Mutational specificity analysis: assay for mutations in the yeast SUP4-o gene.

Mutational specificity analysis can yield valuable insights into processes that generate genetic change or maintain genetic stability. Powerful diagnostic tools for such analysis have been created by combining genetic assays for mutation with DNA sequencing. Here, steps for isolating spontaneous mutations in the yeast (Saccharomyces cerevisiae) suppressor tRNA gene SUP4-o as a prelude to sequence characterization are described (modifications of this protocol can be used to study induction of mutations by various physical or chemical agents).

Age and exposure to arsenic alter base excision repair transcript levels in mice.

Arsenic (As) induces DNA-damaging reactive oxygen species. Most oxidative DNA damage is countered by base excision repair (BER), the capacity for which may be reduced in older animals. We examined whether age and consumption of As in lactational milk or drinking water influences BER gene transcript levels in mice.

A role for PCNA2 in translesion synthesis by Arabidopsis thaliana DNA polymerase eta.

Eukaryotic DNA polymerase eta (Poleta) confers ultraviolet (UV) resistance by catalyzing translesion synthesis (TLS) past UV photoproducts. Poleta has been studied extensively in budding yeast and mammalian cells, where its interaction with monoubiquitylated proliferating cell nuclear antigen (PCNA) is necessary for its biological activity. Recently, in collaboration with other investigators, our laboratory demonstrated that Arabidopsis thaliana Poleta is required for UV resistance in plants.

UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis.

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants.

Arabidopsis thaliana Y-family DNA polymerase eta catalyses translesion synthesis and interacts functionally with PCNA2.

Upon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase eta (Poleta), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPoleta contributes to UV resistance.

Plant responses to UV radiation and links to pathogen resistance.

Increased incident ultraviolet (UV) radiation due to ozone depletion has heightened interest in plant responses to UV because solar UV wavelengths can reduce plant genome stability, growth, and productivity. These detrimental effects result from damage to cell components including nucleic acids, proteins, and membrane lipids. As obligate phototrophs, plants must counter the onslaught of cellular damage due to prolonged exposure to sunlight.

Arabidopsis homologue of human transcription factor IIH/nucleotide excision repair factor p44 can function in transcription and DNA repair and interacts with AtXPD.

Eukaryotic general transcription factor (TF) IIH is composed of 10 proteins, seven of which are also required for nucleotide excision repair (NER) of UV radiation-induced DNA damage in human cells and yeast. Plant homologues of the human TFIIH subunits XPB and XPD that function in NER have been isolated but none has been shown to operate in transcription. Here we address the capabilities of Arabidopsis thaliana AtGTF2H2 and AtXPD, homologues of the essential interacting human/yeast TFIIH components p44/Ssl1 and XPD/Rad3, respectively.

Components of nucleotide excision repair and DNA damage tolerance in Arabidopsis thaliana.

As obligate phototrophs, and despite shielding strategies, plants sustain DNA damage caused by UV radiation in sunlight. By inhibiting DNA replication and transcription, such damage may contribute to the detrimental effects of UV radiation on the growth, productivity, and genetic stability of higher plants. However, there is evidence that plants can reverse UV-induced DNA damage by photoreactivation or remove it via nucleotide excision repair.

Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant.

The Saccharomyces cerevisiae RAD1 and human XPF genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination. An Arabidopsis thaliana gene (AtRAD1) encoding the orthologous plant protein has been identified recently. Here we report the isolation of three structurally distinct AtRAD1 cDNAs from A.