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.

DNA lesions caused by UV radiation are highly recombinogenic. In wild-type cells, the recombinogenic effect of UV partially reflects the processing of UV-induced pyrimidine dimers into DNA gaps or breaks by the enzymes of the nucleotide excision repair (NER) pathway. In this study, we show that unprocessed pyrimidine dimers also potently induce recombination between homologs. In NER-deficient rad14 diploid strains, we demonstrate that unexcised pyrimidine dimers stimulate crossovers, noncrossovers, and break-induced replication events. The same dose of UV is about six-fold more recombinogenic in a repair-deficient strain than in a repair-proficient strain. We also examined the roles of several genes involved in the processing of UV-induced damage in NER-deficient cells. We found that the resolvase Mus81p is required for most of the UV-induced inter-homolog recombination events. This requirement likely reflects the Mus81p-associated cleavage of dimer-blocked replication forks. The error-free post-replication repair pathway mediated by Mms2p suppresses dimer-induced recombination between homologs, possibly by channeling replication-blocking lesions into recombination between sister chromatids.

Expansion of a trinucleotide (CGG) repeat element within the 5' untranslated region (5'UTR) of the human FMR1 gene is responsible for a number of heritable disorders operating through distinct pathogenic mechanisms: gene silencing for fragile X syndrome (>200 CGG) and RNA toxic gain-of-function for FXTAS (∼ 55-200 CGG). Existing models have focused almost exclusively on post-transcriptional mechanisms, but co-transcriptional processes could also contribute to the molecular dysfunction of FMR1. We have observed that transcription through the GC-rich FMR1 5'UTR region favors R-loop formation, with the nascent (G-rich) RNA forming a stable RNA:DNA hybrid with the template DNA strand, thereby displacing the non-template DNA strand. Using DNA:RNA (hybrid) immunoprecipitation (DRIP) of genomic DNA from cultured human dermal fibroblasts with both normal (∼ 30 CGG repeats) and premutation (55

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.