Germ line DNA mismatch repair mutations in MLH1 and MSH2 underlie the vast majority of hereditary non-polyposis colon cancer. Four mammalian homologues of Escherichia coli MutL heterodimerize to form three distinct complexes: MLH1/PMS2, MLH1/MLH3, and MLH1/PMS1. Although MLH1/PMS2 is generally thought to have the major MutL activity, the precise contributions of each MutL heterodimer to mismatch repair functions are poorly understood. Here, we show that Mlh3 contributes to mechanisms of tumor suppression in the mouse. Mlh3 deficiency alone causes microsatellite instability, impaired DNA-damage response, and increased gastrointestinal tumor susceptibility. Furthermore, Mlh3;Pms2 double-deficient mice have tumor susceptibility, shorter life span, microsatellite instability, and DNA-damage response phenotypes that are indistinguishable from Mlh1-deficient mice. Our data support previous results from budding yeast that show partial functional redundancy between MLH3 and PMS2 orthologues for mutation avoidance and show a role for Mlh3 in gastrointestinal and extragastrointestinal tumor suppression. The data also suggest a mechanistic basis for the more severe mismatch repair-related phenotypes and cancer susceptibility in Mlh1-versus Mlh3-or Pms2-deficient mice. Contributions by both MLH1/MLH3 and MLH1/PMS2 complexes to mechanisms of mismatch repair-mediated tumor suppression, therefore, provide an explanation why, among MutL homologues, only germ line mutations in MLH1 are common in hereditary nonpolyposis colon cancer. (Cancer Res 2005; 65(19): 8662-70)
Phenylketonuria (PKU) due to recessively inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia, which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake. In this study, liver-tropic recombinant AAV2/8 vectors were used to deliver CRISPR/Cas9 machinery and facilitate correction of the Pah enu2 allele by homologous recombination. Additionally, a non-homologous end joining (NHEJ) inhibitor, vanillin, was co-administered with the viral drug to promote homologydirected repair (HDR) with the AAV-provided repair template. This combinatorial drug administration allowed for lifelong, permanent correction of the Pah enu2 allele in a portion of treated hepatocytes of mice with PKU, yielding partial restoration of liver PAH activity, substantial reduction of blood phenylalanine, and prevention of maternal PKU effects during breeding. This work reveals that CRISPR/Cas9 gene editing is a promising tool for permanent PKU gene editing.
DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the hMLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wildtype NF1 gene. Somatic NF1 mutations were also de-tected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/-mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.
We developed a cell division-activated Cre-lox system for stochastic recombination of loxP-flanked loci in mice. Cre activation by frameshift reversion is modulated by DNA mismatch-repair status and occurs in individual cells surrounded by normal tissue, mimicking spontaneous cancer-causing mutations. This system should be particularly useful for delineating pathways of neoplasia, and determining the developmental and aging consequences of specific gene alterations.Valuable mouse cancer models exist that combine conditional expression of the Cre recombinase with various loxP-flanked tumor suppressor or oncogene alleles 1 . In these model systems, 'cancer' is induced by gene alteration ubiquitously throughout the target tissue or in a selected cell type. But this does not accurately mimic the natural process of sporadic cancer initiation and progression. Two Cre-lox models that facilitate stochastic cancer gene alterations in isolated cells rely on homologous recombination to induce activation of an oncogene 2 , or to induce inactivation of one or more tumor suppressor genes on the same chromosome 3 . The former system is restricted in that it is only applicable to activation of an oncogenic K-ras allele 2 . The latter more flexible system uses engineered loxP-FRT sites to induce mitotic recombination of individual chromosomes containing modified genes 3 . Here we report a highly versatile system that features Cre-mediated stochastic genetic changes in single cells or cell lineages in normal tissue. The system can be applied to any loxP-flanked allele, is dependent on cell division and can be