Abstract
Oxidative DNA damage is a threat to genome stability. Using a genetic system in yeast that allows detection of mitotic recombination, we found that the frequency of crossovers is greatly elevated when cells are treated with hydrogen peroxide (H2O2). Using a combination of microarray analysis and genomic sequencing, we mapped the breakpoints of mitotic recombination events and other chromosome rearrangements at a resolution of about 1 kb. Gene conversions and crossovers were the two most common types of events, but we also observed deletions, duplications, and chromosome aneuploidy. In addition, H2O2-treated cells had elevated rates of point mutations (particularly A to T/T to A and C to G/G to C transversions) and small insertions/deletions (in/dels). In cells that underwent multiple rounds of H2O2 treatments, we identified a genetic alteration that resulted in improved H2O2 tolerance by amplification of the CTT1 gene that encodes cytosolic catalase T. Lastly, we showed that cells grown in the absence of oxygen have reduced levels of recombination. This study provided multiple novel insights into how oxidative stress affects genomic instability and phenotypic evolution in aerobic cells.
Highlights
Reactive oxidative species (ROS), including ·O2, H2O2 and ·OH, are produced within eukaryotic cells, largely as a consequence of electron transport in the mitochondria during aerobic growth [1]
We use microarray analysis and genomic sequencing to characterize the effects of H2O2 on a number of different types of changes throughout the yeast genome
We examined the effects of acute exposure of G1-synchronized yeast cells to H2O2 on the rates of mitotic crossovers, gene conversion events, large deletions/duplications, and ploidy changes; both selected events on chromosome IV and unselected events throughout the genome were mapped
Summary
Reactive oxidative species (ROS), including ·O2, H2O2 and ·OH, are produced within eukaryotic cells, largely as a consequence of electron transport in the mitochondria during aerobic growth [1]. The levels of ROS are low under normal growth conditions, exposure of cells to certain environmental conditions including ultraviolet light [3], heat shock [4], certain pathogens [5] and several types of chemicals [6] lead to oxidative stress and damage to multiple species of biological macromolecules. Oxidative damage results in more than 80 different types of base damage, as well as single-strand nicks and doublestrand breaks (DSBs) [7]. Both single-strand nicks and DSBs stimulate mitotic recombination [8]. In a study of H2O2-induced genomic alterations on chromosome V, Hayashi and Umezu [10] showed a dosedependent elevation of chromosome loss, crossovers, and gene conversion events by H2O2
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