Adenomatous polyposis coli truncation mutations in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced intestinal tumours of multiple intestinal neoplasia mice

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces intestinal tumours in C57BL/6J-multiple intestinal neoplasia (Min)/+ mice. The main mechanism for PhIP-induced tumour induction in Min/+ mice is loss of the wild-type adenomatous polyposis coli (Apc) allele, i.e. loss of heterozygosity (LOH). In this study, single injections of either 10, 17.5 or 25 mg/kg PhIP on days 3-6 after birth all increased the mean number of small intestinal tumours two to three-fold, from 37.7 in controls to 124.8 in the PhIP-treated Min/+ mice. In total, we analysed 292 small intestinal tumours and 253 of these had LOH. The frequency of LOH in the Apc gene was 88, 93, 83 and 84% in tumours of 0, 10, 17.5 and 25 mg/kg PhIP-treated mice, respectively. Therefore, these lower doses of PhIP did not reduce the frequency of LOH, as found in our previous study with a single injection of 50 mg/kg PhIP (Mutat. Res. 1-2 (2002) 157). In the second part of this study, we wanted to characterise Apc truncation mutations from tumour samples apparently retaining the Apc wild-type allele from this and two previous experiments with PhIP-exposed Min/+ mice. In the first half of exon 15 in Apc, we verified 25 mutations from 804 tumour samples of PhIP-treated mice. Of these were 60% G-->T transversions, and 16% G deletions, indicating that these are the predominant types of PhIP-induced truncation mutations in the Apc gene in Min/+ mice. Most of the mutations were located between codon 989 and 1156 corresponding to the first part of the beta-catenin binding region. We also identified two Apc truncation mutations from 606 spontaneously formed intestinal tumours from untreated Min/+ mice, one C-->T transition and one T insertion, which were different from those induced by PhIP.     

A role for the MutL mismatch repair Mlh3 protein in immunoglobulin class switch DNA recombination and somatic hypermutation

Class switch DNA recombination (CSR) and somatic hypermutation (SHM) are central to the maturation of the Ab response. Both processes involve DNA mismatch repair (MMR). MMR proteins are recruited to dU:dG mispairs generated by activation-induced cytidine deaminase-mediated deamination of dC residues, thereby promoting S-S region synapses and introduction of mismatches (mutations). The MutL homolog Mlh3 is the last complement of the mammalian set of MMR proteins. It is highly conserved in evolution and is essential to meiosis and microsatellite stability. We used the recently generated knockout mlh3(-/-) mice to address the role of Mlh3 in CSR and SHM. We found that Mlh3 deficiency alters both CSR and SHM. mlh3(-/-) B cells switched in vitro to IgG and IgA but displayed preferential targeting of the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif by Sgamma1 and Sgamma3 breakpoints and introduced more insertions and fewer donor/acceptor microhomologies in Smu-Sgamma1 and Smu-Sgamma3 DNA junctions, as compared with mlh3(+/+) B cells. mlh3(-/-) mice showed only a slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged J(H)4 gene. However, the residual mutations were altered in spectrum. They comprised a decreased proportion of mutations at dA/dT and showed preferential RGYW/WRCY targeting by mutations at dC/dG. Thus, the MMR Mlh3 protein plays a role in both CSR and SHM.     

Regional mutagenicity of heterocyclic amines in the intestine: mutation analysis of the cII gene in lambda/lacZ transgenic mice

