Different phenotypic consequences of simultaneous versus stepwise Apc loss.

APC is considered a gatekeeper for colorectal cancer (CRC). Cells with heterozygous APC mutations have altered expression profiles suggesting that the first APC hit may help set the stage for subsequent transformation. Therefore, we measured transformation efficiency following what we have designated as 'simultaneous' versus 'stepwise' Apc loss. We combined a conditional Apc allele (Apc(CKO)) with a Cre reporter gene and an out-of-frame Cre allele (Pms2(cre)) that stochastically becomes functional by a frameshift mutation in single cells. Loss of one Apc allele (Apc(CKO/+)) had little consequence, whereas simultaneous loss of both Apc alleles (Apc(CKO/CKO)) resulted in increased clonal expansion (crypt fission), consistent with the gatekeeper function of Apc. Interestingly, our analyses showed that most of the Apc-deficient crypts in Apc(CKO/CKO) mice appeared normal, with morphological transformation, including ß-catenin deregulation, occurring in only 17% of such crypts. To determine whether transformation efficiency was different following stepwise Apc loss, we combined Apc(CKO) with a germline mutant allele, either Apc(Min) or Apc(1638N). Transformation efficiency following stepwise Apc loss (Apc(Min/CKO) or Apc(1638N/CKO)) was increased five-fold and essentially all of the Apc-deficient cells were dysplastic. In summary, our data suggest that the gatekeeper function of Apc consists of two roles, clonal expansion and morphological transformation, because simultaneous Apc loss frequently leads to occult clonal expansion without morphological transformation, whereas stepwise Apc loss more often results in visible neoplasia. Finally, that Apc-deficient cells in certain scenarios can retain a normal phenotype is unexpected and may have clinical implications for surveillance strategies to prevent CRC.

Mlh1 deficiency accelerates myeloid leukemogenesis in neurofibromatosis 1 (Nf1) heterozygous mice.

Defects in DNA mismatch repair (MMR) have been implicated in the genesis of a diverse set of human cancers. Recent studies have suggested that one of the targets of MMR is the neurofibromatosis 1 (NF1) gene. To evaluate the contribution of Mlh1 MMR deficiency to Nf1 tumorigenesis, Mlh1-/-;Nf1+/- mice were generated. All Mlh1-/-;Nf1+/- mice (n=21) were dead by 260 days compared to none of the Nf1+/- mice. In all, 50% of the Mlh1-/-;Nf1+/- mice were dead at 150 days compared to 252 days for Mlh1-/- mice. Nine of the Mlh1-/-;Nf1+/- mice were found to harbor intrathoracic NOS2-immunoreactive myeloid leukemias similar to the hematopoietic malignancies observed in older Nf1+/- mice. As expected, significant microsatellite instability was observed in six of six tumors and neurofibromin expression was lost in all tumors analysed. These results suggest that MMR deficiency can accelerate myeloid leukemogenesis in Nf1+/- mice, presumably by inactivating Nf1 gene expression.

Evidence for the lack of mismatch-repair directed antirecombination during mouse meiosis.

Meiotic recombination was studied in DNA mismatch repair (MMR)-deficient mice using a strain carrying a Pms2 knockout mutation. Using single-sperm typing, recombination was analyzed over five intervals on four chromosomes in four Pms2 -/- animals. A total of 1936 meioses were studied and compared to 1848 meioses from three Pms2 +/+ controls. A smaller study was carried out on a single interval in each of two chromosomes in an MMR-deficient mouse homozygous for the Msh2 knockout mutation. A total of 792 meioses were examined in the Msh2 -/- and 880 meioses in the Msh2 +/+ animal. Recombination fractions were not significantly different in either of the MMR-deficient mouse strains when compared to MMR-proficient controls. Our results appear to conflict with mouse embryonic stem (ES) cell gene-targeting experiments where MMR plays a major role in determining the efficiency of homologous recombination between nonidentical sequences. A number of possibilities could explain the apparent lack of a significant effect on meiosis.

Interactions of Exo1p with components of MutLalpha in Saccharomyces cerevisiae.

