Ultraviolet Mutagenesis Coupled with Next-Generation Sequencing as a Method for Functional Interrogation of Powdery Mildew Genomes.

Powdery mildews are obligate biotrophic fungal pathogens causing important diseases of plants worldwide. Very little is known about the requirements for their pathogenicity at the molecular level. This is largely due to the inability to culture these organisms in vitro or to modify them genetically. Here, we describe a mutagenesis procedure based on ultraviolet (UV) irradiation to accumulate mutations in the haploid genome of the barley powdery mildew pathogen Blumeria graminis f. sp. hordei. Exposure of B. graminis f. sp. hordei conidia to different durations of UV-C radiation (10 s to 12 min) resulted in a reduced number of macroscopically visible fungal colonies. B. graminis f. sp. hordei colony number was negatively correlated with exposure time and the total number of consecutive cycles of UV irradiation. Dark incubation following UV exposure further reduced fungal viability, implying that photoreactivation is an important component of DNA repair in B. graminis f. sp. hordei. After several rounds of UV mutagenesis, we selected two mutant isolates in addition to the parental B. graminis f. sp. hordei K1 isolate for whole-genome resequencing. By combining automated prediction of sequence variants and their manual validation, we identified unique UV-induced mutations in the genomes of the two isolates. Most of these mutations were in the up- or downstream regions of genes or in the intergenic space. Some of the variants detected in genes led to predicted missense mutations. As an additional insight, our bioinformatic analyses revealed a complex population structure within supposedly clonal B. graminis f. sp. hordei isolates.

Repair of multiple simultaneous double-strand breaks causes bursts of genome-wide clustered hypermutation.

A single cancer genome can harbor thousands of clustered mutations. Mutation signature analyses have revealed that the origin of clusters are lesions in long tracts of single-stranded (ss) DNA damaged by apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, raising questions about molecular mechanisms that generate long ssDNA vulnerable to hypermutation. Here, we show that ssDNA intermediates formed during the repair of gamma-induced bursts of double-strand breaks (DSBs) in the presence of APOBEC3A in yeast lead to multiple APOBEC-induced clusters similar to cancer. We identified three independent pathways enabling cluster formation associated with repairing bursts of DSBs: 5' to 3' bidirectional resection, unidirectional resection, and break-induced replication (BIR). Analysis of millions of mutations in APOBEC-hypermutated cancer genomes revealed that cancer tolerance to formation of hypermutable ssDNA is similar to yeast and that the predominant pattern of clustered mutagenesis is the same as in resection-defective yeast, suggesting that cluster formation in cancers is driven by a BIR-like mechanism. The phenomenon of genome-wide burst of clustered mutagenesis revealed by our study can play an important role in generating somatic hypermutation in cancers as well as in noncancerous cells.

Single-nucleotide resolution dynamic repair maps of UV damage in Saccharomyces cerevisiae genome.

We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts in the Saccharomyces cerevisiae genome. We find that these photoproducts are removed from the genome primarily by incisions 13-18 nucleotides 5' and 6-7 nucleotides 3' to the UV damage that generate 21- to 27-nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand at early time points followed by predominantly nontranscribed strand repair at later stages. We have also characterized the excision repair level as a function of the transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism.

Genome-wide high-resolution mapping of UV-induced mitotic recombination events in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae and most other eukaryotes, mitotic recombination is important for the repair of double-stranded DNA breaks (DSBs). Mitotic recombination between homologous chromosomes can result in loss of heterozygosity (LOH). In this study, LOH events induced by ultraviolet (UV) light are mapped throughout the genome to a resolution of about 1 kb using single-nucleotide polymorphism (SNP) microarrays. UV doses that have little effect on the viability of diploid cells stimulate crossovers more than 1000-fold in wild-type cells. In addition, UV stimulates recombination in G1-synchronized cells about 10-fold more efficiently than in G2-synchronized cells. Importantly, at high doses of UV, most conversion events reflect the repair of two sister chromatids that are broken at approximately the same position whereas at low doses, most conversion events reflect the repair of a single broken chromatid. Genome-wide mapping of about 380 unselected crossovers, break-induced replication (BIR) events, and gene conversions shows that UV-induced recombination events occur throughout the genome without pronounced hotspots, although the ribosomal RNA gene cluster has a significantly lower frequency of crossovers.

