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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 NHEJ deficient yeast. Normalized ADE2 revertant frequency for the mPing (blue) and 14T32 (red) elements in control (JIM17) and NHEJ mutant yeast strains (a). Error bars indicate the standard error for 6 replicates. Repaired excision sites from control and rad50 yeast strains (b). Lowercase letters indicate the bases derived from the TSD (mPing) or TIRs and TSDs (14T32)
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Fig1: Transposition assays in NHEJ deficient yeast. Normalized ADE2 revertant frequency for the mPing (blue) and 14T32 (red) elements in control (JIM17) and NHEJ mutant yeast strains (a). Error bars indicate the standard error for 6 replicates. Repaired excision sites from control and rad50 yeast strains (b). Lowercase letters indicate the bases derived from the TSD (mPing) or TIRs and TSDs (14T32)

Mentions: The yeast transposition assay used for these experiments measures the rate at which the ADE2 gene is repaired in-frame following excision of the TE (Additional file 1) [14, 15, 20, 21]. Traditionally, these assays have been performed in haploid yeast lacking an ADE2 homologous template for HR repair of the excision site. Under these conditions the excision site should be repaired only by NHEJ. Performing transposition assays with mPing and 14T32 in haploid yeast strains lacking the NHEJ pathway proteins KU70, MRE11, or RAD50 showed that these proteins are required for efficient repair of the excision sites of both elements (Fig. 1a). Almost no ADE2 revertant colonies were obtained in the ku70 strain, as KU70 is a highly conserved protein involved in the initial binding of the double stranded breaks [22]. For both elements, the rad50 strain showed a higher DNA repair rate than the mre11 strain. This is consistent with a previous study indicating that MRE11 function is more important for repair than RAD50 even though these two proteins function together in the MRX complex to process double stranded breaks before ligation [22, 23]. These results also indicated RAD50 plays a more important role in excision site repair for the 14T32 element than the mPing element [92 % vs. 56 % decrease in repair efficiency (Fig. 1a)]. However, some of this change could be due to a difference in the amount of repair products that result in reading frame disruption. Analysis of excision sites produced in the rad50 background showed that the mPing excision sites were still repaired precisely, while the 14T32 excision sites had more bases deleted (less precise repair) compared to the control (Fig. 1b). This difference in repair efficiency and quality observed for the two elements in the rad50 strain provides evidence that there are important differences in the nature of the double strand breaks produced by these two elements.Fig. 1


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 NHEJ deficient yeast. Normalized ADE2 revertant frequency for the mPing (blue) and 14T32 (red) elements in control (JIM17) and NHEJ mutant yeast strains (a). Error bars indicate the standard error for 6 replicates. Repaired excision sites from control and rad50 yeast strains (b). Lowercase letters indicate the bases derived from the TSD (mPing) or TIRs and TSDs (14T32)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Transposition assays in NHEJ deficient yeast. Normalized ADE2 revertant frequency for the mPing (blue) and 14T32 (red) elements in control (JIM17) and NHEJ mutant yeast strains (a). Error bars indicate the standard error for 6 replicates. Repaired excision sites from control and rad50 yeast strains (b). Lowercase letters indicate the bases derived from the TSD (mPing) or TIRs and TSDs (14T32)
Mentions: The yeast transposition assay used for these experiments measures the rate at which the ADE2 gene is repaired in-frame following excision of the TE (Additional file 1) [14, 15, 20, 21]. Traditionally, these assays have been performed in haploid yeast lacking an ADE2 homologous template for HR repair of the excision site. Under these conditions the excision site should be repaired only by NHEJ. Performing transposition assays with mPing and 14T32 in haploid yeast strains lacking the NHEJ pathway proteins KU70, MRE11, or RAD50 showed that these proteins are required for efficient repair of the excision sites of both elements (Fig. 1a). Almost no ADE2 revertant colonies were obtained in the ku70 strain, as KU70 is a highly conserved protein involved in the initial binding of the double stranded breaks [22]. For both elements, the rad50 strain showed a higher DNA repair rate than the mre11 strain. This is consistent with a previous study indicating that MRE11 function is more important for repair than RAD50 even though these two proteins function together in the MRX complex to process double stranded breaks before ligation [22, 23]. These results also indicated RAD50 plays a more important role in excision site repair for the 14T32 element than the mPing element [92 % vs. 56 % decrease in repair efficiency (Fig. 1a)]. However, some of this change could be due to a difference in the amount of repair products that result in reading frame disruption. Analysis of excision sites produced in the rad50 background showed that the mPing excision sites were still repaired precisely, while the 14T32 excision sites had more bases deleted (less precise repair) compared to the control (Fig. 1b). This difference in repair efficiency and quality observed for the two elements in the rad50 strain provides evidence that there are important differences in the nature of the double strand breaks produced by these two elements.Fig. 1

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