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Precise repair of mPing excision sites is facilitated by target site duplication derived microhomology.

Gilbert DM, Bridges MC, Strother AE, Burckhalter CE, Burnette JM, Hancock CN - Mob DNA (2015)

Bottom Line: These small insertions or deletions known as "footprints" can potentially disrupt coding or regulatory sequences.In contrast, Tourist-like MITEs and the associated PIF/Pong/Harbinger elements generally excise precisely, returning the genome to its original state.Our data suggests that Tourist-like elements excise with staggered cleavage of the TSDs, which provides microhomology that facilitates precise repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Geology, University of South Carolina Aiken, 471 University Parkway, Aiken, SC 29801 USA.

ABSTRACT

Background: A key difference between the Tourist and Stowaway families of miniature inverted repeat transposable elements (MITEs) is the manner in which their excision alters the genome. Upon excision, Stowaway-like MITEs and the associated Mariner elements usually leave behind a small duplication and short sequences from the end of the element. These small insertions or deletions known as "footprints" can potentially disrupt coding or regulatory sequences. In contrast, Tourist-like MITEs and the associated PIF/Pong/Harbinger elements generally excise precisely, returning the genome to its original state. The purpose of this study was to determine the mechanisms underlying these excision differences, including the role of the host DNA repair mechanisms.

Results: The transposition of the Tourist-like element, mPing, and the Stowaway-like element, 14T32, were evaluated using yeast transposition assays. Assays performed in yeast strains lacking non-homologous end joining (NHEJ) enzymes indicated that the excision sites of both elements were primarily repaired by NHEJ. Altering the target site duplication (TSD) sequences that flank these elements reduced the transposition frequency. Using yeast strains with the ability to repair the excision site by homologous repair showed that some TSD changes disrupt excision of the element. Changing the ends of mPing to produce non-matching TSDs drastically reduced repair of the excision site and resulted in increased generation of footprints.

Conclusions: Together these results indicate that the difference in Tourist and Stowaway excision sites results from transposition mechanism characteristics. The TSDs of both elements play a role in element excision, but only the mPing TSDs actively participate in excision site repair. Our data suggests that Tourist-like elements excise with staggered cleavage of the TSDs, which provides microhomology that facilitates precise repair. This slight modification in the transposition mechanism results in more efficient repair of the double stranded break, and thus, may be less harmful to host genomes by disrupting fewer genes.

No MeSH data available.


Related in: MedlinePlus

Transposition assays with non-matching TSDs. Normalized ADE2 revertant frequencies for 14T32-T7 (a) and mPing (b) elements with altered TSDs. Blue bars indicate the rate in CB101 (capable of both NHEJ and HR), while red bars indicate the rate in DG21B9 (only capable of HR). Values were normalized to the wild-type TSD (left column). Error bars indicate the standard error
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Fig5: Transposition assays with non-matching TSDs. Normalized ADE2 revertant frequencies for 14T32-T7 (a) and mPing (b) elements with altered TSDs. Blue bars indicate the rate in CB101 (capable of both NHEJ and HR), while red bars indicate the rate in DG21B9 (only capable of HR). Values were normalized to the wild-type TSD (left column). Error bars indicate the standard error

Mentions: The fact that multiple bases are equally acceptable in the middle position of mPing’s TSDs allowed experiments to determine if homology between the two TSDs facilitates repair of mPing excision sites. Yeast transposition assays comparing mPing constructs with matching TSDs (TTA/TTA and TAA/TAA) and non-matching TSDs (TTA/TAA and TAA/TTA) were performed (Fig. 5, Additional file 3). As shown in CB101 (Fig. 5b) or JIM17 (Additional file 3) yeast strains, the mPing elements with non-matching TSDs showed significantly lower transposition than those with matching TSDs. Performing this assay in the DG21B9 strain, which is only capable of repair by HR did not show this effect, with all TSD combinations showing a similar number of ADE2 revertant colonies (Fig. 5b). Together these results indicate that the reduction in ADE2 revertant colonies for non-matching TSDs is caused by reduced or inaccurate NHEJ repair efficiency. For comparison, similar experiments using the 14T32-T7 element showed that non-matching TSDs produced a similar effect in both NHEJ competent (CB101) and NHEJ deficient (DG21B9) strains (Fig. 5a). This indicates that changing the TSDs of 14T32-T7 only affected its excision and not the repair of the excision site.Fig. 5


Precise repair of mPing excision sites is facilitated by target site duplication derived microhomology.