A role for SUMO in nucleotide excision repair.

The two Siz/PIAS SUMO E3 ligases Siz1 and Siz2 are responsible for the vast majority of sumoylation in Saccharomyces cerevisiae. We found that siz1Δ siz2Δ mutants are sensitive to ultra-violet (UV) light. Epistasis analysis showed that the SIZ genes act in the nucleotide excision repair (NER) pathway, and suggested that they participate both in global genome repair (GGR) and in the Rpb9-dependent subpathway of transcription-coupled repair (TCR), but have minimal role in Rad26-dependent TCR. Quantitative analysis of NER at the single-nucleotide level showed that siz1Δ siz2Δ is deficient in repair of both the transcribed and non-transcribed strands of the DNA. These experiments confirmed that the SIZ genes participate in GGR. Their role in TCR remains unclear. It has been reported previously that mutants deficient for the SUMO conjugating enzyme Ubc9 contain reduced levels of Rad4, the yeast homolog of human XPC. However, our experiments do not support the conclusion that SUMO conjugation affects Rad4 levels. We found that several factors that participate in NER are sumoylated, including Rad4, Rad16, Rad7, Rad1, Rad10, Ssl2, Rad3, and Rpb4. Although Rad16 was heavily sumoylated, elimination of the major SUMO attachment sites in Rad16 had no detectable effect on UV resistance or removal of DNA lesions. SUMO attachment to most of these NER factors was significantly increased by DNA damage. Furthermore, SUMO-modified Rad4 accumulated in NER mutants that block the pathway downstream of Rad4, suggesting that SUMO becomes attached to Rad4 at a specific point during its functional cycle. Collectively, these results suggest that SIZ-dependent sumoylation may modulate the activity of multiple proteins to promote efficient NER.

Saccharomyces cerevisiae Rad16 mediates ultraviolet-dependent histone H3 acetylation required for efficient global genome nucleotide-excision repair.

In yeast, global genome nucleotide-excision repair (GG-NER) requires a protein complex containing Rad7 and Rad16. Rad16 is a member of the switch/sucrose nonfermentable superfamily, and it is presumed that chromatin remodelling is its primary function during repair. We show that RAD16 is required for ultraviolet-dependent hyperacetylation of histone H3 (Lys 9 and Lys 14) at the MFA2 promoter and throughout the genome. The yeast repressor complex Ssn6-Tup1 represses many genes including MFA2. TUP1 deletion results in constitutive hyperacetylation of histone H3, nucleosome disruption and derepression of gene transcription in Tup1-regulated genes. GG-NER in the MFA2 promoter proceeds more rapidly in tup1Delta alpha-cells compared with wild type, even when transcription is inhibited. We show that elevated histone H3 acetylation levels in the MFA2 promoter in tup1Delta alpha-cells result in Rad7- and Rad16-independent GG-NER, and that Rad16 mediates the ultraviolet-induced acetylation of histone H3, necessary for efficient GG-NER.

[Deletion of two genes from the genome of fission yeast Schizosaccharomyces pombe. Genetic manipulation and phenotype study]. / Deletia a doua gene din genomul drojdiei fisipare Schizosaccharomyces pombe. Manipulare genetica si studiu fenotipic.

The aim of this study was to delete two genes from the genome of the fission yeast S. pombe in order to search for their functions in the cell. These genes are SPAC869.02c (MRI) and SPBC21C3.19 (MR2) and previous studies reported their significant induction after gamma irradiation. We carried out the deletions of the two genes and we replaced them with the selection marker ura4. Among the phenotype characteristics we tested the viability, the sexual behaviour and the radiosensitivity to ultraviolet and gamma irradiation. Our results indicate that MR1-deleted strain is sensitive to both UV and gamma irradiation, while the survival of the irradiated MR2-deleted strain doesn't appear to be influenced by the deletion. This suggests an involvement of MR1 gene in the adaptive response triggered by these types of genotoxic aggression. The comparison of MR1-d and MR2-d with the double deleted strains containing the deletion of MR1 or MR2 combined with the deletion of sty1 or rad3 genes led to a surprising result: the double mutants MR1-d sty1-d and MR1-d rad3-d were more resistant to both UV and gamma irradiation than the simple deleted strains sty1-d and rad3-d, respectively. This suggests a possible contribution of MR1 gene to the lethal process taking place in irradiated cells.