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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

Gene conversion tract analysis.HR events using the DR-white.mu assay were isolated, and the GCT direction and length were determined. Briefly, Sce.white was amplified from y+w+(HR+) isolates and then sequenced for the conversion of polymorphisms to the iwhite.mu donor sequence. Minimal conversion lengths are displayed for (a) 45 msh6–/+ HR events and (b) 35 msh6–/– HR events, including the last polymorphism converted. Discontinuous conversion is indicated by a thin line. The distance converted (bp) to the left and to the right of the SacI site (0) is given. Data are representative of four independent experiments.
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f4: Gene conversion tract analysis.HR events using the DR-white.mu assay were isolated, and the GCT direction and length were determined. Briefly, Sce.white was amplified from y+w+(HR+) isolates and then sequenced for the conversion of polymorphisms to the iwhite.mu donor sequence. Minimal conversion lengths are displayed for (a) 45 msh6–/+ HR events and (b) 35 msh6–/– HR events, including the last polymorphism converted. Discontinuous conversion is indicated by a thin line. The distance converted (bp) to the left and to the right of the SacI site (0) is given. Data are representative of four independent experiments.

Mentions: To explore at which step in the HR pathway that MMR machinery functions to suppress recombination between diverged sequences, GCTs were analyzed in y+w+ (HR+) DR-white.mu flies. The average GCT of msh6–/+ and msh6–/– flies were 264.0 ± 57.7 bp and 296.7 ± 65.3 bp respectively (Fig. 4; P > 0.05, by unpaired t test). Of all the msh6–/+ HR repair events (n = 45), 14 (31.1%) of the GCTs were limited to the SacI site, 19 (42.2%) were unidirectional, and 12 (26.7%) were bidirectional (Fig. 4). The unidirectional GCTs did not display a preference to either the left or right. Of all the msh6–/–HR repair events analyzed (n = 35), 12 (34.3%) were limited to the SacI site, 16 (45.7%) were unidirectional, and 7 (20%) were bidirectional (Fig. 4). msh6–/–HR repair events displayed directional gene conversion preference, with 13 (81.3%) of the unidirectional GCTs converting to the right side; however, this was not significant compared to the distribution of the direction of unidirectional tracts in msh6–/+ HR events (P > 0.05, Fisher’s exact test). Discontinuous GCTs were also observed, where conversion along the entire length of the gene conversion tract did not occur. Two discontinuous tracts were observed in msh6–/+ flies and 4 were observed in msh6–/– flies (P > 0.05, Fisher’s exact test; Fig. 4).


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

Do AT, LaRocque JR - Sci Rep (2015)

Gene conversion tract analysis.HR events using the DR-white.mu assay were isolated, and the GCT direction and length were determined. Briefly, Sce.white was amplified from y+w+(HR+) isolates and then sequenced for the conversion of polymorphisms to the iwhite.mu donor sequence. Minimal conversion lengths are displayed for (a) 45 msh6–/+ HR events and (b) 35 msh6–/– HR events, including the last polymorphism converted. Discontinuous conversion is indicated by a thin line. The distance converted (bp) to the left and to the right of the SacI site (0) is given. Data are representative of four independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Gene conversion tract analysis.HR events using the DR-white.mu assay were isolated, and the GCT direction and length were determined. Briefly, Sce.white was amplified from y+w+(HR+) isolates and then sequenced for the conversion of polymorphisms to the iwhite.mu donor sequence. Minimal conversion lengths are displayed for (a) 45 msh6–/+ HR events and (b) 35 msh6–/– HR events, including the last polymorphism converted. Discontinuous conversion is indicated by a thin line. The distance converted (bp) to the left and to the right of the SacI site (0) is given. Data are representative of four independent experiments.
Mentions: To explore at which step in the HR pathway that MMR machinery functions to suppress recombination between diverged sequences, GCTs were analyzed in y+w+ (HR+) DR-white.mu flies. The average GCT of msh6–/+ and msh6–/– flies were 264.0 ± 57.7 bp and 296.7 ± 65.3 bp respectively (Fig. 4; P > 0.05, by unpaired t test). Of all the msh6–/+ HR repair events (n = 45), 14 (31.1%) of the GCTs were limited to the SacI site, 19 (42.2%) were unidirectional, and 12 (26.7%) were bidirectional (Fig. 4). The unidirectional GCTs did not display a preference to either the left or right. Of all the msh6–/–HR repair events analyzed (n = 35), 12 (34.3%) were limited to the SacI site, 16 (45.7%) were unidirectional, and 7 (20%) were bidirectional (Fig. 4). msh6–/–HR repair events displayed directional gene conversion preference, with 13 (81.3%) of the unidirectional GCTs converting to the right side; however, this was not significant compared to the distribution of the direction of unidirectional tracts in msh6–/+ HR events (P > 0.05, Fisher’s exact test). Discontinuous GCTs were also observed, where conversion along the entire length of the gene conversion tract did not occur. Two discontinuous tracts were observed in msh6–/+ flies and 4 were observed in msh6–/– flies (P > 0.05, Fisher’s exact test; Fig. 4).

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