Precise repair of mPing excision sites is facilitated by target site duplication derived microhomology

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.

Keywords: Excision site repair; Target site duplication; mPing.

Fig. 1

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)

Fig. 2

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

Fig. 3

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

Fig. 4

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

Fig. 5

Transposition assays with non-matching TSDs. Normalized ADE2 revertant frequencies for 14T32-T7 (a) and mPing (b) elements with altered 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 wild-type TSD (left column). Error bars indicate the standard error

Fig. 6

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