Construction and Analysis of a Genome-wide Insertion Library in Schizosaccharomyces Pombe Reveals Novel Aspects of DNA Repair
Author | : Yanhui Li |
Publisher | : |
Total Pages | : 184 |
Release | : 2015 |
ISBN-10 | : OCLC:906165251 |
ISBN-13 | : |
Rating | : 4/5 (51 Downloads) |
Book excerpt: In the post-genomic era, utilizing yeast deletion libraries for high-throughput phenotypic screening accelerates gene function studies. However, haploid deletion collections are still insufficient to reveal all gene functions because only non-essential genes are knocked out. Mutants in essential genes and a large number of functionally uncharacterized non-coding genes are not included. Moreover, haploid deletion collections only contain null mutants. A resource that allows rapid phenotypic screening of many kinds of mutants (e.g. null, partial or altered function) in all genes is needed. We constructed a defined, DNA barcode-tagged Hermes transposon insertion mutant library in S. pombe. The collection contained 4,095 haploid insertion mutant strains. Most strains (~90%) carry single transposon insertions. The insertions were distributed among 368 essential genes, 2,470 non-essential genes and 1,159 non-coding genes. The library contains a wide variety of mutations than the available gene deletion library. Insertions are distributed among open reading framesas well as 5' and 3' regulatory regions of genes. Phenotypic screening of selected mutants in the presence of the topoisomerase I inhibitor CPT revealed that some insertion mutants have the predicted phenotypes while some have unexpected phenotypes. This library therefore represents an important resource for the international S. pombe community. The construction of similar transposon insertion libraries in other organisms is labor-intensive. We developed a novel three-dimensional pooling strategy and a multiplexed high-throughput analysis pipeline to sequence the transposon insertion sites and DNA barcodes from thousands of samples at once. The approach greatly reduced the effort and was cost-effective. It can be applied to any insertion element, and will accelerate the construction of sequenced insertion mutant libraries in a wide variety of model systems.As a first step in the development of the Hermes transposon as a genetic tool for large-scale mutagenesis, we analyzed its insertion and excision behavior in the context of the S. pombe genome, which led to novel insights into DNA damage repair by Non-homologous End joining (NHEJ) processing in S. pombe. Excision of the Hermes transposon from the S. pombe genome leaves a DNA double-strand break capped by hairpin ends that must be processed and ligated by cellular enzymes. We found that repair was through NHEJ and required the Mre11-Rad50-Nbs1 (MRN) and, to a lesser extent, Ctp1. The MRN complex has important roles in DNA damage signaling, homologous recombination repair and telomere replication. The role of MRN in NHEJ is unclear. We found that NHEJ in S. pombe did not require Mre11 nuclease function, but did require Mre11 dimerization function, suggesting that the primary role of Mre11 may be to tether the broken DNA ends in NHEJ.