosidase C-Terminal to Fragment li-Galactostdase N-Terminal a Protein X-Gal cleavage * • No X-Gal cleavage so bacterial colony remains white Accumulation of the X-Gal product results in blue colonies Figure 1.3: The N and C - terminal portions of p-galactosidase cleave X-ga l to produce blue colonies. Figure adapted from: http://cmgm.stanford.edu/biochem201/Slides/ 12 1.6 Thesis goals 1. To quantitate the contribution of M S H 6 to M M R mediated mutation surveillance in an in vivo setting. 2. Determine the mutation spectra of M S H 6 deficient mice to further understand the sub-set of mutations that M S H 6 , or M u t S a is able to recognize and repair. In so doing we can further understand the nature of tumors deficient in M S H 6 . 1.7 Thesis summary This thesis is composed of two parts. In the first section (Chapter 3), we undertook a quantitative assessment of the mutation frequency in D N A from small intestine epithelial cells from Msh6~A deficient mice. Using the B i g Blue ™ (Stratagene) lacl based transgenic shuttle-phage mutation we chose to study epithelia from the small intestinal cells as this is a site of tumorigenesis in Msh6~'~ mice. Also , this is a highly proliferative tissue, an essential requirement for the fixation of mutations (Bielas and Heddle 2000). We demonstrated a 41-fold increase in the mutant frequency compared to controls. Our data quantifies the importance of M S H 6 relative to obligate partners in M M R , such as M S H 2 and M L H 1 , which, when inactivated have been linked to the familial cancer syndrome, H N P C C . In the second section (Chapter 4) of this thesis, we set out to define the sequence spectrum of Msh6~'' mice. Our mutation spectrum gathered from an in vivo setting support a role for M S H 6 and therefore M u t S a in suppression of base mispair mutations, in particular G : C -> A : T transitions. Our spectra is consistent with the established role of M S H 6 in recognition and subsequent repair of G:T mismatches, the pre-mutagenic lesion leading to G : C A : T mutations. It is also of interest to correlate the mutation spectrum in MMR-deficient mice with the tumor spectrum. Our data demonstrates that an increase in mutation frequency caused largely by base mispairs and to a lesser extent +1/-1 IDLs is sufficient to cause an increase in lymphomas and to some extent gastrointestinal tumors. 13 Chapter 2 Materials and methods 2.1 Transgenic mice M S H 6 deficient mice, generated through gene targeting of embryonic stem cells, were provided by W . Edelmann (Edelmann, Yang et al. 1997). B i g Blue™ mice were obtained from Stratagene (La Jolla, C A ) . A l l mice were viral antibody-free, and maintained in a barrier facility according to University of Calgary guidelines. 2.1.2 Breeding strategies Msh6 + /~ (Edelmann, Yang et al. 1997) mice were crossed with B i g Blue™ mice carrying the lacl gene to generate M s h 6 + / + / l a c l + , Msh6 + / " / l a c l + and Msh6 _ /" / l a c l + mice. The resulting mice were on a C57BL/6 background. Hemizygous B i g Blue™ mice carry approximately 40 copies of a X L I Z shuttle vector containing a lacl gene, in a head-to-tail orientation at a single integration site near the brown locus on chromosome 4. 2.1.3 Chemicals and reagents A l l chemicals unless otherwise stated were obtained either from Sigma Aldr ich or Merck. 2.1.4 Preparation of tail DNA for genotype testing of mice For genotyping, tail clips obtained from anesthesized mice were digested overnight at 55° C in 300LI1 of lysis buffer (1.0 mg /ml of proteinase K , 50 m M Tr i s -HCl p H 7.5, 50 m M E D T A , 5% SDS). 100 u.1 of 10 M ammonium acetate was added to the digested tail prep. After spinning at 14 000 rpm (Eppendorf 5415D micro-centrifuge) for 1 min, the supernatant was decanted into a clean labeled tube. D N A was precipitated with 1 ml of 100% ethanol and then washed with 1 ml of 70%o ethanol. The pelleted D N A was allowed to air dry until slightly opaque. D N A was then left to rehydrate in 100 u.1 of 10 m M Tris p H 8.0 at room temperature overnight. 1 u.1 of the digested tail prep was used for genotyping 2.1.5 Determination of the Msh6 genotype 14 A three primer P C R assay was used to distinguish the wildtype from the targeted allele (Edelmann et al., 1997). Primers were from Operon (Qiagen). Msh6 A ( T T A C C T C C T C C A C T G A C G T G ) and Msh6 D ( C A A G C C C C T T T C T T T G T T T G ) were used to amplify a 470 bp product from the wildtype allele. Msh6 A and Msh6 B ( T G G A A G G A T T G G A G C T A C G G ) were used to amplify a 400 bp product from the targeted allele. Reagents required for P C R reactions were obtained from Pharmacia Biotech Inc. P C R was performed in a ~ 25 [ iL reaction containing: 17.5 jxL distilled/deonized water, 2.5 i i L 10X P C R Buffer, 2.5 u L dNTP (1.25 m M ) , 1 p:L (100 (J.M) each of primer Msh6A, Msh6B, Msh6D, 0.2 |J,L Taq polymerase and 1 i i L of digested tail prep. A l l P C R reactions were carried out either in a 9700 GeneAmp (Applied Biosystems) or a Genius (Techne) thermocycler. Cycling conditions were, 35 cycles of 94° C for 1 min, 55° C for 1 min, 72° C for 1 min followed by 5 min at 72° C . P C R products were separated in a 3% agarose gel and visualized as described in section 2.1.5. 2.1.6 Determination of the lacl genotype Mice carrying the lacl transgene mice were identified by P C R using primers -11 ( G A C A C C A T C G A A T G G T G C ) and 487 ( C T G G T C A G A G A C A T C A A G ) which amplify a product of ~ 500 bp (Andrew, Pownall et al. 1996) P C R was performed in a 25 \\L reaction containing: 16 p,L distilled/deonized water, 2.5 p L 10X P C R buffer, 2 p X dNTP (1.25mM), 1 pX (50 | i M ) each of primers -11 and 487, 0.2 p X o f Taq D N A polymerase and 2 p X o f digested tail prep. P C R cycling conditions were, 35 cycles of 95° C for 45 sees, 61° C for 1 min, and 72° C for 1 min. P C R products were separated in a 2% agarose gel and visualized as described in section 2.1.5. 2.1.7 Agarose gel electrophoresis D N A fragments from P C R products were resolved by electrophoresis at 100-150 V using a Power Pack 300 (Bio-Rad) in an agarose gel (2-3 %) in T A E buffer (40 m M Tris-acetate, 1 m M E D T A ) . Agarose gels contained ethidium bromide (Molecular Probes) at a concentration of 2.5 p X / 100 m L agarose / T A E solution. D N A was visualized by U V illumination and the image was captured using a Fluor-S™ Max Multilmager (Bio-Rad) and Quantity One software (Fig. 2.1). 15 2.1.8 Obtaining mouse tissues Mice between the ages of 41 and 55 days were sacrificed by carbon-dioxide inhalation. The tissues were promptly removed, flash frozen on dry ice and stored at - 8 0 ° C until used for D N A isolation. 2.1.9 Isolation of small intestine epithelial cells The small intestine of each mouse was flushed out with sterile phosphate-buffered saline (Gibco Invitrogen Corporation), then inverted with a probe. Inverted small intestine was placed in a chilled (4° C) sterile 75 m M KC1 and 20 m M E D T A and pulled up and down several times using a needleless 5 m L syringe to separate epithelial cells from the intestinal wall (Baross-Francis, Makhani et al. 2001). Cells were pelleted at 1500 rpm and resuspended in 3 m L of K C 1 / E D T A . The solution containing the epithelial cells was flash-frozen on dry ice before storage at - 8 0 ° C. 2.2 Measurement of lacl mutational frequency 2.2.1 Isolation of genomic DNA Frozen tissue was transferred to a chilled 7 m L Wheaton Dounce tissue grinder containing 3ml of 4° C douncing buffer (6 m M N a 2 H P 0 4 , 10 m M E D T A , pH8.0). The tissue was homogenized using a Wheaton pestle B and transferred to a 50 ml conical tube. 3 ml of Proteinase K solution (2 mg/mL Proteinase K , 2 % SDS, and 100 m M E D T A , p H 7.5) was pre-heated for 5 min at 55° C and added to 1.5 m L of small intestine epithelial cell emulsion. After mixing by inverting the tube a few times, the mixture was incubated in a 55° C water bath for a minimum of 3 hours up to overnight. A n equal volume o f buffer saturated phenol / choloroform (1:1) was added and the mixture was gently inverted until an emulsion formed. Emulsions were centrifuged at 4000 rpm for 10 minutes (Eppendorf Centrifuge 581 OR) and the aqueous phase was transferred to a new polypropylene tube with a wide-bore transfer pipet. The phenol / chloroform extraction was repeated two more times followed by an extraction with 5 m L of chloroform. Two volumes of 100% ethanol were added to the final aqueous phase and mixed by gentle swirling until a visible 16 cu A) MSH6 PCR z < < - ^ ^ ^ L <. > < < \ < ^ Q + + > > > > + i i ? r + + 4 7 5 b p 4 0 0 b p & B) /ac/ PCR <T3 Q - + - + + + + + - + + + + - + wiw miiBi mini iiiiiilftiJMiai^MrflBnMwaiilh 500 bp Figure 2.1: Agarose gels resolving P C R fragments from the Msh6 and the / a c / P C R reactions 17 D N A precipitate formed. The D N A precipitate was transferred to a sterile 1.5 m L microcentrifuge tube and allowed to dry until the D N A became opaque. Genomic D N A was rehydrated in 80-300 U.L of 10 m M Tris p H 8.0 for a minimum of 12 hours at room temperature, then stored at -20° C (Kohler, Provost et al. 1990). 2.2.2 Maintenance of the SCS-8 cells The Escherichia coli SCS-8 (recAl, endAl, mcrA, A(mcrBC-hsdRMS-mrr), A(argF-lac)\3\69, <^S0dlacZAM15, Tnl0(tet r) host strain (Stratagene) was maintained by streaking a colony onto a B i g Blue ™ media agar plate with 5 U.L (5 p,g/mL) tetracycline. The bacterial streak plate was incubated overnight at 37° C and stored at 4° C for up to four weeks. 2.2.3 Preparing the SCS-8 plating culture To prepare the SCS-8 plating culture, 20 m L of sterile Luria Bertani broth (Sambrook, Fritsch et al. 1989) supplemented with 250 U.L of maltose (20% w/v) / M g S 0 4 ( I M ) was inoculated with a single SCS-8 colony and grown at 37° C overnight with shaking at 200 rpm. Cells were harvested at 4000 rpm for 10 min (Eppendorf Centrifuge 5810 R) , resuspended in sterile 10 m M MgSC»4 at an OD600 ~5.0 and stored at 4° C for up to two weeks (Kohler, Provost et al. 1990; Kohler, Provost etal . 1991). 2.2.4 Performing the packaging reaction The A, shuttle vector containing the lacl gene was recovered from mouse genomic D N A using Transpack in vitro A, packaging extract (Stratagene). 8 U.L of genomic D N A (adjusted to 0.5 mg/mL) was incubated with the orange tube packaging extract (Stratagene) for 90 min at 30° C. 12 U.L of the blue tube packaging extract (Stratagene) was then added and incubated for 90 min at 30° C . The packaging reaction was terminated by addition of 972 |0,L of S M buffer (10 m M N a C l , 8 m M M g S O 4 5 0 m M Tris HC1, p H 7.5, 0.01% gelatin). The terminated reaction was kept at 4° C until used for plating. If the entire packaging reaction was not plated on the same day, 50 U.L of chloroform was added per ml of packaged D N A sample, gently vortexed and stored at 4° C for up to one week (Kohler, Provost et al. 1991). 18 2.2.5 Plating the packaged DNA samples Rescued phage were plated on SCS-8 (Stratagene) bacterial lawns containing X-ga l , an analog of lactose (Invitrogen Life Technologies). 1.5 m L of OD6oo~2.0 SCS-8 culture were aliquoted into a 50 m L conical tube for each packaging reaction to be plated. 200-350 u L of packaging reaction was added to each tube of SCS-8 host cell aliquot and incubated for 37° C for 20 min, with gentle shaking every 5 min. 30-35 m L of molten B i g Blue™ top agar (containing 1.5 mg/mL X-ga l {Invitrogen Life Technologies}) was added to each tube, mixed by swirling and immediately plated onto 25 cm x 25 cm assay tray containing B i g Blue ™ bottom agar. These dishes were incubated overnight at 37° C (Kohler, Provost et al. 1991). 2.2.6 Screening and analyzing assay trays Plates were examined for the presence of blue mutant plaques on a background of non-mutant plaques by two individuals. The ratio of blue plaques to colorless plaques was taken as a measure of lacl mutant frequencies (Kohler, Provost et al. 1991). Mutant frequencies were determined from 4-10 packaging reactions per sample or until a total of 200 000 plaques per mouse tissue had been screened. 2.2.7 Verifying putative mutant plaques Blue mutant plaques were picked from the assay trays with wide-bore pipet tips and transferred to sterile microcentrifuge tubes containing 500 u L S M buffer and 50 ixL chloroform. 