期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：48
页码：13809-13814
DOI：10.1073/pnas.1617110113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceRepeated activation of the same DNA replication origin, termed "rereplication," is one developmental strategy to increase gene copies for high levels of protein production. However, it also generates DNA double-strand breaks and can lead to genome instability. We present evidence for competition between different pathways of double-strand break repair during rereplication in Drosophila follicle cells. Loss of DNA polymerase {theta} (Pol {theta}), which operates in an error-prone repair mechanism named "microhomology-mediated end joining," impedes the progress of rereplication forks at a specific genomic locus. Pol {theta}-mediated repair is also used in the absence of classical end joining, but only at certain regions. Our findings suggest that genomic context has a major impact on genomic stability and mutagenesis in rereplicating DNA. Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. In Drosophila follicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase {theta}/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context.
