Whole-Genome Sequencing of Suppressor DNA Mixtures Identifies Pathways That Compensate for Chromosome Segregation Defects in Schizosaccharomyces pombe

G3: Genes, Genomes, Genetics

article

Xingya Xu¹  Li Wang¹  Mitsuhiro Yanagida¹ 

[1] G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa 904-0495, Japan

关键词: Schizosaccharomyces pombe;    temperature sensitive mutant;    Suppressor screen;    next-generation sequencing;    chromosome segregation;   

DOI  :  10.1534/g3.118.200048

学科分类：社会科学、人文和艺术（综合）

来源: Genetics Society of America

【 摘 要 】

Suppressor screening is a powerful method to identify genes that, when mutated, rescue the temperature sensitivity of the original mutation. Previously, however, identification of suppressor mutations has been technically difficult. Due to the small genome size of Schizosaccharomyces pombe , we developed a spontaneous suppressor screening technique, followed by a cost-effective sequencing method. Genomic DNAs of 10 revertants that survived at the restrictive temperature of the original temperature sensitive (ts) mutant were mixed together as one sample before constructing a library for sequencing. Responsible suppressor mutations were identified bioinformatically based on allele frequency. Then, we isolated a large number of spontaneous extragenic suppressors for three ts mutants that exhibited defects in chromosome segregation at their restrictive temperature. Screening provided new insight into mechanisms of chromosome segregation: loss of Ufd2 E4 multi-ubiquitination activity suppresses defects of an AAA ATPase, Cdc48. Loss of Wpl1, a releaser of cohesin, compensates for the Eso1 mutation, which may destabilize sister chromatid cohesion. The segregation defect of a ts histone H2B mutant is rescued if it fails to be deubiquitinated by the SAGA complex, because H2B is stabilized by monoubiquitination.

【 授权许可】

CC BY|CC BY-NC