1 u L of the eluted phage was mixed with 200 uJL of SCS-8 plating culture in a 15 m L conical tube and incubated at 37° C for 20 min with gentle shaking every 5 min. 3 m L of molten B i g Blue ™ top agar containing 1.5 mg/mL X-ga l (Invitrogen Life Technologies) was added to each tube and the tube contents were poured into a 100 mm petri dish containing B i g Blue ™ bottom agar. The plates were incubated overnight at 37° C. The mutant plaques that replated with a blue phenotype were isolated with a wide-bore pipet tip and transferred to 200 u L S M buffer mixed with 25 u L chloroform. The tubes were then stored at 4° C. The eluted phage templates were later used for amplification of mutant lacl genes (Andrew, Pownall et al. 1996). 19 2.2.8 Statistical analysis of mutant frequencies Mean mutant frequencies resulting from 5 animals per group were compared using the Student's two tailed t-test. Significant differences were determined at a 95% level of confidence (p<0.05) using Microsoft excel. 2.3 Determination of lacl mutation spectrum 2.3.1 Amplification of mutant lacl genes Following isolation and verification of single mutant clones, the 1.2 kb lacl genes were amplified by P C R of phage templates using the following lacl primers: -11 ( G A C A C C A T C G A A T G G T G C ) and 1201 ( A C A A T T C C A C A C A A C A T ) (Andrew, Pownall et al. 1996). P C R was performed in a 50 U.L reaction containing 32.5 p L of distilled/deonized water, 5 u L 10X P C R Buffer, 5 p X dNTP (1.25mM), 2 u L each of primer -11 and primer 1201 (50 ixM) and 0.4 p L Taq polymerase. The P C R cycling conditions were 95° C for 3 min, followed by 35 cycles of 95 0 C for 45 sec, 61° C for 1 min, 72° C for 1 min followed by 72° C for 3 min. 2.3.2. Purification of lacl template for sequencing D N A was purified using QIAquick D N A purification kit (Qiagen) (Andrew, Pownall et al. 1996) with the following modifications: products were eluted with buffer E B diluted 1:5 in mi l l i -Q water, tubes were finger vortexed after elution to ensure homogeneity. 5 pJL of the end product was separated in a 2 % agarose gel to check quality and the remainder of the purified D N A was used for sequence analysis. 2.3.3 Sequencing of the lacl mutants Templates obtained from lacl mutants were sequenced using forward primer F l ( G A C A C C A T C G A A T G G T G C ) and R l ( C T G G T C A G A G A C A T C A A G ) . If no mutations were found within the amplified region, primer F2 ( G C T G C C T G C A C T A A T G T T C C G ) , R2 ( A T C G T C G T A T C C C A C T A C C G ) , and/or F3 ( C A T G C A A A T G C T G A A T G A G G ) were used to 20 amplify a different region of the lacl gene (Andrew, Pownall et al. 1996) (Fig.2.2). Sequencing reactions were performed at either the University of Calgary, or the Center for Molecular Medicine and Therapeutics, University of British Columbia sequencing facilities using A B I 377 instruments (Perkin-Elmer). 2.3.4 Sequence analysis for mutation detection Alignment of the consensus and mutant lacl sequences and detection of mutations was performed using the Multal in software (Corpet 1988) (Fig 2.3). 21 -236 AGC GTC GAT TTT TGT GAT GCT CGT CAG GGG GGC GGA GCC TAT GGA AAA ACG CCA GCA ACG CGG CCT TTT TAC -164 GGT TCC TGG CCT TTT GCT GGC CTT TTG CTC ACA TGT TCT TTC CTG CGT TAT CCC CTG ATT CTG TGG ATA ACC -92 GTA TTA CCG CCA TGC ATA CTA GTC TCG AGT ACG TAG GTA CCC GAC ACC ATC GAA TGG TGC AAA ACC TTT CGC -20 GGT ATG GCA TGA TAG CGC CCG GAA GAG AGT CAA TTC AGG GTG GTG AAT GTG AAA CCA GTA ACG TTA TAC GAT 53 GTC GCA GAG TAT GCC GGT GTC TCT TAT CAG ACC GTT TCC CGC GTG GTG AAC CAG GCC AGC CAC GTT TCT GCG 125 AAA ACG CGG GAA AAA GTG GAA GCG GCG ATG GCG GAG CTG AAT TAC ATT CCC AAC CGC GTG GCA CAA CAA CTG 197 GCG GGC AAA CAG TCG TTG CTG ATT GGC GTT GCC ACC TCC AGT CTG GCC CTG CAC GCG CCG TCG CAA ATT GTC 269 GCG GCG ATT AAA TCT CGC GCC GAT CAA CTG GGT GCC AGC GTG GTG GTG TCG ATG GTA GAA CGA AGC GGC GTC 341 GAA GCC TGT AAA GCG GCG GTG CAC AAT CTT CTC GCG CAA CGC GTC AGT GGG CTG ATC ATT AAC TAT CCG CTG 413 GAT GAC CAG GAT GCC ATT GCT GTG GAA GCT GCC TGC ACT AAT GTT CCG GCG TTA TTT CTT GAT GTC TCT GAC 485 CAG ACA CCC ATC AAC AGT ATT ATT TTC TCC CAT GAA GAC GGT ACG CGA CTG GGC GTG GAG CAT CTG GTC GCA 557 TTG GGT CAC CAG CAA ATC GCG CTG TTA GCG GGC CCA TTA AGT TCT GTC TCG GCG CGT CTG CGT CTG GCT GGC 629 TGG CAT AAA TAT CTC ACT CGC AAT CAA ATT CAG CCG ATA GCG GAA CGG GAA GGC GAC TGG AGT GCC ATG TCC 701 GGT TTT CAA CAA ACC ATG CAA ATG CTG AAT GAG GGC ATC GTT CCC ACT GCG ATG CTG GTT GCC A AC GAT CAG 773 ATG GCG CTG GGC GCA ATG CGC GCC ATT ACC GAG TCC GGG CTG CGC GTT GGT GCG GAT ATC TCG GTA GTG GGA 845 TAC GAC GAT ACC GAA GAC AGC TCA TGT TAT ATC CCG CCG TTA ACC ACC ATC AAA CAG GAT TTT CGC CTG CTG 917 GGG C A A ACC AGC GTG GAC CGC TTG CTG C A A CTC TCT CAG GGC CAG GCG GTG AAG GGC A AT CAG CTG TTG CCC 989 GTC TCA CTG GTG AAA AGA AAA ACC ACC CTG GCG CCC AAT ACG CAA ACC GCC TCT CCC CGC GCG TTG GCC GAT 1061 TCA TTA ATG CAG CTG GCA CGA CAG GTT TCC CGA CTG GAA AGC GGG CAG TGA GCG CAA CGC AAT TAA TGT GAG 1133 TTA GCT CAC TCA TTA GGC ACC CCA GGC TTT ACA CTT TAT GCT TCC GGC TCG TAT GTT GTG TGG AAT TGT GAG 1205 CGG ATA ACA ATT TCA CAC AGG AAA CAG CTA TGA CCA TGA TTA CGG ATT CAC TGG CCG TCG TTT TAC AAC GTC 1277 GTG ACT GGG AAA ACC CTG GCG TTA CCC AAC TTA ATC GCC TTG CAG CAC ATC CCC CTT TCG CCA GCT GGC GTA 1349 ATA GCG A Figure 2.2: Sequence of the lacl gene. Figure source: http://eden.ceh.uvic.ca/bigblue.htm 22 7-16Rlrevc 7-16F1 WT Consensus 7-16Rlrevc 7-16F1 WT Consensus AGAGAGTCAA TTCAGGGTGG TGAATGTGAA ACCAGTAACG TTATACGATG TCGCAGAGTA AGAGAGTCAA TTCAGGGTGG TGAATGTGAA ACCAGTAACG TTATACGATG TCGCAGAGTA AGAGAGTCAA TTCAGGGTGG TGAATGTGAA ACCAGTAACG TTATACGATG TCGCAGAGTA AGAGAGTCAA TTCAGGGTGG TGAATGTGAA ACCAGTAACG TTATACGATG TCGCAGAGTA 301 360 TGCCGGTGTC TCTTATCAGA CCGTTCCCCG CGTGGTGAAC CAGGCCAGCC ACGTTTCTGC TGCCGGTGTC TCTTATCAGA CCGTTCCCCG CGTGGTGAAC CAGGCCAGCC ACGTTTCTGC TGCCGGTGTC TCTTATCAGA CCGTTTCCCG CGTGGTGAAC CAGGCCAGCC ACGTTTCTGC TGCCGGTGTC TCTTATCAGA CCGTTCCCCG CGTGGTGAAC CAGGCCAGCC ACGTTTCTGC 7-16Rlrevc 7-16F1 WT Consensus 7-16Rlrevc 7-16F1 