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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 altered but matching TSDs. ADE2 revertant rates for 14T32-T7 (a) and mPing (b) elements with altered but matching 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 control TSDs (TA/TA for 14T32-T7 and TAA/TAA for mPing) for each yeast strain separately. Error bars represent standard error
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Fig3: Transposition assays with altered but matching TSDs. ADE2 revertant rates for 14T32-T7 (a) and mPing (b) elements with altered but matching 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 control TSDs (TA/TA for 14T32-T7 and TAA/TAA for mPing) for each yeast strain separately. Error bars represent standard error

Mentions: Previous studies have shown that Mariner-like elements require the TSD (TA on both ends) for transposition in vitro [24]. In this study, we confirmed the importance of the conserved TSD for the 14T32-T7 element by changing the TSDs and performing yeast transposition assays. In CB101, changing both bases of the TSDs from TA/TA (5′/3′) to AT/AT almost completely inhibited transposition, while changing just one base (TT/TT or AA/AA) allowed transposition, but at highly reduced rates (Fig. 3a). This experiment was also performed in DG21B9 (HR competent, NHEJ deficient) to confirm that this decrease in activity was due to inhibited excision and not inhibited excision site repair. Figure 3a shows that in DG21B9 alteration of the 14T32-T7 TSDs produced a comparable decrease in activity to the one observed in CB101 yeast. Thus, the drop in activity upon changing the TSDs is likely due to a decrease in excision, and not due to changes in the efficiency of NHEJ. Other researchers have shown that the Mariner-like transposase proteins bind to the TIRs and not the TSDs [25, 26]. Therefore, the TSDs do not likely play a role in binding, but instead play a role in the catalytic mechanism that cleaves the element from the genome.Fig. 3


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 altered but matching TSDs. ADE2 revertant rates for 14T32-T7 (a) and mPing (b) elements with altered but matching 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 control TSDs (TA/TA for 14T32-T7 and TAA/TAA for mPing) for each yeast strain separately. Error bars represent standard error
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Transposition assays with altered but matching TSDs. ADE2 revertant rates for 14T32-T7 (a) and mPing (b) elements with altered but matching 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 control TSDs (TA/TA for 14T32-T7 and TAA/TAA for mPing) for each yeast strain separately. Error bars represent standard error
Mentions: Previous studies have shown that Mariner-like elements require the TSD (TA on both ends) for transposition in vitro [24]. In this study, we confirmed the importance of the conserved TSD for the 14T32-T7 element by changing the TSDs and performing yeast transposition assays. In CB101, changing both bases of the TSDs from TA/TA (5′/3′) to AT/AT almost completely inhibited transposition, while changing just one base (TT/TT or AA/AA) allowed transposition, but at highly reduced rates (Fig. 3a). This experiment was also performed in DG21B9 (HR competent, NHEJ deficient) to confirm that this decrease in activity was due to inhibited excision and not inhibited excision site repair. Figure 3a shows that in DG21B9 alteration of the 14T32-T7 TSDs produced a comparable decrease in activity to the one observed in CB101 yeast. Thus, the drop in activity upon changing the TSDs is likely due to a decrease in excision, and not due to changes in the efficiency of NHEJ. Other researchers have shown that the Mariner-like transposase proteins bind to the TIRs and not the TSDs [25, 26]. Therefore, the TSDs do not likely play a role in binding, but instead play a role in the catalytic mechanism that cleaves the element from the genome.Fig. 3

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