Differential Usage of Alternative Pathways of Double-Strand Break Repair in Drosophila

Abstract

Double-strand DNA breaks can be repaired by any of several alternative mechanisms that differ greatly in the nature of the final repaired products. We used a reporter construct, designated ''Repair reporter 3'' (Rr3), to measure the relative usage of these pathways in Drosophila germ cells. The method works by creating a double-strand break at a specific location such that expression of the red fluorescent protein, DsRed, in the next generation can be used to infer the frequency at which each pathway was used. A key feature of this approach is that most data come from phenotypic scoring, thus allowing large sample sizes and considerable precision in measurements. Specifically, we measured the proportion of breaks repaired by (1) conversion repair, (2) nonhomologous end joining (NHEJ), or (3) single-strand annealing (SSA). For conversion repair, the frequency of mitotic crossing over in the germ line indicates the relative prevalence of repair by double Holliday junction (DHJ) formation vs. the synthesis-dependent strand annealing (SDSA) pathway. We used this method to show that breaks occurring early in germ-line development were much more frequently repaired via single-strand annealing and much less likely to be repaired by end joining compared with identical breaks occurring later in development. Conversion repair was relatively rare when breaks were made either very early or very late in development, but was much more frequent in between. Significantly, the changes in relative usage occurred in a compensatory fashion, such that an increase in one pathway was accompanied by decreases in others. This negative correlation is interpreted to mean that the pathways for double-strand break repair compete with each other to handle a given breakage event.

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

“…Repair of P-element-induced DNA breaks occurs predominantly through two distinct DSB repair pathways: NHEJ or a variant of classical homologous recombinational repair, SDSA (18)(19)(20)(21) The choice between these two pathways is dictated by cell-cycle location, the availability of pathway substrates, and tissue type (22,23). Because the IRBP homozygous mutant males are sterile, we cannot at present use any of the established DNA repair reporter strains to determine in which DNA repair pathway IRBP18 and Xrp1 participates.…”

“…Repair of P-element-induced DNA breaks typically occurs through two distinct DSB repair pathways: nonhomologous end joining (NHEJ) or a variant of classical homologous recombinational repair, termed "Synthesis-Dependent Strand Annealing" (SDSA) (18)(19)(20)(21) The choice between these two pathways is dictated by cellcycle location, the availability of pathway substrates, and tissue type (22,23).…”

“…The flanking donor site DNA is released after P-element cleavage (16) and needs to be efficiently repaired by endogenous DNA repair mechanisms. Engineered somatic mobilization of as few as 15-20 small nonautonomous P elements by transposase leads to a temperature-dependent pupal lethality due to extensive DNA damage and apoptosis (17).Repair of P-element-induced DNA breaks typically occurs through two distinct DSB repair pathways: nonhomologous end joining (NHEJ) or a variant of classical homologous recombinational repair, termed "Synthesis-Dependent Strand Annealing" (SDSA) (18-21) The choice between these two pathways is dictated by cellcycle location, the availability of pathway substrates, and tissue type (22,23).In addition to containing sequences critical for transposasemediated DNA cleavage, the 31-bp TIRs of the P element have been postulated to be important for DNA repair (24), perhaps through protein-DNA interactions. To date, the identity and functional role of endogenous Drosophila proteins bound to the 31-bp TIRs in the regulation of P-element transposition is undetermined.…”

Inviting instability: Transposable elements, double-strand breaks, and the maintenance of genome integrity

“…With the availability of NHEJ -which theoretically necessitates less sequence loss on average than SSA-throughout the cell cycle, invocation of SSA would seldom seem desirable for the cell. However, experimental evidence increasingly points to a competition between multiple repair pathways whose outcome may be determined by a number of cell-state-dependent host factors [43,44].…”

“…The generation of DSBs by L1 products, which are used by L1, Alu, and SVA insertions, probably alerts the cell DSB signaling system and recruits DNA repair machinery to the insertion loci. This raises the possibility that heterogeneity in pathway preference, both at the level of cell cycle phase [57] and across tissue types and developmental stages [44], could greatly impact the relative efficiency of retrotransposon insertions.…”