WT Consensus 7-16Rlrevc 7-16F1 WT Consensus •16Rlrevc 7-16F1 WT 361 420 GAAAACGCGG GAAAAAGTGG AAGCGGCGAT GGCGGAGCTG AATTACATTC CCAACCGCGT GAAAACGCGG GAAAAAGTGG AAGCGGCGAT GGCGGAGCTG AATTACATTC CCAACCGCGT GAAAACGCGG GAAAAAGTGG AAGCGGCGAT GGCGGAGCTG AATTACATTC CCAACCGCGT GAAAACGCGG GAAAAAGTGG AAGCGGCGAT GGCGGAGCTG AATTACATTC CCAACCGCGT 421 480 GGCACAACAA CTGGCGGGCA AACAGTCGTT GCTGATTGGC GTTGCCACCT CCAGTCTGGC GGCACAACAA CTGGCGGGCA AACAGTCGTT GCTGATTGGC GTTGCCACCT CCAGTCTGGC GGCACAACAA CTGGCGGGCA AACAGTCGTT GCTGATTGGC GTTGCCACCT CCAGTCTGGC GGCACAACAA CTGGCGGGCA AACAGTCGTT GCTGATTGGC GTTGCCACCT CCAGTCTGGC 481 540 CCTGCACGCG CTCGTCGCAA ATTGTCGCGG CGATTAAATC TCGCGCCGAT CAACTGGGTG CCTGCACGCG C-CGTCGCAA ATTGTCGCGG CGATTAAATC TCGCGCCGAT CAACTGGGTG CCTGCACGCG C-CGTCGCAA ATTGTCGCGG CGATTAAATC TCGCGCCGAT CAACTGGGTG CCTGCACGCG C.CGTCGCAA ATTGTCGCGG CGATTAAATC TCGCGCCGAT CAACTGGGTG 541 600 CCAGCGTGGT GGTGTCGATG GTAGAACGAA GCGGCGTCGA AGCTCTGTAA AGCGGCGGTG CCAGCGTGGT GGTGTCGATG GTAGAACGAA GCGGCGTCGA AGC-CTGTAA AGCGGCGGTG CCAGCGTGGT GGTGTCGATG GTAGAACGAA GCGGCGTCGA AGC-CTGTAA AGCGGCGGTG Consensus CCAGCGTGGT GGTGTCGATG GTAGAACGAA GCGGCGTCGA AGC.CTGTAA AGCGGCGGTG 601 660 7-16Rlrevc CACAATCTTC TCGCGCAACG CGTCAGTGGG CTGATCATTA ACTATCCGCT GGATGACCAG 7-16F1 CACAATCTTC TCGCGCAACG CGTCAGTGGG CTGATCATTA ACTATCCGCT GGATGACCAG WT CACAATCTTC TCGCGCAACG CGTCAGTGGG CTGATCATTA ACTATCCGCT GGATGACCAG Consensus CACAATCTTC TCGCGCAACG CGTCAGTGGG CTGATCATTA ACTATCCGCT GGATGACCAG 661 720 7-16Rlrevc GATGCCATTG TCTGTGGAAG CTGCCTGCAC TAATGTTCCG GCGTTA 7-16F1 GATGCCATTG -CTGTGGAAG CTGCCTGCAC TAATGTTCCG GCGTTATTTC TTGATGTCTC WT GATGCCATTG -CTGTGGAAG CTGCCTGCAC TAATGTTCCG GCGTTATTTC TTGATGTCTC Consensus GATGCCATTG .CTGTGGAAG CTGCCTGCAC TAATGTTCCG GCGTTAtttc t t g a t g t c t c 721 780 Figure 2.3 : Alignment of mutant forward sequence, mutant reverse and complement and wildtype lacl gene. Site of confirmed mutation is indicated by enlarged font. Figure source : Multiple sequence alignment with hierarchical clustering F. Corpet, 1988, Nucl . Acids Res., 16 (22), 10881-10890 23 Chapter 3 DNA from small intestinal epithelial cells deficient in MSH6 have an increase in mutation frequency 3.1 Introduction A tumorigenic phenotype is often associated with genomic instability. This may be manifested by any of, or a combination of: chromosomal instability, microsatellite instability or an increase in the frequency of point mutations. Thus, malignancy evolves from a single cellular genotype/phenotype to a heterogeneous population of progeny with a common initiating mutation (Loeb 2001).. Pre-mutagenic lesions leading to point mutations and microsatellite instability are recognized and repaired by M M R . The importance of this pathway in preventing mutagenesis is highlighted by the fact that mutations in M M R components primarily, hMSH2, hMLHl and to a lesser extent hMSH6, hPMS2 and hPMSl are associated with the familial cancer syndrome H N P C C (Peltomaki 2001). Affected individuals inherit a germline mutation in one allele and upon somatic mutations of the second allele, develop malignancies, predominantly of the proximal colon, endometrium and ovary (Kolodner 2000). M M R deficiency in mice results in a cancer susceptibility phenotype with an increase in neoplasms, primarily thymic and gastrointestinal in origin. A s previously mentioned, D N A from M M R deficient tissues also exhibits an increase in mutation frequency caused by both frameshifts and point mutations, as assayed by in vivo mutational reporter systems (Andrew, Reitmair et al. 1997; Andrew, X u et al. 2000; Baross-Francis, Makhani et al. 2001). Recognition of damage by mismatch repair is mediated by two protein complexes, M u t S a and MutS p. The cytoplasmic and nuclearplasmic concentrations of these two protein complexes is partially regulated by competition for unbound M S H 2 by M S H 6 and M S H 3 . The ratio of M S H 6 to M S H 3 is about 10:1 in cytoplasmic extracts (Marra, Iaccarino et al. 1998), making M u t S a the predominant participant in M M R mediated damage recognition. While unpartnered M S H 6 or M S H 3 is highly susceptible to proteolytic degradation, heterodimeric M u t S a and MutS(3 are resistant (Marra, Iaccarino et al. 1998). 24 Given the dominance of M u t S a in recognition of M M R substrates it would be expected that a cancer susceptibility syndrome similar H N P C C would be seen when one hMSH6 allele was inactivated. Clinical statistics, however, are not in accordance with this as the number of diagnosed malignancies caused by an M S H 6 deficiency is relatively small. (Peltomaki 2001). This could be explained by difficulty in diagnosing this cancer predisposition syndrome or, alternatively, by M S H 6 not being a major contributor to M M R mediated mutation surveillance. Data from cell lines deficient in M M R components suggest that M S H 6 has a non-redundant role in recognition of pre-mutagenic lesions and therefore mutation prevention (Palombo, Gallinari et al. 1995; Papadopoulos, Nicolaides et al. 1995;Glaab and Tindall 1997;Ohzeki, Tachibana et al. 1997;Umar, Risinger et al. 1998;Marra, Iaccarino et al. 1998); however, data quantifying the contribution of M S H 6 to M M R mediated mutation surveillance vary considerably depending on the experimental approach employed (Glaab and Tindall 1997; Umar, Risinger et al. 1998;Ohzeki, Tachibana et al. 1997). Glaab and Tindall, (1997) found an M S H 6 deficient cell line to have a mutation frequency of 216 * 10"6. Umar and Risinger (1998) using a micro-cell chromosome transfer technique found hMSH6 ~'~ I hMSH3 + / " cells to have a mutation rate of 9 * 10"6. The role of M S H 6 in prevention of tumorigenesis is complicated further by a discrepancy in the tumor spectra of mice deficient in M S H 6 that were generated by two independent research groups. De Wind et al, (1999) introduced a disruption cassette into exon4 of Msh6 in 129/OLA ES cells. Mice analyzed for experiments were hybrids of mouse strains 129/OLA and F V B . Targeted mice had an increase in thymic lymphomas but no increase in intestinal neoplasias. Edelmann et al, (1997) disrupted Msh6 at exon 4 using W W 6 E S cells injected into C57BL/6 blastocysts. Homozygous M S H 6 deficient hybrids had an increase in spontaneous tumors, most notably B and T cell lymphomas, as well as gastrointestinal tumors. In order to quantitate the contribution of M S H 6 to M M R mediated mutation surveillance in an in vivo setting, we obtained mice deficient in M S H 6 (Edelmann, Yang et al. 1997) and crossed them with mice carrying a passive lacl forward mutational target (Stratagene™). For our analysis we chose epithilial cells from the small intestine, to further understand the contribution of M S H 6 in preventing gastrointestinal tumors and, possibly to shed some light on the relationship between M S H 6 deficiency and H N P C C . 