Naoko Shima

A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice

Mcm4 (minichromosome maintenance–deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2–MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4Chaos3 encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4−) caused preimplantation lethality, McmChaos3/− embryos died late in gestation, indicating that Mcm4Chaos3 is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4Chaos3/Chaos3 females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.

The translesion DNA polymerase θ plays a dominant role in immunoglobulin gene somatic hypermutation

Immunoglobulin (Ig) somatic hypermutation (SHM) critically underlies the generation of high‐affinity antibodies. Mutations can be introduced by error‐prone polymerases such as polymerase ζ (Rev3), a mispair extender, and polymerase η, a mispair inserter with a preference for dA/dT, while repairing DNA lesions initiated by AID‐mediated deamination of dC to yield dU:dG mismatches. The partial impairment of SHM observed in the absence of these polymerases led us to hypothesize a main role for another translesion DNA polymerase. Here, we show that deletion in C57BL/6J mice of the translesion polymerase θ, which possesses a dual nucleotide mispair inserter–extender function, results in greater than 60% decrease of mutations in antigen‐selected V186.2DJH transcripts and greater than 80% decrease in mutations in the Ig H chain intronic JH4‐iEμ sequence, together with significant alterations in the spectrum of the residual mutations. Thus, polymerase θ plays a dominant role in SHM, possibly by introducing mismatches while bypassing abasic sites generated by UDG‐mediated deglycosylation of AID‐effected dU, by extending DNA past such abasic sites and by synthesizing DNA during dU:dG mismatch repair.

The Mouse Genomic Instability Mutation chaos1 Is an Allele of Polq That Exhibits Genetic Interaction with Atm

ABSTRACT chaos1 (for chromosome aberrations occurring spontaneously 1) is a recessive mutation that was originally identified in a phenotype-based screen for chromosome instability mutants in mice. Mutant animals exhibit significantly higher frequencies of spontaneous and radiation- or mitomycin C-induced micronucleated erythrocytes, indicating a potential defect in homologous recombination or interstrand cross-link repair. The chaos1 allele was genetically associated with a missense mutation in Polq, which encodes DNA polymerase θ. We demonstrate here that chaos1 is a mutant allele of Polq by using two genetic approaches: chaos1 mutant phenotype correction by a bacterial artificial chromosome carrying wild-type Polq and a failed complementation test between chaos1 and a Polq-disrupted allele generated by gene targeting. To investigate the potential involvement of Polq in DNA double-strand break repair, we introduced chaos1 into an Atm (for ataxia telangiectasia mutated)-deficient background. The majority (∼90%) of double-homozygous mice died during the neonatal period. Surviving double mutants exhibited synergistic phenotypes such as severe growth retardation and enhanced chromosome instability. However, remarkably, double mutants had delayed onset of thymic lymphoma, significantly increasing life span. These data suggest a unique role of Polq in maintaining genomic integrity, which is probably distinctive from the major homologous recombination pathway regulated by ATM.

The cII Locus in the Muta " Mouse System

Here, we report the first application and characterization of the cII locus as a mutational target for use with the Muta™Mouse system for quantifying somatic mutations in vivo. This locus can be analyzed for mutations using positive selection and is identical in sequence to the cII in the Big Blue® Mouse. The cII displays similar spontaneous (5.5 × 10–5) and induced mutation frequencies when compared to the lacZ gene in the small intestine of MutaMice treated with ENU (N‐ethyl‐N‐nitrosourea). After acute treatment with 250 mg/kg ENU (ip) the mutant frequencies were 127 × 10–5 at the cII and 147 × 10–5 at the lacZ loci, reaching a maximal mutant frequency 10 days posttreatment and remaining constant thereafter. These data prove that this transgene is genetically neutral, conferring neither selective advantage nor disadvantage on the host cells. The cII dose response curve was linear (R2 = 0.93) comparable to the lacZ after treatments with 0, 50, 150, or 250 mg/kg ENU. Use of the cII locus (0.3 kb) addresses the single most significant drawback associated with the MutaMouse system, namely the inability to obtain sequence spectra efficiently, due to the large size of the lacZ gene (3.0 kb). Moreover, a less obvious application, but nevertheless of considerable importance, is the easy identification of jackpot mutations, without sequencing. The cII, identical in both sequence and origin on the transgenic constructs used in producing the Big Blue and MutaMouse systems, provides the first transgenic locus common to the two widely used in vivo mutagenesis assays. Environ. Mol. Mutagen. 34:201–207, 1999

Forward genetic screens for meiotic and mitotic recombination-defective mutants in mice.

The goal of understanding the function of all mammalian genes is best accomplished through mutational analyses. Although the sequence of the mouse genome is now available and many genes have been identified, it is not possible to ascribe functions accurately to these genes in silico. Gene targeting using embryonic stem cells is ideal for analysis of individual genes selected on the basis of sequence features, but it is impractical for identifying novel genes involved in particular biological processes. Phenotype-based random mutagenesis of the genome is well suited for this goal. In the mouse, N-ethyl-N-nitrosourea (ENU) induces point mutations at a high frequency in the mouse germline. In this chapter, we describe methods for detecting and characterizing recombination mutations in mice produced by ENU mutagenesis. Potential meiotic recombination mutants are identified in a hierarchical fashion, by performing a screen for infertility, then gonad histology to determine whether meiotic arrest occurs, and finally by immunohistochemical analysis of meiotic chromosome with a battery of antibody markers. Screening for mutations potentially required for recombinational repair of DNA damage in somatic cells is performed using a flow cytometry-based micronucleus assay. Both strategies have proved effective in identifying desired classes of mutations.