Transgenic mouse assays have revealed that the mouse intestine, despite its resistance to carcinogenesis, is sensitive to the mutagenicity of some heterocyclic amines (HCAs). Little is known, however, about the level and localization of that sensitivity. We assessed the mutagenicity of four orally administered (20 mg/kg per day for 5 days) HCAs-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) hydrochloride, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) acetate-in the intestine of male MutaMice. Two weeks after the last administration, we isolated epithelium from the small intestine, cecum, and colon and analyzed lacZ and cII transgene mutations. PhIP increased the lacZ mutant frequency (MF) in all the samples, and in the small intestine, cII and lacZ MFs were comparable. In the cII gene, G:C to T:A and G:C to C:G transversions were characteristic PhIP-induced mutations (which has also been reported for the rat colon, where PhIP is carcinogenic). In the small intestine, PhIP increased the cII MF to four-fold that of the control, but IQ, MeIQ, and Trp-P-2 did not have a significant mutagenic effect. In the cecum, cII MFs induced by IQ and MeIQ were 1.9 and 2.7 times those in the control, respectively. The MF induced by MeIQ in the colon was 3.1 times the control value. Mutagenic potency was in the order PhIP>MeIQ>IQ; Trp-P-2 did not significantly increase the MF in any tissue. The cecum was the most susceptible organ to HCA mutagenicity.     

One dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induces tumours in Min/+ mice by truncation mutations or LOH in the Apc gene

The C57BL/6J-Min/+ (multiple intestinal neoplasia) mouse has a heterozygous nonsense Apc(Min) (adenomatous polyposis coli) mutation, and numerous adenomas spontaneously develop in the intestine. Neonatal exposure of Min/+ mice to the food carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (one injection of 50mg/kg) increased the number of small intestinal tumours about three- and two-fold, respectively. The number of colonic tumours was only increased in males. We examined whether the wild-type Apc allele was affected in intestinal tumours induced by either PhIP or IQ. In spontaneously formed and in IQ-induced small intestinal and colonic tumours from these mice, the main mechanism for tumour induction was loss of wild-type Apc allele, i.e. loss of heterozygosity (LOH). In contrast to the IQ-induced (84% LOH) and spontaneously (88% LOH) formed tumours, only 55% of the PhIP-induced small intestinal tumours from males showed LOH. Tumours that apparently had retained the wild-type Apc allele were further analysed for the presence of truncated Apc proteins by the in vitro synthesised protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in two of the 12 PhIP-induced tumours in segment 2 (codons 686-1217), and two of five IQ-induced tumours, one in segment 2 and the other in segment 3 (codons 1099-1693). Three of these four mutations, all in segment 2 of the Apc gene, were confirmed by sequencing. The PhIP-induced mutations were detected at codon 1125 (C deletion) and 1130 (G-T transversion), and the IQ-induced mutation was at codon 956 (C-T transition). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc allele. These results show that one injection of either PhIP or IQ induces intestinal tumours in the Min/+ mice by inactivation of the wild-type Apc allele either by causing LOH or truncation mutations.     

Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes

Oxidative damage of nucleotides within DNA or precursor pools caused by oxygen radicals is thought to play an important role in spontaneous mutagenesis, as well as carcinogenesis and aging. In particular, 8-oxodGTP and 2-OHdATP are potent mutagenic substrate for DNA synthesis. Mammalian MTH1 catalyzes hydrolysis of these mutagenic substrates, suggesting that it functions to prevent mutagenesis caused by these oxidized nucleotides. We have established MTH1(-/-) mice lacking the 8-oxodGTPase activity, which were shown to be susceptible to lung, liver and stomach cancers. To examine in vivo mutation events due to the MTH1-deficiency, a reporter gene, rpsL of Escherichia coli, was introduced into MTH1(-/-) mice. Interestingly, the net frequency of rpsL(-) forward mutants showed no apparent increase in MTH1(-/-) mice as compared to MTH1(+/+) mice. However, we found differences between these two genotypes in the class- and site-distributions of the rpsL(-) mutations recovered from the mice. Unlike MutT-deficient E. coli showing 1000-fold higher frequency of A:T-->C:G transversion than the wild type cells, an increase in frequency of A:T-->C:G transversion was not evident in MTH1 nullizygous mice. Nevertheless, the frequency of single-base frameshifts at mononucleotide runs was 5.7-fold higher in spleens of MTH1(-/-) mice than in those of wild type mice. Since the elevated incidence of single-base frameshifts at mononucleotide runs is a hallmark of the defect in MSH2-dependent mismatch repair system, this weak site-specific mutator effect of MTH1(-/-) mice could be attributed to a partial sequestration of the mismatch repair function that may act to correct mispairs with the oxidized nucleotides. Consistent with this hypothesis, a significant increase in the frequency of G:C-->T:A transversions was observed with MTH1(-/-) MSH2(-/-) mice over MSH2(-/-) mice alone. These results suggest a possible involvement of multiple anti-mutagenic pathways, including the MTH1 protein and other repair system(s), in mutagenesis caused by the oxidized nucleotides.     

Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.     

Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis

To examine involvement of mismatch repair system in alkylation-induced apoptosis and mutagenesis, cell lines defective in the Mgmt gene encoding a DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase, and/or the Mlh1 gene encoding a protein involved in mismatch repair were established from gene-targeted mice. Mgmt(-/-) cells are hypersensitive to the killing effect of N-methyl-N-nitrosourea (MNU) and this effect of MNU was overcome by introducing an additional mutation in the Mlh1 gene. Mgmt(-/-)Mlh1(-/-) cells are more resistant to MNU than are wild-type cells. When the human Mgmt cDNA sequence with a strong promoter was introduced, the wild-type cells acquired the same high level of resistance to MNU as that of Mgmt(-/-)Mlh1(-/-) cells. Although no apparent increase in MNU-induced mutant frequency was observed in such methyltransferase-overproducing wild-type cells, mutant frequency of Mgmt(-/-)Mlh1(-/-) cells became 10-fold higher after being treated with MNU. Mgmt(-/-)Mlh1(+/-) cells carrying approximately half the normal level of MLH1 protein showed a normal level of spontaneous mutant frequency, yet were still highly responsive to the mutagenic effect of the alkylating carcinogen. This haploinsufficient character of Mlh1 mutation was also observed in cell survival assays; Mgmt(-/-)Mlh1(+/-) cells were as resistant to MNU as were Mgmt(-/-)Mlh1(-/-) cells. While caspase-3 was induced in Mgmt(-/-)Mlh1(+/+) cells after treatment with MNU, no induction occurred in Mgmt(-/-)Mlh1(+/-) cells or in Mgmt(-/-)Mlh1(-/-) cells. The cellular content of MLH1 protein seems to be critical for determining if damaged cells enter into either a death or mutation-inducing pathway. The haploinsufficient phenotype of Mlh1-heterozygous cells may be explained by competition in heterodimer formation between MLH1 homologues.     

Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates

DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wild-type cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.     

A role for Mlh3 in somatic hypermutation

Somatic hypermutation (SHM) and class switch recombination (CSR) allow B cells to make high affinity antibodies of various isotypes. Both processes are initiated by activation-induced cytidine deaminase (AID) to generate dG:dU mismatches in the immunoglobulin genes that are resolved differently in SHM and CSR to introduce point mutations and recombination, respectively. The MutL homolog MLH3 has been implicated in meiosis and DNA mismatch repair (MMR). Since it interacts with MLH1, which plays a role in SHM and CSR, we examined these processes in Mlh3-deficient mice. Although deficiencies in other MMR proteins result in defects in SHM, Mlh3(-/-) mice exhibited an increased frequency of mutations in their immunoglobulin variable regions, compared to wild type littermates. Alterations of mutation spectra were observed in the Jh4 flanking region in Mlh3(-/-) mice. Nevertheless, Mlh3(-/-) mice were able to switch to IgG3 or IgG1 with similar frequencies to control mice. This is the first instance where a loss of a DNA repair protein has a positive impact on the rate of SHM, suggesting that Mlh3 normally inhibits the accumulation of mutations in SHM.     