25 3.2 Results Elevated lacl mutant frequencies in MSH6"'" DNA Mutation frequencies were determined for D N A from small intestine epithelial cells from MSH6-deficient and wildtype control mice using the B i g Blue™ /ac/-based transgenic system. MSH6-deficient mice (Table 3.1) exhibited a 41-fold increase in mutation frequency over controls (Table 3.2). Analysis of the mutation spectra of randomly selected lacl mutants derived from MSH6-deficient mice revealed a high incidence of repetitively isolated mutations (average 37 %) (Table 3.1). Mutation frequencies from MSH6-deficient mice also showed significantly higher variability, both in terms of the incidence of repetitively isolated mutations, and mutation frequencies, compared to lacl mutants isolated from small intestinal epithelial cells of Mlhl'1', BC-1 mice (Baross-Francis et al., 2001). Our results from small intestine epithelial cell D N A strongly suggest a critical role for M S H 6 in the recognition and repair of a subset o f pre-mutagenic lesions. 3.3 Discussion The current model of eukaryotic M M R suggests that two heterodimeric protein complexes, M u t S a and MutSp\ initiate M M R by recognizing mismatches and IDLs (Buermeyer, Deschenes et al. 1999). To evaluate the specific contribution of M u t S a to mutation prevention in vivo, we obtained the mutant frequencies and mutation spectra from the D N A of small intestinal epithelial cells derived from MshS1' mice using the /acT-based B i g Blue™ mutation reporter system. We show that this Msh6~'~ tissue exhibits mutation frequency inductions wel l above those of control mice. Interestingly, the spontaneous mutation frequency of MSH6-deficient small intestinal epithelial cells (41 +/- 16) was greater (p-value: 0.145) than that observed in M L H 1 deficient small intestinal epithelial cells (30 +/- 4) (Baross-Francis, Makhani et al. 2001) (Fig.3.1). This is intriguing as M L H 1 is an obligate partner in M M R . In an M L H 1 deficient background one would expect that a complete loss of M M R activity would result in a relative increase in the mutation frequency over genetic defects which only partially block M M R ; the case for M S H 6 deficiency as MutS^-mediated damage recognition and repair would still be present. A possible explanation for this discrepancy is the use of different reporter systems for mutational analysis: analysis of M L H 1 -deficient tissues carried out previously employed the BC-1 lacl based 26 Mouse Age Sex Total pfu Number Mutant (days) of mutants frequency (xin-5) Repetitive Mutants Corrected mutant frequency (xlO 5) 1 48 F 249245 78 31.29 35 20.34 2 46 F 200215 87 43.45 48 22.60 4 46 M 298126 197 66.08 28 47.58 5 55 F 263674 276 104.67 46 56.52 6 49 F 218297 123 56.35 19 45.64 7 49 M 228514 230 100.65 45 55.36 A V G 49 243012 165 67.08 37 41.34 SD 3.31 35088 81.22 29.98 12 15.98 Table 3.1: Spontaneous lacl mutant frequencies in D N A isolated from Msh6~'~ lacf small intestine epithelial cells Abbreviations: pfu= plaque forming unit; A V G = average; SD = standard deviation 27 Mouse Age Total pfu Number of Mutant mutants frequency (xl0 s ) 1 41 254220 6 2.4 2 41 220540 2 0.9 3 41 203160 1 0.5 4 41 225320 1 0.4 5 41 209160 2 1.0 A V G 41 222480 2.4 1.0 SD 0 19811 2.1 0.8 Table 3.2: Spontaneous lacl mutant frequencies of D N A isolated from small intestinal epithelial cells of control B i g Blue T M mice Abbreviations: pfu= plaque forming unit; A V G = average; SD = standard deviation 28 60 • o 7 40 MSH6 -/- MLH1 -/- Big Blue Controls Genotype Figure 3.1: Comparison of B i g Blue, M S H 6 and M L H 1 deficient lacl mutation frequencies in D N A from small intestine epithelial cells. Mutation frequencies for control and M S H 6 deficient mice were derived from B i g Blue mutation system, M L H 1 deficient mutation frequencies were obtained using the BC-1 transgenic system (Andrew, Pownall et al. 1996). 29 transgenic system, (Andrew, Pownall et al. 1996), whereas the current data was generated using the lacl based B i g Blue™ system. Zhang et al, (2001) examined spontaneous mutation frequencies in liver of B i g Blue and BC-1 transgenic mice. Their experiments addressed the question of whether or not the chromosomal context of the transgene or strain differences in the murine hosts could act as a source of variability between the two different in vivo mutation assays. N o difference was observed for wildtype mice. A s M M R is a ubiquitous mutation surveillance mechanism, it is possible that strain dependent differences in spontaneous mutation frequency would only be apparent in a state of M M R deficiency. Mismatch repair could effectively mask genetic variables which could influence the spontaneous mutation frequency. A comparison of the mutation frequencies between M S H 6 and M L H 1 deficient small intestine epithelial cells is complicated further by the use of the lacl gene as a mutational target. The sequence of lacl makes it an ideal target for testing the frequency of single base substitutions and short IDLs (+1/-1 nt). This reporter, however, exhibits a paucity of repeats, the longest being A 5 tracts. The short length of these repeats renders it sub-optimal as a substrate for spontaneous D N A polymerase slippage events; events that are expected to increase the mutation frequency of M L H 1 deficient tissues more than M S H 6 deficient tissues. A n in vivo transgenic reporter system such as the supF gene (Andrew, X u et