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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 in yeast with altered DNA repair potentials. ADE2 revertant frequencies for the mPing and 14T32-T7 elements in yeast strains with different DNA repair mechanisms available for excision site repair (a). JIM17 repairs by NHEJ, CB101 is capable of both HR and NHEJ, and DG21B9 can only repair by HR. Frequencies were normalized to the activity of each transposable element in JIM17. Error bars represent standard error. Sequences identified at the mPing (5′ TAA/3′ TAA TSDs) excision sites by restriction site analysis and sequencing (b). Underlined sequences indicate the HpaI and HaeIII sites used for analysis. Red bases are unique to the ADE2* template. *indicates the excision site was repaired by HR using the ADE2* template
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Fig2: Transposition assays in yeast with altered DNA repair potentials. ADE2 revertant frequencies for the mPing and 14T32-T7 elements in yeast strains with different DNA repair mechanisms available for excision site repair (a). JIM17 repairs by NHEJ, CB101 is capable of both HR and NHEJ, and DG21B9 can only repair by HR. Frequencies were normalized to the activity of each transposable element in JIM17. Error bars represent standard error. Sequences identified at the mPing (5′ TAA/3′ TAA TSDs) excision sites by restriction site analysis and sequencing (b). Underlined sequences indicate the HpaI and HaeIII sites used for analysis. Red bases are unique to the ADE2* template. *indicates the excision site was repaired by HR using the ADE2* template

Mentions: Performing yeast transposition assays in a yeast strain that provides a partial ADE2 template is an effective strategy to evaluate whether HR can be used for excision site repair. This approach has been used to study the Ac element (hAT superfamily, also creates footprints upon excision) where it was reported that when a template is available, about half of the excision sites are repaired by HR [12]. In this study, we employed similar methodology to determine if mPing and the hyperactive OsMar 14T32-T7 excision sites are repaired by HR; we used the CB101 yeast strain that contains a partial ADE2 template called ADE2*. This experiment showed that while no significant difference in the rate of ADE2 revertant colonies is observed with or without the ADE2* template, CB101 seems to show slightly lower average ADE2 revertants (Fig. 2). This may be due to competition between the two repair pathways or some unknown genetic change present in CB101. This slight difference did not affect our experiments because we were able to normalize within strains. To determine if HR was occurring in this strain, we analyzed 96 mPing excision sites and fifteen 14T32-T7 excision sites by PCR and digestion with HaeIII (present in ADE2* but not in the original ADE2). Under these conditions, none of the excision sites in either element contained the HaeIII site, and thus, were not repaired by HR at detectable levels (Fig. 2b). This indicates that even when a homologous template is present, the predominant repair pathway for these excision sites is NHEJ.Fig. 2


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 in yeast with altered DNA repair potentials. ADE2 revertant frequencies for the mPing and 14T32-T7 elements in yeast strains with different DNA repair mechanisms available for excision site repair (a). JIM17 repairs by NHEJ, CB101 is capable of both HR and NHEJ, and DG21B9 can only repair by HR. Frequencies were normalized to the activity of each transposable element in JIM17. Error bars represent standard error. Sequences identified at the mPing (5′ TAA/3′ TAA TSDs) excision sites by restriction site analysis and sequencing (b). Underlined sequences indicate the HpaI and HaeIII sites used for analysis. Red bases are unique to the ADE2* template. *indicates the excision 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

Fig2: Transposition assays in yeast with altered DNA repair potentials. ADE2 revertant frequencies for the mPing and 14T32-T7 elements in yeast strains with different DNA repair mechanisms available for excision site repair (a). JIM17 repairs by NHEJ, CB101 is capable of both HR and NHEJ, and DG21B9 can only repair by HR. Frequencies were normalized to the activity of each transposable element in JIM17. Error bars represent standard error. Sequences identified at the mPing (5′ TAA/3′ TAA TSDs) excision sites by restriction site analysis and sequencing (b). Underlined sequences indicate the HpaI and HaeIII sites used for analysis. Red bases are unique to the ADE2* template. *indicates the excision site was repaired by HR using the ADE2* template
Mentions: Performing yeast transposition assays in a yeast strain that provides a partial ADE2 template is an effective strategy to evaluate whether HR can be used for excision site repair. This approach has been used to study the Ac element (hAT superfamily, also creates footprints upon excision) where it was reported that when a template is available, about half of the excision sites are repaired by HR [12]. In this study, we employed similar methodology to determine if mPing and the hyperactive OsMar 14T32-T7 excision sites are repaired by HR; we used the CB101 yeast strain that contains a partial ADE2 template called ADE2*. This experiment showed that while no significant difference in the rate of ADE2 revertant colonies is observed with or without the ADE2* template, CB101 seems to show slightly lower average ADE2 revertants (Fig. 2). This may be due to competition between the two repair pathways or some unknown genetic change present in CB101. This slight difference did not affect our experiments because we were able to normalize within strains. To determine if HR was occurring in this strain, we analyzed 96 mPing excision sites and fifteen 14T32-T7 excision sites by PCR and digestion with HaeIII (present in ADE2* but not in the original ADE2). Under these conditions, none of the excision sites in either element contained the HaeIII site, and thus, were not repaired by HR at detectable levels (Fig. 2b). This indicates that even when a homologous template is present, the predominant repair pathway for these excision sites is NHEJ.Fig. 2

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