Effect of conjugated linoleic acid on the formation of spontaneous and PhIP-induced mutation in the colon and cecum of rats

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has been reported to inhibit chemically induced mammary and colon carcinogenesis in rodents. In a preliminary experiment, we found that CLA significantly reduced the induction of mutations by the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the distal colon in male rats. Here, the chemopreventive properties of CLA were further evaluated by assessing its effect on PhIP-induced mutation and aberrant crypt foci (ACF) in both male and female rats. CLA (1%, w/w) was added to the diet (1) from weaning to 50-day-old, or (2) starting 1 week prior to exposure to PhIP. The 50-day-old Big Blue^® and F344 rats were then exposed to 100 ppm PhIP for 47 days. No sex differences were observed in mutagenic response to the various treatments in either the distal colon or cecum. The mutation frequency (MF) in the cecum and the distal colon from control animals is 4.3±1.3 and 5.3±1.4×10⁻⁵, respectively showing no statistically significant difference. Administration of PhIP induced a four-fold increase in the MF in the cecum and a seven-fold increase in the distal colon compared to the corresponding controls. Supplementation of the diet with CLA lowered the PhIP-induced MF in the distal colon by 23% (P<0.03), but had no effect in the cecum. The PhIP-induced ACF, determined 9 weeks after the termination of treatment with PhIP, were 0.75 ACF/rat, with 1.7 aberrant crypts /ACF in the colon of male rats, all located in the distal colon. This induction was completely inhibited by the addition of CLA.     

Apoptosis and mutation in the murine small intestine: loss of Mlh1- and Pms2-dependent apoptosis leads to increased mutation in vivo

The mismatch repair (MMR) protein Msh2 has been shown to function in the apoptotic response to alkylating agents in vivo. Here, we extend these studies to the MutL homologues (MLH) Mlh1 and Pms2 by analysing the apoptotic response within the small intestine of gene targeted strains. We demonstrate significant differences between Msh2, Mlh1 and Pms2 mutations in influencing apoptotic signalling following 50mg/kg N-methyl-nitrosourea (NMNU), with no obvious reliance upon either Mlh1 or Pms2. However, following exposure to 100mg/kg temozolomide or lower levels of NMNU (10mg/kg) both Mlh1- and Pms2-dependent apoptosis was observed, indicating that the apoptotic response at these levels of DNA damage is dependent on the MutL homologues. Given our ability to observe a MutLalpha dependence of the apoptotic response, we tested whether perturbations of this response directly translate into increases in mutation frequency in vivo. We show that treatment with temozolomide or 10mg/kg NMNU significantly increases mutation in both the Mlh1 and Pms2 mutant mice. At higher levels of NMNU, where the apoptotic response is independent of Mlh1 and Pms2, no gene dependent increase in mutation frequency was observed. These results argue that the MutSalpha and MutLalpha are not equally important in their ability to signal apoptosis. However, when MMR does mediate apoptosis, perturbation of this response leads to long-term persistence of mutant cells in vivo.     