modulated by DNA mismatch-repair status.To construct an inactive but revertible Cre allele, we first engineered an 11-bp A·T run in a modified version of Cre 4 without altering the nuclear localization signal (Supplementary Methods online). We added an extra A·T base pair, creating a +1 bp out-of-frame Cre allele, which we termed 12A-Cre. To ensure efficient expression at the intended target locus, we added a splice acceptor and internal ribosome entry site to 12A-Cre. We cloned the modified 12A-Cre plus a neo module between homology arms of the DNA mismatch repair gene Pms2 (ref. 5) to generate a Pms2-Cre targeting vector. Targeting resulted in an out-of-frame Cre gene under the control of the Pms2 promoter, which is expressed in several cell types, including stem cells of the mouse intestine 6 . Targeting to Pms2 removed exon 2, creating a null allele, which we refer to as Pms2 cre (Fig. 1a). Because of mismatch-repair deficiency, Pms2 cre/cre mice should have increased frequency of −1 bp frameshifts 7 and hence increased Cre reversion relative to Pms2 cre/+ mice. Therefore, Cre activation frequency can be modulated appropriately for a particular study by breeding Pms2 cre/+ or Pms2 cre/cre mice. Notably, this system should
Null mutations in DNA mismatch repair (MMR) genes elevate both base substitutions and insertions/ deletions in simple sequence repeats. Data suggest that during replication of simple repeat sequences, polymerase slippage can generate single-strand loops on either the primer or template strand that are subsequently processed by the MMR machinery to prevent insertions and deletions, respectively. In the budding yeast Saccharomyces cerevisiae and mammalian cells, MMR appears to be more efficient at repairing mispairs comprised of loops on the template strand compared to loops on the primer strand. We identified two novel yeast pms1 alleles, pms1-G882E and pms1-H888R, which confer a strong defect in the repair of "primer strand" loops, while maintaining efficient repair of "template strand" loops. Furthermore, these alleles appear to affect equally the repair of 1-nucleotide primer strand loops during both leading-and lagging-strand replication. Interestingly, both pms1 mutants are proficient in the repair of 1-nucleotide loop mispairs in heteroduplex DNA generated during meiotic recombination. Our results suggest that the inherent inefficiency of primer strand loop repair is not simply a mismatch recognition problem but also involves Pms1 and other proteins that are presumed to function downstream of mismatch recognition, such as Mlh1. In addition, the findings reinforce the current view that during mutation avoidance, MMR is associated with the replication apparatus.DNA mismatch repair (MMR) contributes to genomic integrity by repairing mismatches generated during replication, by chemical damage, and as "heteroduplex" intermediates during recombination (7,28,31,35,44). In addition, the MMR system in higher eukaryotes plays a role in response to DNA damage (3,6,7,62). Inherited MMR defects lead to a mutator phenotype, which in humans and mice is associated with increased cancer susceptibility (5,7,13,16,38,50). The MMR system of Escherichia coli has been reconstituted in vitro with purified proteins, including the dedicated proteins MutS, MutL, and MutH (44,56). The MutS protein, a homodimer, first binds the mispair, followed by recruitment of MutL, the endonuclease MutH, the UvrD helicase, four exonucleases, DNA polymerase, and ligase. Together with transient Dammediated hemimethylation, these proteins impose strand specificity that leads to specific repair of the newly replicated strand (10,25,26,43,44,74).In the budding yeast Saccharomyces cerevisiae, six MutS homologues (Msh proteins Msh1 to Msh6) and four MutL homologues (Mlh proteins, Mlh1 to Mlh3, and Pms1) function in various MMR transactions (7,28,31,35). Unlike E. coli, the MutS and MutL activities of budding yeast and mammals are each comprised of heterodimers. Mismatches in nuclear DNA replication intermediates are recognized by the Msh2/Msh6 and Msh2/Msh3 heterodimers, which have partial functional overlap (7,35,42). Msh2/Msh6 operates in the repair of basebase mispairs and 1-nucleotide "insertion/deletion" loops (28,32,41), while Msh2/Msh3 functions ...
Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.
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