Previously, we reported evidence suggesting that Saccharomyces cerevisiae MutLalpha, composed of Mlh1p and Pms1p, was a functional member of the gyrase b/Hsp90/MutL (GHL) dimeric ATPase superfamily characterized by highly conserved ATPase domains. Similar to other GHL ATPases, these putative ATPase domains of MutLalpha may be important for the recruitment and/or activation of downstream effectors. One downstream effector candidate is Exo1p, a 5'-3' double stranded DNA exonuclease that has previously been implicated in DNA mismatch repair (MMR). Here we report yeast two-hybrid results suggesting that Exo1p can interact physically with MutLalpha through the Mlh1p subunit. We also report epistasis analysis involving MutLalpha ATPase mutations combined with exo1Delta. One interpretation of our genetic results is that MutLalpha ATPase domains function to direct Exo1p and other functionally redundant exonucleases during MMR. Finally, our results show that much of the increase in spontaneous mutation observed in an exo1Delta strain is REV3-dependent, in turn suggesting that Exo1p is also involved in one or more MMR-independent mutation avoidance pathways.

Elevated mutant frequencies and increased C : G-->T : A transitions in Mlh1-/- versus Pms2-/- murine small intestinal epithelial cells.

Mutations in DNA mismatch repair (MMR) genes are associated with increased genomic instability and susceptibility to cancer. Mice rendered deficient in either Mlh1 or Pms2 as a result of gene targeting are prone to tumorigenesis, particularly, lymphomas. In addition, although Mlh1-/- mice also develop small intestinal adenomas and adenocarcinomas, Pms2-/- animals remain free of such tumors. To establish whether this phenotypic dichotomy might be associated with a quantitative and/or qualitative difference in genomic instability in these mice, we determined small intestinal epithelial cell DNA mutant frequency and mutation spectrum using a transgenic lambda-phage lacI reporter system. Mutant frequencies obtained from both Mlh1-/- and Pms2-/- mice revealed elevations of 18- and 13-fold, respectively, as compared to their wild-type littermates. Interestingly, we found that C : G-->T : A transitions were significantly elevated in Mlh1-/- mice, accounting in large measure for the 1.5-fold lacI mutant frequency increase seen in these animals. We hypothesize that the increased level of C : G-->T : A mutations may explain, in part, why Mlh1-/- mice, but not Pms2-/- mice, develop small intestinal tumors. Furthermore, the difference in the lacI mutational spectrum of Mlh1-/- and Pms2-/- mice suggests that other MutL-like heterodimers may play important roles in the repair of G : T mispairs arising within murine small intestinal epithelial cells.

Hypermutability to ionizing radiation in mismatch repair-deficient, Pms2 knockout mice.

DNA mismatch repair (MMR) has been shown to play a role in the cytotoxicity of ionizing radiation (IR), as cell lines established from MMR-deficient mice exhibit higher clonogenic survival after IR than do cell lines from wild-type littermates. To test whether this tolerance phenotype would render MMR-deficient animals hypermutable to IR, we compared IR mutagenesis of Pms2-deficient versus wild-type transgenic mice carrying a lambda shuttle vector for mutation detection. In Pms2 nullizygous animals, the mutation frequency in the supFG1 reporter gene was increased from 210 x 10(-5) in untreated animals to 734 x 10(-5) after 6 Gy of IR (an increase of 524 mutants per 10(5)), whereas the frequency in wild-type mice increased from 1.9 x 10(-5) to 10.2 x 10(-5) (an increase of only 8.3 mutants per 10(5)). Similarly, when the lambda cII gene was used as a reporter, the mutation frequency in nullizygous mice was increased from 16.3 x 10(-5) to 42.3 x 10(-5) after IR (an increase of 26.0 x 10(-5)), whereas the frequency in wild-type mice increased from 2.4 x 10(-5) to 9.4 x 10(-5) (an increase of only 7.0 x 10(-5)). The pattern of IR-induced mutations in the MMR-deficient animals was notable for single bp deletions and insertions in mononucleotide repeat sequences, along with a slight increase in transversions. Overall, these results suggest that MMR-deficiency confers hypermutability to IR, and that much of this hypermutability can be attributed to induced instability of simple sequence repeats. Hence, MMR influences not only the survival but also the mutability of cells in response to IR.

MSH-MLH complexes formed at a DNA mismatch are disrupted by the PCNA sliding clamp.

In the yeast Saccharomyces cerevisiae, mismatch repair (MMR) is initiated by the binding of heterodimeric MutS homolog (MSH) complexes to mismatches that include single nucleotide and loop insertion/deletion mispairs. In in vitro experiments, the mismatch binding specificity of the MSH2-MSH6 heterodimer is eliminated if ATP is present. However, addition of the MutL homolog complex MLH1-PMS1 to binding reactions containing MSH2-MSH6, ATP, and mismatched substrate results in the formation of a stable ternary complex. The stability of this complex suggests that it represents an intermediate in MMR that is subsequently acted upon by other MMR factors. In support of this idea, we found that the replication processivity factor proliferating cell nuclear antigen (PCNA), which plays a critical role in MMR at step(s) prior to DNA resynthesis, disrupted preformed ternary complexes. These observations, in conjunction with experiments performed with streptavidin end-blocked mismatch substrates, suggested that PCNA interacts with an MSH-MLH complex formed on DNA mispairs.