al. 2000), which contains a run of C7 and G8, would be more suitable to compare the mutation frequencies of M S H 6 and M L H 1 deficient tissues with respect to the contribution of IDLs to the mutation frequencies. The striking increase in mutation frequency of M S H 6 deficient small intestine epithelial cells relative to B i g Blue™ controls is not unexpected and is supported by data quantifying the incidence of tumors in M S H 6 deficient mice relative to other mice with complete deficiencies in M M R . The MshG*' mice that were generated by de Wind et al., (1999) displayed the same rate of lymphomagenesis as Msh2~'~ mice, but these rarely developed intestinal tumors. They also reported Msh6~'~l Msh3'A mice had a tumor incidence and a tumor spectra identical to Msh2~/~ mice. Edelmann et a/.,(1999) developed an experimental system to test the contribution of individual M M R components to the development of intestinal neoplasia using APC]6iS>i + / \ a germline mutation which accelerates intestinal tumorigenesis. Kuraguchi et al., (2001) quantified tumor incidence in the intestines of Msh3'A, Msh6~'~ and MshS'l Msh3'A mice relative to M M R proficient ^ 4 P C I 6 3 8 N + / " mice and found a 6-7 fold increase in the incidence o f intestinal tumors in M S H 6 deficient mice, and a 10-fold increase in the double knockout mice. 30 Our data and that of others suggest that M S H 6 ' s role in mutation surveillance is comparable to that of M S H 2 and M L H 1 , obligate partners in mismatch repair. The under-representation of hMSH6 related human malignancies could be explained in part by the low degree of microsatellite instability in h M S H 6 deficient tumors, a hallmark of H N P C C tumors and a useful diagnostic tool (Wang, Lasset et al. 1999). Prior to 1996, there had been no reports of HMSH6 mutations leading to a cancer susceptibility syndrome. Presently, hereditary defects in hMSH6 account for 9% of pathogenic mutations reported to the International Collaborative Group on H N P C C (Table 1.2) (Peltomaki 2001). In a study of familial n o n - H N P C C colorectal cancer, Kolodner et al, (1999) found 7.1 % of probands analyzed had germline mutations in HMSH6. Patients with germline mutations in hMSH6 display a late onset cancer susceptibility syndrome with tumors in extra-colonic tissues including the endometrium and to a lesser extent small bowel, ureter and renal pelvis, (Peltomaki 2001) making a definitive diagnosis difficult. Chapter 4 Mutation spectra of MSH6 deficient small intestine epithelial cells 4.1 Introduction Mismatch repair is initiated by the recognition of damaged D N A by two different protein complexes; M u t S a is believed to recognize mismatches and small IDLs (lnt), MutSp, larger IDLs (2-6nts) (Edelmann, Yang et al. 1997; Genschel, Littman et al. 1998). Experiments defining the recognition substrate of M S H 6 and M S H 3 have largely been performed either in vitro, using reconstituted systems (Genschel, Littman et al. 1998; Marra, Iaccarino et al. 1998) and, or in cell lines isolated from human tumor tissues deficient in various components of the M M R pathway (Ohzeki, Tachibana et al. 1997; Umar, Risinger et al. 1998). The results from these experiments vary depending on the experimental approach. The use of cell lines complicates the interpretation of experimental data as transformed cells may harbor non-characterized genetic elements (Loeb 1998). Thus, in vitro results may not be reflective of the actual in vivo substrates of these two heterodimers. Below is a brief description of some of the key experiments defining the recognition substrates of M u t S a and MutSp\ Human h M u t S a and hMutS(3 complexes were isolated and tested for their respective substrate specificity in a cell free system (Genschel, Littman et al. 1998). h M u t S a efficiently repaired base 31 mispairs, short IDLs (lnt) and IDLs between 2-8 nts. hMutSp, on the other hand, did not repair base mispairs, repaired short DDLs at a low rate and repaired 2-8nt IDLs with high efficiency. Ohzeki et al., (1997) defined the spectra of spontaneous mutations at the hprt locus in human colorectal carcinoma cell lines defective in mismatch repair. In an h M S H 6 deficient state, 92% of mutations were base substitutions whereas in cells deficient in h M L H 1, the mutation spectra was composed of 57% base substitutions and 43% frameshifts. These results indicate that h M S H 6 is primarily involved in the repair of base mismatches and that M S H 3 , in the absence of M S H 6 , is able to initiate the repair of frameshift mutations. Using another experimental approach, Umar et al., (1998) used human cell lines devoid of both M S H 3 and M S H 6 . Functional copies of either hMSH3 or hMSH6 were transferred into these cells via micro-cell fusion of chromosomes to determine the recognition substrate of MutSP and M u t S a respectively. Addit ion of a single functional copy of hMSH6 restored repair o f transitions, transversions and +1/-1 frameshifts, as determined by sequencing of mutants at the hprt locus. Addit ion of hMSH3 restored repair of +1/-1 frameshifts and surprisingly, transitions and to a lesser extent transversions; suggestive of overlapping functions for h M S H 6 and h M S F D . While this manuscript was in preparation, Kuraguchi et al, (2001) defined the mutation spectra at the APC locus of gastrointestinal tumors in Msh3~'~, Msh6~f~ and Msh6~'~l Msh3~l~ MshG*' mice on an APC^am +/~ background. They found Msh6~f~ mice had a mutation spectrum dominated by mispairs (93 %) while the spectrum of Msh6'f'l Msh3~'~ mice was composed of 46% substitutions and 54% IDLs. One of the possible caveats with using mouse models to study human disease states is that data drawn from mouse models may not accurately portray the corresponding human condition. To test the conservation of the M M R recognition between mice and humans, Edelmann et al., (1997) characterized extracts from M S H 6 deficient mice. They were defective in the repair of single nucleotide mismatches, while the repair of 1,2 and 4 nt DDLs were unaffected. This finding supported the notion that substrate recognition of M u t S a and MutS(3 in mice correlates with substrate recognition in humans. 