Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH-/- mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.

Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1 (REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1⌬ apn2⌬ strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes. Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase mediates the mutagenic bypass of AP sites.

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases (Pol) and Pol and postreplicational repair mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Here we report our studies with a proliferating cell nuclear antigen (PCNA) mutation, pol30-119, which results from a change of the lysine 164 residue to arginine. It has been shown recently that following treatment of yeast cells with DNA-damaging agents, the lysine 164 residue of PCNA becomes monoubiquitinated in a Rad6-Rad18-dependent manner and that subsequently this PCNA residue is polyubiquitinated via a lysine 63-linked ubiquitin chain in an Mms2-Ubc13-, Rad5-dependent manner. PCNA is also modified by SUMO conjugation at the lysine 164 residue. Our genetic studies with the pol30-119 mutation show that in addition to conferring a defect in Pol-dependent UV mutagenesis and in Pol-dependent TLS, this PCNA mutation inhibits postreplicational repair of discontinuities that form in the newly synthesized strand across from UV lesions. In addition, we provide evidence for the activation of the RAD52 recombinational pathway in the pol30-119 mutant and we infer that SUMO conjugation at the lysine 164 residue of PCNA has a role in suppressing the Rad52-dependent postreplicational repair pathway.Proliferating cell nuclear antigen (PCNA) is the eukaryotic sliding clamp required for processive DNA synthesis. The Saccharomyces cerevisiae POL30 gene encoding PCNA is essential for cell viability (7), and conditional lethal pol30 mutations confer defects in DNA replication (1, 3). PCNA is loaded onto the template-primer junctions of DNA by replication factor C in an ATP-dependent reaction. DNA polymerase ␦ (Pol␦) then binds PCNA and carries out processive DNA synthesis (6, 23). In reconstituted systems containing viral origin sequences, PCNA and Pol␦ carry out replication of both the leading and lagging strands (26,38). In addition to its essential role in DNA replication, PCNA has been shown to be required for various DNA repair processes, including nucleotide excision repair (NER), base excision repair, and mismatch repair (23); more recently, PCNA has been shown to interact physically and functionally with the various translesion synthesis (TLS) polymerases of yeasts and humans (9-11, 13).Genetic studies of S. cerevisiae have indicated that PCNA is involved in RAD6-dependent error-free postreplicative bypass of UV-damaged DNA (35). Postreplicative bypass processes come into play when the DNA replicational machinery encounters a DNA lesion in the template strand and is unable to replicate past the lesion. Replication of damaged DNA templates can occur by error-free damage avoidance processes in which the undamaged complementary sequence is used to accomplish replication through the damaged site (14) or it may involve TLS by a specialized DNA polymerase across from the lesion. The S. cerevisiae RAD6 and RAD18 ...

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitinconjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5⌬ and mms2⌬ mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase (Pol) but not in the absence of REV3-encoded DNA Pol. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Pol, error-free translesion synthesis by Pol, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Pol␦. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.Replication of damaged DNA templates can occur by translesion synthesis. This process is usually mutagenic but can be error free as well (see below). In Escherichia coli, the umuCencoded DNA polymerase V (PolV) promotes mutagenic translesion synthesis (29,35). Error-prone translesion synthesis, however, accounts for only a minor portion of the damage bypass in E. coli, whereas much of the damage bypass occurs by means of error-free mechanisms. Error-free bypass mechanisms come into play when the replication machinery terminates synthesis at the site of a DNA lesion and replication restarts downstream of the lesion. This results in the formation of a gap in the newly synthesized strand across from the DNA lesion (30). In E. coli, this gap is filled in by RecA-dependent recombination mechanisms by which the DNA strand from the undamaged sister duplex is transferred to the gapped strand in the damaged duplex (31). In mammalian cells, a copy choice type of DNA synthesis has been invoked as the major mechanism for the filling in of gaps formed in the newly synthesized strand opposite DNA lesions (11). In this mechanism, the newly synthesized daughter strand of the undamaged complementary sequence is used as the template to bypass the lesion. Once the lesion is bypassed, DNA polymerase switches back to copying the damaged template strand.Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the RAD6 and RAD18 genes in error-free, as well as mutagenic, damage bypass processes (22,28). rad6 and rad18 mutants exhibit a ...

Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD, however the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD.