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The role of Drosophila mismatch repair in suppressing recombination between diverged sequences.

Do AT, LaRocque JR - Sci Rep (2015)

Bottom Line: DNA double-strand breaks (DSBs) must be accurately repaired to maintain genomic integrity.DSBs can be repaired by homologous recombination (HR), which uses an identical sequence as a template to restore the genetic information lost at the break.These findings suggest that MMR has an early role in suppressing recombination between diverged sequences that is conserved in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Science, Georgetown University Medical Center, Washington DC 20057.

ABSTRACT
DNA double-strand breaks (DSBs) must be accurately repaired to maintain genomic integrity. DSBs can be repaired by homologous recombination (HR), which uses an identical sequence as a template to restore the genetic information lost at the break. Suppression of recombination between diverged sequences is essential to the repair of DSBs without aberrant and potentially mutagenic recombination between non-identical sequences, such as Alu repeats in the human genome. The mismatch repair (MMR) machinery has been found to suppress recombination between diverged sequences in murine cells. To test if this phenomenon is conserved in whole organisms, two DSB repair systems were utilized in Drosophila melanogaster. The DR-white and DR-white.mu assays provide a method of measuring DSB repair outcomes between identical and diverged sequences respectively. msh6(-/-) flies, deficient in MMR, were not capable of suppressing recombination between sequences with 1.4% divergence, and the average gene conversion tract length did not differ between msh6(-/+) and msh6(-/-)flies. These findings suggest that MMR has an early role in suppressing recombination between diverged sequences that is conserved in Drosophila.

No MeSH data available.


Related in: MedlinePlus

DR-white and DR-white.mu DSB Repair Assays.(a) The DR-white assay contains two nonfunctional direct repeats of the white gene. The first repeat, Sce.white, is nonfunctional due to the insertion of an I-SceI recognition sequence into the wild-type SacI recognition sequence of white cDNA resulting in a defective white gene. The second repeat, iwhite, is nonfunctional due to 5′ and 3′ truncations. DR-white is targeted using the attB sequence and integration is confirmed using yellow (y + ) transgene expression. DR-white flies are crossed with flies containing the heat-shock inducible I-SceI transgene, followed by heat shock, which results in DSB formation at the I-SceI recognition sequence. One of four repair products will result. White-eyed progeny (y+w–) suggest (i) no DSB or repair by (ii) NHEJ with processing, resulting in loss of the I-SceI recognition sequence. These two outcomes can be distinquished through molecular analysis. (iii) Repair by HR results in restoration of the wild-type SacI site and a red-eyed fly (y+w+). (iv) Yellow-bodied white-eyed (y–w–) progeny indicates repair by SSA. (b) The DR-white.mu assay includes the incorporation of 28 silent polymorphisms on the iwhite sequence, resulting in a sequence divergence of 1.4% between the two direct repeats. The silent polymorphisms allow recombination between diverged sequences to be studied as well as determining the length and direction of gene conversion tracts. Conversion of each of the polymorphisms varies from one repair product to the next (indicated by “?”), and can be determined by molecular analyses.
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f2: DR-white and DR-white.mu DSB Repair Assays.(a) The DR-white assay contains two nonfunctional direct repeats of the white gene. The first repeat, Sce.white, is nonfunctional due to the insertion of an I-SceI recognition sequence into the wild-type SacI recognition sequence of white cDNA resulting in a defective white gene. The second repeat, iwhite, is nonfunctional due to 5′ and 3′ truncations. DR-white is targeted using the attB sequence and integration is confirmed using yellow (y + ) transgene expression. DR-white flies are crossed with flies containing the heat-shock inducible I-SceI transgene, followed by heat shock, which results in DSB formation at the I-SceI recognition sequence. One of four repair products will result. White-eyed progeny (y+w–) suggest (i) no DSB or repair by (ii) NHEJ with processing, resulting in loss of the I-SceI recognition sequence. These two outcomes can be distinquished through molecular analysis. (iii) Repair by HR results in restoration of the wild-type SacI site and a red-eyed fly (y+w+). (iv) Yellow-bodied white-eyed (y–w–) progeny indicates repair by SSA. (b) The DR-white.mu assay includes the incorporation of 28 silent polymorphisms on the iwhite sequence, resulting in a sequence divergence of 1.4% between the two direct repeats. The silent polymorphisms allow recombination between diverged sequences to be studied as well as determining the length and direction of gene conversion tracts. Conversion of each of the polymorphisms varies from one repair product to the next (indicated by “?”), and can be determined by molecular analyses.

Mentions: It is unknown if the role of MMR in suppressing recombination between diverged sequences is conserved in multicellular systems. This question was addressed for the first time in the context of a multicellular organism using the genetically tractable organism Drosophila melanogaster. The DR-white and DR-white.mu assays are capable of detecting the repair pathway of an induced simple DSB (Fig. 2), including HR, NHEJ and SSA9. In the DR-white.mu assay, the donor sequence contains 28 silent polymorphisms, resulting in an increase of sequence divergence to 1.4%. To determine whether MMR plays a role in the suppression of recombination between diverged sequences in Drosophila, the DR-white and DR-white.mu assays were used with msh6 mutant flies20. We found that while HR repair between identical sequences, NHEJ, and SSA were unaffected by MMR deficiency, msh6–/– flies were not capable of suppressing recombination between diverged sequences compared to msh6–/+ heterozygote controls. Futhermore, gene conversion tract (GCT) lengths between msh6–/– and msh6–/+ flies were found to be similar. These findings suggest that MMR suppresses recombination between diverged sequences in Drosophila and the similar GCT lengths suggest that MMR components act on the repair intermediate prior to repair synthesis.