Ionizing radiation-induced apoptosis via separate Pms2- and p53-dependent pathways.

The cytotoxicity of ionizing radiation (IR) has been associated with both the p53 pathway and with DNA mismatch repair (MMR). p53 mediates cell cycle arrest and apoptosis in response to X-ray damage, whereas the MMR complex is thought to recognize damaged bases and initiate a signal transduction pathway that can include phosphorylation of p53. To determine whether p53 and MMR mediate X-ray cytotoxicity via the same pathway, mice with targeted disruptions in either the p53 gene or the MutL homologue MMR gene Pms2 were interbred and primary fibroblasts were established from the progeny with genotypes of either wild type, p53 null, Pms2 null, or double null. Cells with either p53 or Pms2 separately disrupted showed reduced levels of apoptosis after IR in comparison with wild type, but the double null cells showed even lower levels, consistent with nonoverlapping roles for p53 and PMS2 in the X-ray response. In transformed cell lines established from the primary cells at early passage, similar differences in the apoptotic response to IR were seen, and clonogenic survival assays following low dose rate IR further showed that nullizygosity for Pms2 confers increased survival on cells in both wild-type and p53 null backgrounds. These results indicate that both p53 and MMR contribute to X-ray-induced apoptosis and that the role of MMR in the cytotoxicity of IR does not depend on p53.

Analysis of yeast MSH2-MSH6 suggests that the initiation of mismatch repair can be separated into discrete steps.

The yeast MSH2-MSH6 complex is required to repair both base-pair and single base insertion/deletion mismatches. MSH2-MSH6 binds to mismatch substrates and displays an ATPase activity that is modulated by mispairs that are repaired in vivo. To understand early steps in mismatch repair, we analyzed mismatch repair (MMR) defective MSH2-msh6-F337A and MSH2-msh6-340 complexes that contained amino acid substitutions in the MSH6 mismatch recognition domain. While both heterodimers were defective in forming stable complexes with mismatch substrates, only MSH2-msh6-340 bound to homoduplex DNA with an affinity that was similar to that observed for MSH2-MSH6. Additional analyses suggested that stable binding to a mispair is not sufficient to initiate recruitment of downstream repair factors. Previously, we observed that MSH2-MSH6 forms a stable complex with a palindromic insertion mismatch that escapes correction by MMR in vivo. Here we show that this binding is not accompanied by either a modulation in MSH2-MSH6 ATPase activity or an ATP-dependent recruitment of the MLH1-PMS1 complex. Together, these observations suggest that early stages in MMR can be divided into distinct recognition, stable binding, and downstream factor recruitment steps.

Functional studies on the candidate ATPase domains of Saccharomyces cerevisiae MutLalpha.

Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLalpha, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH(2) termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLalpha, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLalpha demonstrated that the NH(2) terminus of MutLalpha undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH(2) termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLalpha undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.

Mutagenesis in PMS2- and MSH2-deficient mice indicates differential protection from transversions and frameshifts.

DNA mismatch repair (MMR) deficiency leads to an increased mutation frequency and a predisposition to neoplasia. 'Knockout' mice deficient in the MMR proteins Msh2 and Pms2 crossed with mutation detection reporter (supF, lacI and cII) transgenic mice have been used to facilitate a comparison of the changes in mutation frequency and spectra. We find that the mutation frequency was consistently higher in Msh2-deficient mice than Pms2-deficient mice. The lacI target gene, which is highly sensitive to point mutations, demonstrated that both Msh2- and Pms2-deficient mice accumulate transition mutations as the predominant mutation. However, when compared with Msh2(-/-) mice, lacI and cII mutants from Pms2-deficient mice revealed an increased proportion of +/-1 bp frameshift mutations and a corresponding decrease in transversion mutations. The supF target gene, which is sensitive to frameshift mutations, and the cII target gene revealed a strong tendency for -1 bp deletions over +1 bp insertions in Msh2(-/-) compared with Pms2(-/-) mice. These data indicate that Msh2 and Pms2 deficiency have subtle but differing effects on mutation avoidance which may contribute to the differences in tumor spectra observed in the two 'knockout' mouse models. These variances in mutation accumulation may also play a role, in part, in the differences seen in prevalence of MSH2 and PMS2 germline mutations in hereditary non-polyposis colorectal cancer patients.