RAD51D protects against MLH1-dependent cytotoxic responses to O6-methylguanine

S_N1-type methylating agents generate O⁶-methyl guanine (O⁶-meG), which is a potently mutagenic, toxic, and recombinogenic DNA adduct. Recognition of O⁶-meG:T mismatches by mismatch repair (MMR) causes sister chromatid exchanges, which are representative of homologous recombination (HR) events. Although the MMR-dependent mutagenicity and toxicity caused by O⁶-meG has been studied, the mechanisms of recombination induced by O⁶-meG are poorly understood. To explore the HR and MMR genetic interactions in mammals, we used the Rad51d and Mlh1 mouse models. Ablation of Mlh1 did not appreciably influence the developmental phenotypes conferred by the absence of Rad51d. Mouse embryonic fibroblasts (MEFs) deficient in Rad51d can only proliferate in p53-deficient background. Therefore, Rad51d^?/?Mlh1^?/? Trp53^?/? MEFs with a combined deficiency of HR and MMR were generated and comparisons between MLH1 and RAD51D status were made. To our knowledge, these MEFs are the first mammalian model system for combined HR and MMR defects. Rad51d-deficient MEFs were 5.3-fold sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) compared to the Rad51d-proficient MEFs. A pronounced G2/M arrest in Rad51d-deficient cells was accompanied by an accumulation of γ-H2AX and apoptosis. Mlh1-deficient MEFs were resistant to MNNG and showed no G2/M arrest or apoptosis at the doses used. Importantly, loss of Mlh1 alleviated sensitivity of Rad51d-deficient cells to MNNG, in addition to reducing γ-H2AX, G2/M arrest and apoptosis. Collectively, the data support the hypothesis that MMR-dependent sensitization of HR-deficient cells is specific for O⁶-meG and suggest that HR resolves DNA intermediates created by MMR recognition of O⁶-meG:T. This study provides insight into recombinogenic mechanisms of carcinogenesis and chemotherapy resulting from O⁶-meG adducts.     

The influence of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZalpha gene of m13mp10

One of the most predominating oxidative DNA damages, both spontaneously formed and after gamma-radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZalpha gene. For that purpose, non-irradiated or gamma-irradiated double-stranded (ds) M13mp10 DNA, with the lacZalpha gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)) as compared to the fpg(-) and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)).     

Age at exposure and Apc status influence the levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts in mouse intestine and liver

We have previously shown that C57BL/6J-Min/+ (multiple intestinal neoplasia) mice, heterozygous for the Min mutation in the adenomatous polyposis coli gene, were more susceptible to intestinal tumorigenesis and had higher intestinal PhIP-DNA adduct levels after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) on day 12 than on day 36 after birth [I.-L. Steffensen, H.A.J. Schut, J.E. Paulsen, A. Andreassen, J. Alexander, Intestinal tumorigenesis in multiple intestinal neoplasia mice induced by the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: perinatal susceptibility, regional variation, and correlation with DNA adducts, Cancer Res. 61 (200l) 8689-8696]. In the present study, we have evaluated further whether this difference in susceptibility is related to adduct formation/removal, cell proliferation, apoptosis or expression of the nucleotide excision repair protein Xeroderma pigmentosum group A (XPA) in the intestines. Min/+ and +/+ (wild-type) mice were given a subcutaneous injection of 50 mg/kgbw PhIP on day 12 or 36, and the levels of PhIP-DNA adducts after 8, 12, 24 h, 3 or 7 days were quantified by use of 32P-postlabelling. In Min/+ mice, adduct levels were significantly higher after exposure on day 12 than on day 36 in the middle (1.5- to 8.5-fold) and distal (1.3- to 6.5-fold) small intestine from 8h to 3 days after administration of PhIP, but not in the colon and proximal small intestine. In the liver - a non-target organ for PhIP - adduct levels were 2.0- to 7.5-fold higher after exposure on day 12 than on day 36 from 8 to 24h after exposure. Adduct levels were generally higher in the middle (1.1- to 1.8-fold) and distal (1.1- to 2.0-fold) small intestines of Min/+ compared with +/+ mice after PhIP exposure on day 12, i.e. in the area of the intestines previously found also to have the highest number of tumors in Min/+ mice. PhIP increased cell proliferation and the number of apoptotic cells in the intestine and liver. However, the higher susceptibility to intestinal tumorigenesis in Min/+ mice exposed to PhIP at early age, or in Min/+ mice compared with +/+ mice, could not be explained by differences in cell proliferation, apoptosis or expression of the XPA repair protein.     