Gilbert DM, Bridges MC, Strother AE, Burckhalter CE, Burnette JM, Hancock CN - Mob DNA (2015)

Transposition assays with non-matching TSDs. Normalized ADE2 revertant frequencies for 14T32-T7 (a) and mPing (b) elements with altered TSDs. Blue bars indicate the rate in CB101 (capable of both NHEJ and HR), while red bars indicate the rate in DG21B9 (only capable of HR). Values were normalized to the wild-type TSD (left column). Error bars indicate the standard error
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4561436&req=5

Fig5: Transposition assays with non-matching TSDs. Normalized ADE2 revertant frequencies for 14T32-T7 (a) and mPing (b) elements with altered TSDs. Blue bars indicate the rate in CB101 (capable of both NHEJ and HR), while red bars indicate the rate in DG21B9 (only capable of HR). Values were normalized to the wild-type TSD (left column). Error bars indicate the standard error
Mentions: The fact that multiple bases are equally acceptable in the middle position of mPing’s TSDs allowed experiments to determine if homology between the two TSDs facilitates repair of mPing excision sites. Yeast transposition assays comparing mPing constructs with matching TSDs (TTA/TTA and TAA/TAA) and non-matching TSDs (TTA/TAA and TAA/TTA) were performed (Fig. 5, Additional file 3). As shown in CB101 (Fig. 5b) or JIM17 (Additional file 3) yeast strains, the mPing elements with non-matching TSDs showed significantly lower transposition than those with matching TSDs. Performing this assay in the DG21B9 strain, which is only capable of repair by HR did not show this effect, with all TSD combinations showing a similar number of ADE2 revertant colonies (Fig. 5b). Together these results indicate that the reduction in ADE2 revertant colonies for non-matching TSDs is caused by reduced or inaccurate NHEJ repair efficiency. For comparison, similar experiments using the 14T32-T7 element showed that non-matching TSDs produced a similar effect in both NHEJ competent (CB101) and NHEJ deficient (DG21B9) strains (Fig. 5a). This indicates that changing the TSDs of 14T32-T7 only affected its excision and not the repair of the excision site.Fig. 5

Bottom Line: These small insertions or deletions known as "footprints" can potentially disrupt coding or regulatory sequences.In contrast, Tourist-like MITEs and the associated PIF/Pong/Harbinger elements generally excise precisely, returning the genome to its original state.Our data suggests that Tourist-like elements excise with staggered cleavage of the TSDs, which provides microhomology that facilitates precise repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Geology, University of South Carolina Aiken, 471 University Parkway, Aiken, SC 29801 USA.

ABSTRACT

Background: A key difference between the Tourist and Stowaway families of miniature inverted repeat transposable elements (MITEs) is the manner in which their excision alters the genome. Upon excision, Stowaway-like MITEs and the associated Mariner elements usually leave behind a small duplication and short sequences from the end of the element. These small insertions or deletions known as "footprints" can potentially disrupt coding or regulatory sequences. In contrast, Tourist-like MITEs and the associated PIF/Pong/Harbinger elements generally excise precisely, returning the genome to its original state. The purpose of this study was to determine the mechanisms underlying these excision differences, including the role of the host DNA repair mechanisms.

Results: The transposition of the Tourist-like element, mPing, and the Stowaway-like element, 14T32, were evaluated using yeast transposition assays. Assays performed in yeast strains lacking non-homologous end joining (NHEJ) enzymes indicated that the excision sites of both elements were primarily repaired by NHEJ. Altering the target site duplication (TSD) sequences that flank these elements reduced the transposition frequency. Using yeast strains with the ability to repair the excision site by homologous repair showed that some TSD changes disrupt excision of the element. Changing the ends of mPing to produce non-matching TSDs drastically reduced repair of the excision site and resulted in increased generation of footprints.

Conclusions: Together these results indicate that the difference in Tourist and Stowaway excision sites results from transposition mechanism characteristics. The TSDs of both elements play a role in element excision, but only the mPing TSDs actively participate in excision site repair. Our data suggests that Tourist-like elements excise with staggered cleavage of the TSDs, which provides microhomology that facilitates precise repair. This slight modification in the transposition mechanism results in more efficient repair of the double stranded break, and thus, may be less harmful to host genomes by disrupting fewer genes.

No MeSH data available.


Related in: MedlinePlus