Mlh1 deficiency enhances several phenotypes of Apc(Min)/+ mice.

Defects in APC and DNA mismatch repair genes are associated with a strong predisposition to colon cancer in humans, and numerous mouse strains with mutations in these genes have been generated. In this report we describe the phenotype of Min/+ Mlh1-/- mice. We find that these doubly mutant mice develop more than three times the number of intestinal adenomas compared to Min/+ Mlh1+/+ or +/- mice but that these tumors do not show advanced progression in terms of tumor size or histological appearance. Full length Apc protein was not detected in the tumor cells from Min/+ Mlh1-/- mice. Molecular analyses indicated that in many tumors from Min/+ Mlh1-/- mice, Apc was inactivated by intragenic mutation. Mlh1 deficiency in Min/+ mice also led to an increase in cystic intestinal crypt multiplicity as well as enhancing desmoid tumorigenesis and epidermoid cyst development. Thus, Mlh1 deficiency influences the somatic events involved in the development of most of the phenotypes associated with the Min mutation. Oncogene (2000).

Different mutator phenotypes in Mlh1- versus Pms2-deficient mice.

Deficiencies in DNA mismatch repair (MMR) result in increased mutation rates and cancer risk in both humans and mice. Mouse strains homozygous for knockouts of either the Pms2 or Mlh1 MMR gene develop cancer but exhibit very different tumor spectra; only Mlh1(-/-) animals develop intestinal tumors. We carried out a detailed study of the microsatellite mutation spectra in each knockout strain. Five mononucleotide repeat tracts at four different chromosomal locations were studied by using single-molecule PCR or an in vivo forward mutation assay. Three dinucleotide repeat loci also were examined. Surprisingly, the mononucleotide repeat mutation frequency in Mlh1(-/-) mice was 2- to 3-fold higher than in Pms2(-/-) animals. The higher mutation frequency in Mlh1(-/-) mice may be a consequence of some residual DNA repair capacity in the Pms2(-/-) animals. Relevant to this idea, we observed that Pms2(-/-) mice exhibit almost normal levels of Mlh1p, whereas Mlh1(-/-) animals lack both Mlh1p and Pms2p. Comparison between Mlh1(-/-) animals and Mlh1(-/-) and Pms2(-/-) double knockout mice revealed little difference in mutator phenotype, suggesting that Mlh1 nullizygosity is sufficient to inactivate MMR completely. The findings may provide a basis for understanding the greater predisposition to intestinal cancer of Mlh1(-/-) mice. Small differences (2- to 3-fold) in mononucleotide repeat mutation rates may have dramatic effects on tumor development, requiring multiple genetic alterations in coding regions. Alternatively, this strain difference in tumor spectra also may be related to the consequences of the absence of Pms2p compared with the absence of both Pms2p and Mlh1p on as yet little understood cellular processes.

The human MLH1 cDNA complements DNA mismatch repair defects in Mlh1-deficient mouse embryonic fibroblasts.

The DNA mismatch repair gene hMLH1 is reported to function in mutation avoidance, cell cycle checkpoint control, the cytotoxicity of various DNA-damaging agents, and transcription-coupled nucleotide excision repair. Formal proof of the involvement of hMLH1 in these processes requires single gene complementation. We have stably expressed hMLH1 from a transfected cDNA in Mlh1-deficient mouse embryonic fibroblasts. Expression of hMLH1 restored normal levels of mPMS2 protein, reduced spontaneous base substitution and microsatellite mutations, increased sensitivity to the toxic effects of 6-thioguanine (6-TG), and restored 6-TG-induced cell cycle arrest. Our studies confirm that hMLH1 has an essential role in the maintenance of genomic stability and the potentiation of 6-TG cytotoxicity and provide a system for detailed structure/function analysis of the hMLH1 protein.

A mutation of the yeast gene encoding PCNA destabilizes both microsatellite and minisatellite DNA sequences.

The POL30 gene of the yeast Saccharomyces cerevisiae encodes the proliferating cell nuclear antigen (PCNA), a protein required for processive DNA synthesis by DNA polymerase delta and epsilon. We examined the effects of the pol30-52 mutation on the stability of microsatellite (1- to 8-bp repeat units) and minisatellite (20-bp repeat units) DNA sequences. It had previously been shown that this mutation destabilizes dinucleotide repeats 150-fold and that this effect is primarily due to defects in DNA mismatch repair. From our analysis of the effects of pol30-52 on classes of repetitive DNA with longer repeat unit lengths, we conclude that this mutation may also elevate the rate of DNA polymerase slippage. The effect of pol30-52 on tracts of repetitive DNA with large repeat unit lengths was similar, but not identical, to that observed previously for pol3-t, a temperature-sensitive mutation affecting DNA polymerase delta. Strains with both pol30-52 and pol3-t mutations grew extremely slowly and had minisatellite mutation rates considerably greater than those observed in either single mutant strain.