32 To clarify the subset of D N A damage which M u t S a recognizes in vivo, we used the B i g Blue™ system to define the mutation spectrum of M S H 6 deficient small intestine epihthilial cells. This spectrum represents the lesions that M u t S a recognizes, excluding those which M S H 3 also recognizes in the absence of M S H 6 . 4.2 Results Mutation spectra The mutation spectrum of Msh6 ~'~ small intestinal epithelial cells was determined by obtaining 1 2 - 1 7 unique lacl mutations per mouse (Table 4.1). Recurrent lacl mutations within each individual mouse were eliminated from the analysis to prevent the potential influence of clonal mutations on the mutation spectra. 'Hot spots', mutations recurring in different mice in the sample cohort, were found at sixteen locations. For example: G56 in the lacl gene was mutated to A in all the mice, and sites C42 and G381 were mutated in four of five mice to T and A , respectively. These mutations are amongst those most often reported in the lacl database (Cariello and Gorelick 1996; Cariello, Douglas et al. 1998). A n average apparent clonality of 37 % lowered our initial average mutant frequency for MSH6~ A tissue from 67 x 10"5 to 41 x 10"5. A s seen in Table 4.1, base transitions dominated the mutation spectra of MSH6-deficient mice, accounting for 84 % of the unique mutations, with G : C A : T transitions accounting for 73 %: 42 % of these were at C p G sites, and 17% at the second G of a G p G , a site favored by agents capable of alkylating the 0 6 position of this base. The remainder of the M S H 6 mutation spectrum was comprised o f 7 % transversions, 9 % +1 / - l frameshifts and an 8 bp insertion. 4.3 Discussion The predominance of G : C -> A : T transitions (73 %) (Table 4.1) in the mutation spectra confirms a specific requirement for M S H 6 in the recognition of lesions, such as G:T mismatches that can result in this type of mutation. A mutation spectra dominated by G:C-> A : T mismatches is consistent with previous in vitro and in vivo data on M S H 6 deficiency (Jiricny and Nystrom-Lahti 2000). Indeed, high affinity binding of M u t S a to G:T mismatches is a defining characteristic of this complex (Palombo, Gallinari et al. 1995). In keeping with this, extracts from cells overexpressing M S H 3 , which leads to the degradation of M S H 6 , were unable to recognize G:T mispairs in bandshift experiments (Marra, Iaccarino et al. 1998). Recently, Kuraguchi et al., (2001), defined the mutation spectra at the APC locus of gastrointestinal tumors 33 Mutation Type Number of % of mutants Total Transitions 68 84 G:C->A:T 59 73 CpG 34 42 GpG 14 17 A:T->G:C 9 11 Transversions 6 7.4 G:C ->T:A 4 5 T:A ->G:C 1 1.2 T:A ->A:T 1 1.2 Deletions/I nsertions 7 8.6 1 bp deletion 5 6.2 1 bp insertion 1 1.2 8 bp insertion 1 1.2 Total 81 100 Table 4.1: Mutation spectrum of the lacl gene within D N A isolated from small intestine epithelial cells ofMshfr1' lacf mice. 34 in MshG1' I APCmm + / " mice and also noted a predominance of G : C ^ A : T mutations (84 %) (Table 4.2). This is an interesting observation, given that Kuraguchi et al, (2001) employed a completely different assay system to arrive at this result. It is also interesting to note that the chromosomal context of the APC and lacl genes are probably very different, the APC gene being transcribed in intestinal tissue while the lacl genes are likely in a heterochromatic state (Cosentino and Heddle 2000). The latter suggests that the lacl gene mutation spectrum is consistent with that of an active locus. The major causes of G : C -> A : T transitions in mammals include: the generation of G:T (or C : A ) mismatches during replication, recombination events, deamination of 5-methylC at C p G sites, deamination of non-methylated cytosines to uracil and methylation of the 0 6 position of guanine which mispairs with thymine (Marra and Schar 1999). A transgenic mouse with a modified lacl, gene with 86 % of C p G sites removed did not have a substantial reduction in the rate of spontaneous mutations or altered frequency of C p G related events at the transgene (Skopek, Marino et al. 1998). These results suggest that another mechanism independent of 5-methyl cytosine deamination at C p G sites is causing the preponderance of G : C -> A : T mutations both in modified and unmodified lacl genes. MSH6-deficient small intestinal epithelial cells had a lower proportion of+1/-1 IDLs compared to M L H - 1 deficient mice (Baross-Francis, Makhani et al. 2001), consistent with the ability of M S H 3 (via MutSP) to efficiently recognize this subset of frameshifts (Edelmann, Yang et al. 1997; Genschel, Littman et al. 1998) (Table 4.3). Similarly, Kuraguchi et al., (2001) found only a small induction in +1 / - l IDLs in mutation spectra of the A P C locus in Msh6~'~ APC{6iSU +/~ -mice (Table 4.2). Our results suggest that M S H 6 and M S H 3 do not recognize the same subset of base damage. More specifically, M S H 3 is unable to recognize mispairs, in particular G:T mismatches, in an M S H 6 deficient setting. In support of the hypothesis that M S H 3 recognizes a narrower range of base damage, cell lines and mice deficient in M S H 3 do not display a mutator phenotype or increase in tumor incidence respectively (Hinz and Meuth 1999; de Wind , Dekker et al. 1999; Kuraguchi, Yang et al. 2001). This suggests M S H 6 compensates for the loss of M S H 3 by recognizing and repairing DDLs, making M u t S a the dominant partner in M M R mediated recognition (Genschel, Littman et al. 1998; Umar, Risinger et al. 1998). 