Homology-directed repair involves multiple strand invasion cycles in fission yeast.

Homology-directed repair of DNA double-strand breaks (DSBs) represents a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). How homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair often involves multiple site-specific and Rad51-dependent colocalization events between the DSB and donor sequence. Upon loss of the RecQ helicase Rqh1 (BLM in humans) we observe rapid repair possibly involving a single strand invasion event, suggesting that multiple strand invasion cycles antagonized by Rqh1 could reflect ongoing SDSA. However, failure to colocalize with the donor sequence and execute repair is also more likely in rqh1Δ cells, possibly reflecting erroneous strand invasion. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.

Increased numbers of nucleoli in a genome-wide RNAi screen reveal proteins that link the cell cycle to RNA polymerase I transcription.

Nucleoli are dynamic nuclear condensates in eukaryotic cells that originate through ribosome biogenesis at loci that harbor the ribosomal DNA. These loci are known as nucleolar organizer regions (NORs), and there are 10 in a human diploid genome. While there are 10 NORs, however, the number of nucleoli observed in cells is variable. Furthermore, changes in number are associated with disease, with increased numbers and size common in aggressive cancers. In the near-diploid human breast epithelial cell line, MCF10A, the most frequently observed number of nucleoli is two to three per cell. Here, to identify novel regulators of ribosome biogenesis we used high-throughput quantitative imaging of MCF10A cells to identify proteins that, when depleted, increase the percentage of nuclei with ≥5 nucleoli. Unexpectedly, this unique screening approach led to identification of proteins associated with the cell cycle. Functional analysis on a subset of hits further revealed not only proteins required for progression through the S and G2/M phase, but also proteins required explicitly for the regulation of RNA polymerase I transcription and protein synthesis. Thus, results from this screen for increased nucleolar number highlight the significance of the nucleolus in human cell cycle regulation, linking RNA polymerase I transcription to cell cycle progression.

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

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.

Rev7 and 53BP1/Crb2 prevent RecQ helicase-dependent hyper-resection of DNA double-strand breaks.

Poly(ADP ribose) polymerase inhibitors (PARPi) target cancer cells deficient in homology-directed repair of DNA double-strand breaks (DSBs). In preclinical models, PARPi resistance is tied to altered nucleolytic processing (resection) at the 5' ends of a DSB. For example, loss of either 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance, although the mechanisms are poorly understood. Long-range resection can be catalyzed by two machineries: the exonuclease Exo1, or the combination of a RecQ helicase and Dna2. Here, we develop a single-cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living S. pombe cells. Using this assay, we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway, thereby preventing hyper-resection. These results suggest that 'rewiring' of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance.

Using LacO arrays to monitor DNA double-strand break dynamics in live Schizosaccharomyces pombe cells.

LacO arrays, when combined with LacI-GFP, have been a valuable tool for studying nuclear architecture and chromatin dynamics. Here, we outline an experimental approach to employ the LacO/LacI-GFP system in S. pombe to assess DNA double-strand break (DSB) dynamics and the contribution of chromatin state to DSB repair. Previously, integration of long, highly repetitive LacO arrays in S. pombe has been a challenge. To address this problem, we have developed a novel approach, based on the principles used for homologous recombination-based genome engineering in higher eukaryotes, to integrate long, repetitive LacO arrays with targeting efficiencies as high as 70 %. Combining this facile LacO/LacI-GFP system with a site-specific, inducible DSB provides a means to monitor DSB dynamics at engineered sites within the genome.