Mammalian DNA mismatch repair.

DNA mismatch repair (MMR) is one of multiple replication, repair, and recombination processes that are required to maintain genomic stability in prokaryotes and eukaryotes. In the wake of the discoveries that hereditary nonpolyposis colorectal cancer (HNPCC) and other human cancers are associated with mutations in MMR genes, intensive efforts are under way to elucidate the biochemical functions of mammalian MutS and MutL homologs, and the consequences of defects in these genes. Genetic studies in cultured mammalian cells and mice are proving to be instrumental in defining the relationship between the functions of MMR in mutation and tumor avoidance. Furthermore, these approaches have raised awareness that MMR homologs contribute to DNA damage surveillance, transcription-coupled repair, and recombinogenic and meiotic processes.

Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2.

Mutations are introduced into rearranged Ig variable genes at a frequency of 10(-2) mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2(-/-) human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.

Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair.

Germline mutations in the human MSH2, MLH1, PMS2 and PMS1 DNA mismatch repair (MMR) gene homologues appear to be responsible for most cases of hereditary non-polyposis colorectal cancer (HNPCC; refs 1-5). An important role for DNA replication errors in colorectal tumorigenesis has been suggested by the finding of frequent alterations in the length of specific mononucleotide tracts within genes controlling cell growth, including TGF-beta receptor type II (ref. 6), BAX (ref. 7) and APC (ref. 8). A broader role for MMR deficiency in human tumorigenesis is implicated by microsatellite instability in a fraction of sporadic tumours, including gastric, endometrial and colorectal malignancies. To better define the role of individual MMR genes in cancer susceptibility and MMR functions, we have generated mice deficient for the murine homologues of the human genes MLH1, PMS1 and PMS2. Surprisingly, we find that these mice show different tumour susceptibilities, most notably, to intestinal adenomas and adenocarcinomas, and different mutational spectra. Our results suggest that a general increase in replication errors may not be sufficient for intestinal tumour formation and that these genes share overlapping, but not identical functions.

Enhanced intestinal adenomatous polyp formation in Pms2-/-;Min mice.

Analysis of two human familial cancer syndromes, hereditary nonpolyposis colorectal cancer and familial adenomatous polyposis, indicates that mutations in either one of four DNA mismatch repair gene homologues or the adenomatous polyposis coli (APC) gene, respectively, are important for the development of colorectal cancer. To further investigate the role of DNA mismatch repair in intestinal tumorigenesis, we generated mice with mutations in both Apc and the DNA mismatch repair gene, Pms2. Whereas Pms2-deficient mice do not develop intestinal tumors, mice deficient in Pms2 and heterozygous for Min, an allele of Apc, develop approximately three times the number of small intestinal adenomas and four times the number of colon adenomas relative to Min and Pms2+/-;Min mice. Although Pms2 deficiency clearly increases adenoma formation in the Min background, histological analysis indicated no clear evidence for progression to carcinoma.

Role of DNA mismatch repair in the cytotoxicity of ionizing radiation.

The DNA mismatch repair (MMR) system in mammalian cells not only serves to correct base mispairs and other replication errors, but it also influences the cellular response to certain forms of DNA damage. Cells that are deficient in MMR are relatively resistant to alkylation damage because, in wild-type cells, the MMR system is thought to promote toxicity via futile repair of alkylated mispairs. Conversely, MMR-deficient cells are sensitive to UV light, possibly due to the requirement for MMR factors in transcription-coupled repair of active genes. MMR deficiency has been associated with familial and sporadic carcinomas of the colon and other sites, and so, we sought to determine the influence of MMR status on cellular response to ionizing radiation, an agent commonly used for cancer therapy. Fibroblast cell lines were established from transgenic mice carrying targeted disruptions of one of three MMR genes in mammalian cells: Pms2, Mlh1, or Msh2. In comparison to wild-type cell lines from related mice, the Pms2-, Mlh1-, or Msh2-nullizygous cell lines were found to exhibit higher levels of clonogenic survival following exposure to ionizing radiation. Because ionizing radiation generates a variety of lesions in DNA, the differences in survival may reflect a role for MMR in processing a subset of these lesions, such as damaged bases. These results both identify a new class of DNA-damaging agents whose effects are modulated by the MMR system and may help to elucidate pathways of radiation response in cancer cells.