35 Mutation Type Number of % of mutants Total Transitions 41 63 G:C->A:T 33 51 CpG 24 37 A:T->G:C 8 12 Transversions 9 14 G:C ->T:A 6 9 T:A ->G:C 0 0 T:A->A:T 2 3 Deletions/Insertions 15 23 1 bp deletion 9 14 1 bp insertion 2 3 Del/Ins > 1 nt 4 6 Total 65 100 Table 4.2: Mutation spectrum of the APC gene within D N A isolated from small intestine epithelial cells ofMshfr1' APC163*™ + / " -mice. Table adapted from: (Kuraguchi, Yang et al. 2001) 36 Mutation Type Number of % of mutants Total Transitions 41 63 G:C->A:T 33 51 CpG 24 37 A:T->G:C 8 12 Transversions 9 14 G:C ->T:A 6 9 T:A ->G:C 0 0 T:A ->A:T 2 3 Deletions/Insertions 15 23 1 bp deletion 9 14 1 bp insertion 2 3 Del/Ins > 1 nt 4 6 Total 65 100 Table 4.3: Mutation spectrum of the lacl gene within D N A isolated from small intestine epithelial cells of Mlhl'1' BC-1 mice. Table adapted from: (Baross-Francis, Makhani et al. 2001) 37 Chapter 5 Discussion 5.1 Summary of thesis We showed a mean 41 fold increase in the mutation frequency in the D N A of M S H 6 deficient small intestine epithelial cells compared to D N A from control cells. This strongly supports a non-redundant role for M S H 6 in mutation surveillance and consequently tumor prevention. We also determined the mutation spectrum of M S H 6 deficient small intestine epithelial cells by sequencing 131 randomly selected mutants. Eliminating repetitive mutants from our analysis of spectra, we showed a dominance of G : C -> A : T mutations in our spectra, consistent with M u t S a demonstrating a high affinity for G:T mismatches. Our results do not support a role for M S H 3 mediated recognition of mispairs, even in absence of M S H 6 . The overall conclusion from the work presented here is that M S H 6 is critical to the recognition of premutagenic lesions, and therefore mutation suppression. Our data suggest, that in the absence of M S H 6 , M S H 3 recognition is limited to larger IDLs (2-8 nts) and to some extent +1/-1 IDLs. Due to the mutator phenotype present in an M S H 6 deficient state and the lack of a mutator phenotype in a state o f M S H 3 deficiency, our data and others suggest M S H 6 ' s role in M M R mediated mutation surveillance to be of similiar import to that of M S H 2 and M L H 1 , obligate partners in M M R . 5.2 DNA mismatch repair-deficiency leads to tumorigenesis via subsequent mutations in oncogenes and tumor suppressors Mutations in mismatch repair genes alone are not sufficient to initiate tumorigenesis; rather, loss of replicative fidelity results in an increased susceptibility to inactivating mutations in key growth control and tumor suppressor genes. Microsatellite instability, a common feature in H N P C C tumors, can lead to alterations in the length of repetitive mononucleotide runs occurring in the coding regions of key regulatory genes. Such genes include: IGF-IIR a tumor suppressor, B A X a promoter of apoptosis, M B D 4 a candidate D N A glycosylase and T G F - B R I I (Peltomaki 2001). Parsons et al., (1995) examined human colorectal cancer tissue with high degree of microsatellite instability and found inactivating mutations within the T G F (3 type II 38 receptor in 100 of 111 cases. Lack of response to T G F - p alone is enough to alter homeostasis of intestinal epithelia as mice deficient in Smad3, a mediator of T G F - p signaling events, have invasive colorectal adenocarcinomas (Zhu, Richardson et al. 1998). A n increase in the frequency of point mutations, as seen in M M R deficient tissues (Andrew, Reitmair et al. 1997; Andrew, X u et al. 2000), has also been shown to inactivate key regulatory genes including: the tumor suppressor p53, the oncogenes K-ras, N-ras and fi-catenin, involved in control of growth and division (Lazar, Grandjouan et al. 1994; Kinzler and Vogelstein 1996; M i y a k i , Iijima et al. 1999; Olschwang, Hamelin et al. 1997). The adenomatous polyposis coli ( A P C ) gene, found inactivated in a form of hereditary colorectal cancer, can also be inactivated by both base substitutions and insertions/deletions (Huang, Papadopoulos et al. 1996; Kinzler and Vogelstein 1996; Olschwang, Hamelin et al. 1997; Kuraguchi, Yang et al. 2001). Loss of replicative fidelity, as seen in a state of M M R deficiency, has wide-reaching implications for genomic stability, resulting in mutagenesis and inevitably, malignancy. 5.3 Potential future directions One of the unexpected findings of this study was a higher mutation frequency in M S H 6 deficient small intestine epithelial cells compared with that of age matched M L H 1 deficient tissue. This apparent discrepancy made it difficult to determine the relative importance of M S H 6 and M S H 3 in M M R mediated mutation prevention. Determining the mutation frequency and spectra of either M L H 1 or M S H 2 , obligate partners in M M R , on the same genetic background as M S H 6 deficient mice using the B i g Blue assay system would allow for more educated inferences on the relative contribution of M S H 6 and M S H 3 to M M R mediated mutation prevention. A perplexing question which arises when determining the mutation spectrum and mutation frequency is how the mutation spectrum in mice deficient in M M R components correlates with their respective tumor spectrum. Baross-Francis et al., (2001) addressed this particular question by determining the mutation frequency and spectrum of mice deficient in P M S 2 , which are predisposed to lymphomas but not gastrointestinal tumors, and M L H 1 deficient mice, which are predisposed to both lymphomas and gastrointestinal tumors. They found P M S 2 deficient mice had a lower mutation frequency (23 +/- 8) than M L H 1 deficient mice (34 +/- 5) and that the mutation spectrum from M L H 1 deficient mice was enriched in G : C - ^ A : T transitions. Using an in vivo mutagenesis assay, such as B i g Blue ™ , it would be interesting to compare the mutation 39 frequency and mutation spectrum of the two different M S H 6 deficient lines (Edelmann, Yang et al. 1997; de Wind , Dekker et al. 1999) correlating mutation frequency and mutation spectra with a susceptibility to gastrointestinal tumors on different inbred mouse backgrounds. Sensitivity to the cytotoxic effects of alkylating agents is determined largely by the functionality of the M M R system and/or levels of the repair protein M G M T . The mutagenic nature of an alkylating agents is largely dependent on its ability to alkylate D N A at the 0 6 position of guanine, a pre-mutagenic lesion which mispairs with thymine at replication. Mice deficient in the M G M T protein have a lower LD50 to alkylating agents (Glassner, Weeda et al. 1999). 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