Analysis of yeast pms1, msh2, and mlh1 mutators points to differences in mismatch correction efficiencies between prokaryotic and eukaryotic cells

Genetic stability relies in part on the efficiency with which post-replicative mismatch repair (MMR) detects and corrects DNA replication errors. In Escherichia coli, endogenous transition mispairs and insertion/deletion (ID) heterologies are corrected with similar efficiencies – but much more efficiently than transversion mispairs – as revealed by mutation rate increases in MMR mutants. To assess the relative efficiencies with which these mismatches are corrected in the yeast Saccharomyces cerevisiae, we examined repair of defined mismatches on heteroduplex plasmids and compared the spectra for >1000 spontaneous SUP4-o mutations arising in isogenic wild-type or MMR-deficient (pms1, mlh1, msh2) strains. Heteroduplexes containing G/T mispairs or ID heterologies were corrected more efficiently than those containing transversion mismatches. However, the rates of single base-pair insertion/deletion were increased much more (82-fold or 34-fold, respectively) on average than the rate of base pair substitutions (4.4-fold), with the rates for total transitions and transversions increasing to similar extents. Thus, the relative efficiencies with which mismatches formed during DNA replication are repaired appear to differ in prokaryotic and eukaryotic cells. In addition, our results indicate that in yeast, and probably other eukaryotes, these efficiencies may not mirror those obtained from an analysis of heteroduplex correction.     

Analysis of yeast pms1, msh2, and mlh1 mutators points to differences in mismatch correction efficiencies between prokaryotic and eukaryotic cells.

Genetic stability relies in part on the efficiency with which post-replicative mismatch repair (MMR) detects and corrects DNA replication errors. In Escherichia coli, endogenous transition mispairs and insertion/deletion (ID) heterologies are corrected with similar efficiencies – but much more efficiently than transversion mispairs – as revealed by mutation rate increases in MMR mutants. To assess the relative efficiencies with which these mismatches are corrected in the yeast Saccharomyces cerevisiae, we examined repair of defined mismatches on heteroduplex plasmids and compared the spectra for >1000 spontaneous SUP4-o mutations arising in isogenic wild-type or MMR-deficient (pms1, mlh1, msh2) strains. Heteroduplexes containing G/T mispairs or ID heterologies were corrected more efficiently than those containing transversion mismatches. However, the rates of single base-pair insertion/deletion were increased much more (82-fold or 34-fold, respectively) on average than the rate of base pair substitutions (4.4-fold), with the rates for total transitions and transversions increasing to similar extents. Thus, the relative efficiencies with which mismatches formed during DNA replication are repaired appear to differ in prokaryotic and eukaryotic cells. In addition, our results indicate that in yeast, and probably other eukaryotes, these efficiencies may not mirror those obtained from an analysis of heteroduplex correction. Received: 15 November 1998 / Accepted: 4 February 1999     

Comparative analysis of meiotic progression in female mice bearing mutations in genes of the DNA mismatch repair pathway

The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.     

Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide are oxidative metabolites that are responsible for the mutagenicity of 1-nitropyrene. In this study, the mutation spectra induced by oxidative metabolites in human cells were determined using a shuttle vector assay. The mutation frequencies induced by 1-nitropyrene 9,10-oxide were 2-3 times higher than those induced by 1-nitropyrene 4,5-oxide. The base substitutions induced by 1-nitropyrene 4,5-oxide were G --> A transitions, G --> C transversions, and G --> T transversions. In the case of 1-nitropyrene 9,10-oxide, G --> A transitions, G --> T transversions, A --> G transitions and G --> C transversions were observed. Most base substitution mutations induced by oxidative metabolites occurred at the guanine sites in the supF gene. These sequence-specific hot spots were commonly identified as 5'-GA sequences for both metabolites. On the other hand, the sequence-specific hot spots at the adenine sites were identified as 5'-CAC sequences for 1-nitropyrene 9,10-oxide. These results suggest that the oxidative metabolites of 1-nitropyrene induce sequence-specific DNA mutations at the guanine and adenine sites at high frequency.     

DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair

The yeast Mlh1–Pms1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1. We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1–Pms1 heterodimer. Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.