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Multiple-pathway analysis of double-strand break repair mutations in Drosophila.

Johnson-Schlitz DM, Flores C, Engels WR - PLoS Genet. (2007)

Bottom Line: We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes.Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others.We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
The analysis of double-strand break (DSB) repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage "decision circuit" in which the DSB is first placed into one of two pools from which a specific pathway is then selected.

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Decision Circuit for Breaks in Rr3(A) The decision process is represented as distinct from any specific biochemical pathway. It is envisioned as proceeding from left to right. The first step is the placement of each DSB into one of two intermediate pools, labeled pool 1 and pool 2. Breaks in pool 1 can be repaired by either NHEJ or SSA, whereas those in pool 2 are handled by either HR-h or SSA. In addition, HR-s (not drawn) restores the I-SceI cut site, enabling another round of DSB formation and repair.(B–G) Hypothesized effects on the decision circuit by mutant genotypes are shown. When one of the transitions in the circuit is inhibited by reduction of a gene product, use of the alternative route is increased. The sizes of circles surrounding the three measured outcomes, NHEJ, SSA, and HR-h, are meant to reflect the relative frequencies. No attempt was made to scale these circles precisely to the data, since each diagram represents several experiments and sets of estimates.(H) Depicts decision circuit for cross 1, which differs from cross 2 by the absence of any opportunity for HR-h. The HR-s repairs are shown explicitly here and assumed to come from pool 2.(I) Hypothesized effect of rad51 mutation in cross 1 is shown. Decreased HR-s repair results in more use of SSA. Since only two outcome types are measured in cross 1, the relative frequency of NHEJ is reduced through increased competition with SSA.
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pgen-0030050-g006: Decision Circuit for Breaks in Rr3(A) The decision process is represented as distinct from any specific biochemical pathway. It is envisioned as proceeding from left to right. The first step is the placement of each DSB into one of two intermediate pools, labeled pool 1 and pool 2. Breaks in pool 1 can be repaired by either NHEJ or SSA, whereas those in pool 2 are handled by either HR-h or SSA. In addition, HR-s (not drawn) restores the I-SceI cut site, enabling another round of DSB formation and repair.(B–G) Hypothesized effects on the decision circuit by mutant genotypes are shown. When one of the transitions in the circuit is inhibited by reduction of a gene product, use of the alternative route is increased. The sizes of circles surrounding the three measured outcomes, NHEJ, SSA, and HR-h, are meant to reflect the relative frequencies. No attempt was made to scale these circles precisely to the data, since each diagram represents several experiments and sets of estimates.(H) Depicts decision circuit for cross 1, which differs from cross 2 by the absence of any opportunity for HR-h. The HR-s repairs are shown explicitly here and assumed to come from pool 2.(I) Hypothesized effect of rad51 mutation in cross 1 is shown. Decreased HR-s repair results in more use of SSA. Since only two outcome types are measured in cross 1, the relative frequency of NHEJ is reduced through increased competition with SSA.

Mentions: Finally, one can detect compensation through the negative correlation between measurements in individual males. As reported previously (see Figure 6A in [28]), random differences in DSB repair pathway usage between individuals can also display compensation. Figure 2E and 2F show this effect for crosses 1 and 2 with the test males from experiments 2 and 4.


Multiple-pathway analysis of double-strand break repair mutations in Drosophila.

Johnson-Schlitz DM, Flores C, Engels WR - PLoS Genet. (2007)

Decision Circuit for Breaks in Rr3(A) The decision process is represented as distinct from any specific biochemical pathway. It is envisioned as proceeding from left to right. The first step is the placement of each DSB into one of two intermediate pools, labeled pool 1 and pool 2. Breaks in pool 1 can be repaired by either NHEJ or SSA, whereas those in pool 2 are handled by either HR-h or SSA. In addition, HR-s (not drawn) restores the I-SceI cut site, enabling another round of DSB formation and repair.(B–G) Hypothesized effects on the decision circuit by mutant genotypes are shown. When one of the transitions in the circuit is inhibited by reduction of a gene product, use of the alternative route is increased. The sizes of circles surrounding the three measured outcomes, NHEJ, SSA, and HR-h, are meant to reflect the relative frequencies. No attempt was made to scale these circles precisely to the data, since each diagram represents several experiments and sets of estimates.(H) Depicts decision circuit for cross 1, which differs from cross 2 by the absence of any opportunity for HR-h. The HR-s repairs are shown explicitly here and assumed to come from pool 2.(I) Hypothesized effect of rad51 mutation in cross 1 is shown. Decreased HR-s repair results in more use of SSA. Since only two outcome types are measured in cross 1, the relative frequency of NHEJ is reduced through increased competition with SSA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851981&req=5

pgen-0030050-g006: Decision Circuit for Breaks in Rr3(A) The decision process is represented as distinct from any specific biochemical pathway. It is envisioned as proceeding from left to right. The first step is the placement of each DSB into one of two intermediate pools, labeled pool 1 and pool 2. Breaks in pool 1 can be repaired by either NHEJ or SSA, whereas those in pool 2 are handled by either HR-h or SSA. In addition, HR-s (not drawn) restores the I-SceI cut site, enabling another round of DSB formation and repair.(B–G) Hypothesized effects on the decision circuit by mutant genotypes are shown. When one of the transitions in the circuit is inhibited by reduction of a gene product, use of the alternative route is increased. The sizes of circles surrounding the three measured outcomes, NHEJ, SSA, and HR-h, are meant to reflect the relative frequencies. No attempt was made to scale these circles precisely to the data, since each diagram represents several experiments and sets of estimates.(H) Depicts decision circuit for cross 1, which differs from cross 2 by the absence of any opportunity for HR-h. The HR-s repairs are shown explicitly here and assumed to come from pool 2.(I) Hypothesized effect of rad51 mutation in cross 1 is shown. Decreased HR-s repair results in more use of SSA. Since only two outcome types are measured in cross 1, the relative frequency of NHEJ is reduced through increased competition with SSA.
Mentions: Finally, one can detect compensation through the negative correlation between measurements in individual males. As reported previously (see Figure 6A in [28]), random differences in DSB repair pathway usage between individuals can also display compensation. Figure 2E and 2F show this effect for crosses 1 and 2 with the test males from experiments 2 and 4.

Bottom Line: We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes.Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others.We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Wisconsin, Madison, Wisconsin, United States of America.

ABSTRACT
The analysis of double-strand break (DSB) repair is complicated by the existence of several pathways utilizing a large number of genes. Moreover, many of these genes have been shown to have multiple roles in DSB repair. To address this complexity we used a repair reporter construct designed to measure multiple repair outcomes simultaneously. This approach provides estimates of the relative usage of several DSB repair pathways in the premeiotic male germline of Drosophila. We applied this system to mutations at each of 11 repair loci plus various double mutants and altered dosage genotypes. Most of the mutants were found to suppress one of the pathways with a compensating increase in one or more of the others. Perhaps surprisingly, none of the single mutants suppressed more than one pathway, but they varied widely in how the suppression was compensated. We found several cases in which two or more loci were similar in which pathway was suppressed while differing in how this suppression was compensated. Taken as a whole, the data suggest that the choice of which repair pathway is used for a given DSB occurs by a two-stage "decision circuit" in which the DSB is first placed into one of two pools from which a specific pathway is then selected.

Show MeSH
Related in: MedlinePlus