The role of Drosophila mismatch repair in suppressing recombination between diverged sequences.

Do AT, LaRocque JR - Sci Rep (2015)

DR-white and DR-white.mu DSB Repair Assays.(a) The DR-white assay contains two nonfunctional direct repeats of the white gene. The first repeat, Sce.white, is nonfunctional due to the insertion of an I-SceI recognition sequence into the wild-type SacI recognition sequence of white cDNA resulting in a defective white gene. The second repeat, iwhite, is nonfunctional due to 5′ and 3′ truncations. DR-white is targeted using the attB sequence and integration is confirmed using yellow (y + ) transgene expression. DR-white flies are crossed with flies containing the heat-shock inducible I-SceI transgene, followed by heat shock, which results in DSB formation at the I-SceI recognition sequence. One of four repair products will result. White-eyed progeny (y+w–) suggest (i) no DSB or repair by (ii) NHEJ with processing, resulting in loss of the I-SceI recognition sequence. These two outcomes can be distinquished through molecular analysis. (iii) Repair by HR results in restoration of the wild-type SacI site and a red-eyed fly (y+w+). (iv) Yellow-bodied white-eyed (y–w–) progeny indicates repair by SSA. (b) The DR-white.mu assay includes the incorporation of 28 silent polymorphisms on the iwhite sequence, resulting in a sequence divergence of 1.4% between the two direct repeats. The silent polymorphisms allow recombination between diverged sequences to be studied as well as determining the length and direction of gene conversion tracts. Conversion of each of the polymorphisms varies from one repair product to the next (indicated by “?”), and can be determined by molecular analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663472&req=5

f2: DR-white and DR-white.mu DSB Repair Assays.(a) The DR-white assay contains two nonfunctional direct repeats of the white gene. The first repeat, Sce.white, is nonfunctional due to the insertion of an I-SceI recognition sequence into the wild-type SacI recognition sequence of white cDNA resulting in a defective white gene. The second repeat, iwhite, is nonfunctional due to 5′ and 3′ truncations. DR-white is targeted using the attB sequence and integration is confirmed using yellow (y + ) transgene expression. DR-white flies are crossed with flies containing the heat-shock inducible I-SceI transgene, followed by heat shock, which results in DSB formation at the I-SceI recognition sequence. One of four repair products will result. White-eyed progeny (y+w–) suggest (i) no DSB or repair by (ii) NHEJ with processing, resulting in loss of the I-SceI recognition sequence. These two outcomes can be distinquished through molecular analysis. (iii) Repair by HR results in restoration of the wild-type SacI site and a red-eyed fly (y+w+). (iv) Yellow-bodied white-eyed (y–w–) progeny indicates repair by SSA. (b) The DR-white.mu assay includes the incorporation of 28 silent polymorphisms on the iwhite sequence, resulting in a sequence divergence of 1.4% between the two direct repeats. The silent polymorphisms allow recombination between diverged sequences to be studied as well as determining the length and direction of gene conversion tracts. Conversion of each of the polymorphisms varies from one repair product to the next (indicated by “?”), and can be determined by molecular analyses.
Mentions: It is unknown if the role of MMR in suppressing recombination between diverged sequences is conserved in multicellular systems. This question was addressed for the first time in the context of a multicellular organism using the genetically tractable organism Drosophila melanogaster. The DR-white and DR-white.mu assays are capable of detecting the repair pathway of an induced simple DSB (Fig. 2), including HR, NHEJ and SSA9. In the DR-white.mu assay, the donor sequence contains 28 silent polymorphisms, resulting in an increase of sequence divergence to 1.4%. To determine whether MMR plays a role in the suppression of recombination between diverged sequences in Drosophila, the DR-white and DR-white.mu assays were used with msh6 mutant flies20. We found that while HR repair between identical sequences, NHEJ, and SSA were unaffected by MMR deficiency, msh6–/– flies were not capable of suppressing recombination between diverged sequences compared to msh6–/+ heterozygote controls. Futhermore, gene conversion tract (GCT) lengths between msh6–/– and msh6–/+ flies were found to be similar. These findings suggest that MMR suppresses recombination between diverged sequences in Drosophila and the similar GCT lengths suggest that MMR components act on the repair intermediate prior to repair synthesis.

Bottom Line: DNA double-strand breaks (DSBs) must be accurately repaired to maintain genomic integrity.DSBs can be repaired by homologous recombination (HR), which uses an identical sequence as a template to restore the genetic information lost at the break.These findings suggest that MMR has an early role in suppressing recombination between diverged sequences that is conserved in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Science, Georgetown University Medical Center, Washington DC 20057.

ABSTRACT
DNA double-strand breaks (DSBs) must be accurately repaired to maintain genomic integrity. DSBs can be repaired by homologous recombination (HR), which uses an identical sequence as a template to restore the genetic information lost at the break. Suppression of recombination between diverged sequences is essential to the repair of DSBs without aberrant and potentially mutagenic recombination between non-identical sequences, such as Alu repeats in the human genome. The mismatch repair (MMR) machinery has been found to suppress recombination between diverged sequences in murine cells. To test if this phenomenon is conserved in whole organisms, two DSB repair systems were utilized in Drosophila melanogaster. The DR-white and DR-white.mu assays provide a method of measuring DSB repair outcomes between identical and diverged sequences respectively. msh6(-/-) flies, deficient in MMR, were not capable of suppressing recombination between sequences with 1.4% divergence, and the average gene conversion tract length did not differ between msh6(-/+) and msh6(-/-)flies. These findings suggest that MMR has an early role in suppressing recombination between diverged sequences that is conserved in Drosophila.

No MeSH data available.


Related in: MedlinePlus