Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells

Abstract

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 θ/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. oogenesis | break-induced replication | DNA repair | NHEJ | fork collision

TheDrosophila melanogasterPIF1 helicase promotes survival during replication stress and processive DNA synthesis during double-strand gap repair

“…Drosophila stocks were maintained on standard cornmeal agar medium at 25°C. The pif1 167 null mutant was created via imprecise excision of a P-element, (P{EPgy2}Pif1 EY10295 , Bloomington Stock 17658) located in the 5' untranslated region of CG3238 (ALEXANDER et al 2016). The deletion removes nucleotides 64-1759 (relative to the transcription start site) and inserts the sequence CTGTTATTTCATCATG at the deletion breakpoint.…”

“…However, unlike the yeast Pif1 proteins, Drosophila PIF1 does not appear to have a mitochondrial targeting sequence, suggesting that it may not function in mitochondrial maintenance. To investigate the roles of PIF1 in Drosophila, we created the pif1 167 null mutant allele via imprecise excision of a P element (EY10295) located in the 5' untranslated region of CG3238, which resulted in the removal of the majority of the helicase domain (ALEXANDER et al 2016). Flies homozygous for this deletion were viable and apparently healthy, although females displayed greatly reduced fertility.…”

Replication fork instability and the consequences of fork collisions from rereplication

“…NHEJ is required for repair of these DSBs, as the absence of the NHEJ factor Lig4 inhibits fork progression across the DAFCs (Alexander et al 2015). Additionally, the MMEJ component Pol θ is required for complete fork progression at only some of the DAFCs, revealing that the use of this pathway for rereplication DSB repair is site-specific (Alexander et al 2016). In contrast, the absence of the HR components Brca2 and Rad51 enhances fork movement (Alexander et al 2016).…”

“…Additionally, the MMEJ component Pol θ is required for complete fork progression at only some of the DAFCs, revealing that the use of this pathway for rereplication DSB repair is site-specific (Alexander et al 2016). In contrast, the absence of the HR components Brca2 and Rad51 enhances fork movement (Alexander et al 2016). These results suggest that HR is active but cannot productively contribute to DSB repair before the end of amplification, which occurs over a 7.5-h time in development.…”

“…There is a possible contribution of BIR at the DAFCs because replication fork progression is reduced in pol32 and pif1 mutants (Alexander et al 2016), but because these two proteins participate in other aspects of fork progression, it cannot be excluded that these functions account for the mutant phenotype (Alexander et al 2016). Together, the analysis of DAFCs suggests that DSB repair pathway choice following rereplication is governed by genomic position, reaction kinetics, and repair pathway competition.…”