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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

Model of Tourist-like and Stowaway-like MITE transposition. mPing (a) and 14T32-T7 (b) elements are represented by black boxes, with the TSDs (3 bp and 2 bp respectively) created upon insertion shown as letters. Excision of the mPing element produces TSD derived 5′ overhangs that result in precise repair, while 14T32 excision leaves element derived overhangs that results in footprint production
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Fig4: Model of Tourist-like and Stowaway-like MITE transposition. mPing (a) and 14T32-T7 (b) elements are represented by black boxes, with the TSDs (3 bp and 2 bp respectively) created upon insertion shown as letters. Excision of the mPing element produces TSD derived 5′ overhangs that result in precise repair, while 14T32 excision leaves element derived overhangs that results in footprint production

Mentions: Based on these initial experiments, we hypothesized that a difference in the double stranded breaks created by the mPing and 14T32 elements results in their excision site differences. Analysis of repaired excision sites shows that Mariner-like transposase proteins produce staggered DNA cleavage within the element, leaving behind some of the TIR sequences (Fig. 1b, Fig. 4) [15]. In contrast, our model for PIF/Pong/Harbinger transposition is that they are mobilized by staggered cleavage of the TSDs, producing three bases of microhomology that facilitates NHEJ (Fig. 4). Based on this, we predicted that changing the TSDs in such a way as to disrupt the microhomology would affect the quality and efficiency of mPing’s excision site repair.Fig. 4


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)

Model of Tourist-like and Stowaway-like MITE transposition. mPing (a) and 14T32-T7 (b) elements are represented by black boxes, with the TSDs (3 bp and 2 bp respectively) created upon insertion shown as letters. Excision of the mPing element produces TSD derived 5′ overhangs that result in precise repair, while 14T32 excision leaves element derived overhangs that results in footprint production
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Model of Tourist-like and Stowaway-like MITE transposition. mPing (a) and 14T32-T7 (b) elements are represented by black boxes, with the TSDs (3 bp and 2 bp respectively) created upon insertion shown as letters. Excision of the mPing element produces TSD derived 5′ overhangs that result in precise repair, while 14T32 excision leaves element derived overhangs that results in footprint production
Mentions: Based on these initial experiments, we hypothesized that a difference in the double stranded breaks created by the mPing and 14T32 elements results in their excision site differences. Analysis of repaired excision sites shows that Mariner-like transposase proteins produce staggered DNA cleavage within the element, leaving behind some of the TIR sequences (Fig. 1b, Fig. 4) [15]. In contrast, our model for PIF/Pong/Harbinger transposition is that they are mobilized by staggered cleavage of the TSDs, producing three bases of microhomology that facilitates NHEJ (Fig. 4). Based on this, we predicted that changing the TSDs in such a way as to disrupt the microhomology would affect the quality and efficiency of mPing’s excision site repair.Fig. 4

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