Synergisms detected among methyl methanesulfonate, ethyl methanesulfonate and X-rays in inducing somatic mutations in the stamen hairs of Tradescantia clone BNL 4430

Abstract Synergisms among methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and X-rays in inducing somatic mutations were examined in the stamen hairs of Tradescantia clone BNL 4430, a blue-pink heterozygote. The young inflorescence-bearing shoots with roots cultivated in the nutrient solution circulating growth chamber were used as tester plants. The mutagenicity of each single mutagen, MMS, EMS or X-rays, was first examined and a dose-response relation for each of them was determined. Induced somatic pink mutation frequency per hair-cell division increased with increasing MMS, EMS and X-ray doses, with slopes of 1.283, 1.293 and 1.252, respectively, on log-log graphs, confirming the existence of similar patterns of the three dose-response curves. MMS was found, however, to be approximately 20 times more mutagenic than EMS. As for synergism among the different mutagens, it was judged to be acting synergistically when an observed mutation frequency after applying two different mutagens was statistically significantly higher than the mutation frequency expected from the additive effects of the two mutagens. Clear synergistic effects in inducing mutations were confirmed between MMS and X-rays, EMS and X-rays, and also between MMS and EMS, mostly in the higher dose ranges of these mutagens. Synergistic effects in causing loss of reproductive integrity of hair cells were also observed among these three mutagens.

Mutagenic synergism detected between dimethyl sulfate and X-rays but not found between N-methyl-N-nitrosourea and X-rays in the stamen hairs of Tradescantia clone BNL 4430

Mutagenic interactions with X-rays of two monofunctional alkylating agents, dimethyl sulfate (DMS) and N-methyl-N-nitrosourea (MNU), were studied in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. The young inflorescence-bearing shoots with roots cultivated in the nutrient solution circulating growth chamber were used as tester plants. Synergism between two different mutagens was judged to have occurred when the mutation frequency observed after applying the two mutagens concurrently was statistically significantly higher than the mutation frequency expected from the additive effects of the two mutagens. Clear synergistic effects in inducing somatic pink mutations were detected with all combinations of doses of DMS and X-rays examined, even in a relatively low X-ray dose range (down to 299 mGy), resembling those confirmed earlier between ethyl methanesulfonate (EMS) and X-rays, but somewhat differing from the synergisms observed earlier between methyl methanesulfonate (MMS) and X-rays. On the other hand, no mutagenic synergism was detected between MNU and X-rays, even in a relatively high X-ray dose range (up to 862 mGy). The presence or absence of mutagenic synergisms of these alkylating agents with X-rays could be related to the action mechanism of each alkylating agent.

Genetic screen for chromosome instability in mice: Mcm4 and breast cancer.

We recently isolated a hypomorphic mutation of Mcm4 in a phenotype-based screen for chromosome instability in mice. This mutation, named Chaos3 (Chromosome aberrations occurring spontaneously 3), causes exclusively mammary adenocarcinomas in ~80% of homozygous females. Mcm4 encodes a subunit of the MCM2-7 complex, the replication-licensing factor and the replicative helicase. The Mcm4Chaos3 mutation appears to destabilize the MCM2-7 complex, causing impaired DNA replication. These findings demonstrate, for the first time, the causative role of an Mcm mutation in cancer development. Furthermore, this raises the possibility that hypomorphic mutations in MCM2-7 genes may increase breast cancer risk in humans.

A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression

Abstract Accumulating evidence suggests that dormant DNA replication origins play an important role in the recovery of stalled forks. However, their functional interactions with other fork recovery mechanisms have not been tested. We previously reported intrinsic activation of the Fanconi anemia (FA) pathway in a tumor-prone mouse model (Mcm4chaos3) with a 60% loss of dormant origins. To understand this further, we introduced a null allele of Fancc (Fancc−), encoding a member of the FA core complex, into the Mcm4chaos3 background. Primary embryonic fibroblasts double homozygous for Mcm4chaos3 and Fancc− (Mcm4chaos3/chaos3;Fancc−/−) showed significantly increased levels of markers of stalled/collapsed forks compared to either single homozygote. Interestingly, a loss of dormant origins also increased the number of sites in which replication was delayed until prophase, regardless of FA pathway activation. These replication defects coincided with substantially elevated levels of genome instability in Mcm4chaos3/chaos3;Fancc−/− cells, resulting in a high rate of perinatal lethality of Mcm4chaos3/chaos3;Fancc−/− mice and the accelerated tumorigenesis of surviving mice. Together, these findings uncover a specialized role of dormant origins in replication completion while also identifying important functional overlaps between dormant origins and the FA pathway in maintaining fork progression, genome stability, normal development and tumor suppression.

Somatic mutation in the mammary gland: influence of time and estrus.

A critical factor in the quantitation of mutation induction in vivo is the time interval between treatment and sampling. In order to study mutagenesis in the mammary epithelium, the cell type in which breast cancer arises, we have measured the manifestation time, the minimum time required for the maximum mutant frequency to be achieved, in this tissue. The F1 LacZ transgenic mice (Muta MousexSWR) were treated with N-ethyl-N-nitrosourea (ENU) at 50 mg/kg for five consecutive days and then sampled at 1, 2, 4, 6, 9, or 12 weeks after the last treatment. The LacZ- mutant frequency reached a maximum at 4 weeks post-treatment and did not vary significantly thereafter. Dlb-1- mutations in the small intestine reached a maximum at 2 weeks after treatment and did not vary significantly thereafter. Since the stage of estrus cycle during carcinogen exposure influences the mammary tumor incidence and latency, it was expected that it would also affect mutation induction. To test this, F1 LacZ mice in the estrus or di-estrus stage were treated with an acute dose of 250 mg/kg ENU and sampled 10-13 weeks post-treatment. No statistical difference between the two groups was found, indicating that the effect of estrus on carcinogenesis is not due to variation in the sensitivity of the stage of the mammary gland to mutation.