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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

mPing excision sites for non-matching TIRs. Sequences identified at the mPing excision sites by restriction site analysis and sequencing in JIM17 (NHEJ only) and CB101 (HR and NHEJ). Lowercase letters indicate inserted sequences and a base change is in red. * indicates that the site was repaired by HR using the ADE2* template
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Fig6: mPing excision sites for non-matching TIRs. Sequences identified at the mPing excision sites by restriction site analysis and sequencing in JIM17 (NHEJ only) and CB101 (HR and NHEJ). Lowercase letters indicate inserted sequences and a base change is in red. * indicates that the site was repaired by HR using the ADE2* template

Mentions: Analysis of the excision sites from non-matching TSDs by restriction digest and sequencing was performed to determine how these sites were repaired. Figure 6 shows that in JIM17 and CB101 the excision site produced by an element with TAA/TTA TSDs was repaired fairly precisely with most excision sites only showing one of the TSDs. However, two of the excision sites retained both TSDs, consistent with staggered cleavage at the TSDs that was repaired by NHEJ without microhomology. In contrast, we found that repair of the excision site produced by an element with TTA/TAA TSDs was repaired less precisely in JIM17, or exclusively by HR repair using the ADE2* template in CB101 (Fig. 6). This result suggests that the staggered ends created by the TTA/TAA combination were not as easily joined by NHEJ pathway as the TAA/TTA combination. Since a 5′ overhang would create a different set of mismatched bases than a 3′overhang at the excision site (Table 1), we compared our results to the expected base pairing for each non-matching TSD. Based on this result, we propose that mPing’s TSDs cleavage produces a 5′ overhang (Fig. 4, Table 1). A three base 5′ overhang would result in the TAA/TTA TSDs forming a T:T (pyrimidine:pyrimidine) pairing at the middle base of the overhang, a more compatible pairing than the A:A (purine:purine) base paring created by the TTA/TAA TSDs.Fig. 6


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)

mPing excision sites for non-matching TIRs. Sequences identified at the mPing excision sites by restriction site analysis and sequencing in JIM17 (NHEJ only) and CB101 (HR and NHEJ). Lowercase letters indicate inserted sequences and a base change is in red. * indicates that the site was repaired by HR using the ADE2* template
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: mPing excision sites for non-matching TIRs. Sequences identified at the mPing excision sites by restriction site analysis and sequencing in JIM17 (NHEJ only) and CB101 (HR and NHEJ). Lowercase letters indicate inserted sequences and a base change is in red. * indicates that the site was repaired by HR using the ADE2* template
Mentions: Analysis of the excision sites from non-matching TSDs by restriction digest and sequencing was performed to determine how these sites were repaired. Figure 6 shows that in JIM17 and CB101 the excision site produced by an element with TAA/TTA TSDs was repaired fairly precisely with most excision sites only showing one of the TSDs. However, two of the excision sites retained both TSDs, consistent with staggered cleavage at the TSDs that was repaired by NHEJ without microhomology. In contrast, we found that repair of the excision site produced by an element with TTA/TAA TSDs was repaired less precisely in JIM17, or exclusively by HR repair using the ADE2* template in CB101 (Fig. 6). This result suggests that the staggered ends created by the TTA/TAA combination were not as easily joined by NHEJ pathway as the TAA/TTA combination. Since a 5′ overhang would create a different set of mismatched bases than a 3′overhang at the excision site (Table 1), we compared our results to the expected base pairing for each non-matching TSD. Based on this result, we propose that mPing’s TSDs cleavage produces a 5′ overhang (Fig. 4, Table 1). A three base 5′ overhang would result in the TAA/TTA TSDs forming a T:T (pyrimidine:pyrimidine) pairing at the middle base of the overhang, a more compatible pairing than the A:A (purine:purine) base paring created by the TTA/TAA TSDs.Fig. 6

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