Contribution of telomerase RNA retrotranscription to DNA double-strand break repair during mammalian genome evolution

Abstract

Background: In vertebrates, tandem arrays of TTAGGG hexamers are present at both telomeres and intrachromosomal sites (interstitial telomeric sequences (ITSs)). We previously showed that, in primates, ITSs were inserted during the repair of DNA double-strand breaks and proposed that they could arise from either the capture of telomeric fragments or the action of telomerase.

Results: An extensive comparative analysis of two primate (Homo sapiens and Pan troglodytes) and two rodent (Mus musculus and Rattus norvegicus) genomes allowed us to describe organization and insertion mechanisms of all the informative ITSs present in the four species. Two novel observations support the hypothesis of telomerase involvement in ITS insertion: in a highly significant fraction of informative loci, the ITSs were introduced at break sites where a few nucleotides homologous to the telomeric hexamer were exposed; in the rodent genomes, complex ITS loci are present in which a retrotranscribed fragment of the telomerase RNA, far away from the canonical template, was inserted together with the telomeric repeats. Moreover, mutational analysis of the TTAGGG arrays in the different species suggests that they were inserted as exact telomeric hexamers, further supporting the participation of telomerase in ITS formation.

Conclusion: These results strongly suggest that telomerase was utilized, in some instances, for the repair of DNA double-strand breaks occurring in the genomes of rodents and primates during evolution. The presence, in the rodent genomes, of sequences retrotranscribed from the telomerase RNA strengthens the hypothesis of the origin of telomerase from an ancient retrotransposon.

Figure 1

Length of ITSs. Comparison of ITS length in (a) the two primate and (b) the two rodent species.

Figure 2

Microhomology between break sites and inserted telomeric repeats. Telomeric repeats are in red; in the empty ancestral loci the nucleotides in register with the inserted telomeric repeats are boxed. (a) Mouse specific ITS at the MMU12qA1 locus; an AGGG tetranucleotide from the orthologous rat empty locus RNO6q15 is in register with the inserted telomeric repeats. (b) Rat specific ITS at RNO14q21; a GGG trinucleotide from the orthologous mouse locus MMU3qB3 is in register with the inserted telomeric repeats. (c) The human specific ITS at HSA11q24 was inserted together with seven random nucleotides; a TA dinucleotide from the orthologous chimpanzee PTR9 locus is in register with the inserted telomeric repeats.(d) The insertion of the chimpanzee specific ITS at PTR22 occurred together with a 7 bp deletion; an AGG trinucleotide from the orthologous human locus HSA21q22 is in register with the inserted telomeric repeats.

Figure 3

Organization of TERC-ITS loci. RNA sequences are in italic. The RNA sequences involved in the events and the DNA sequences corresponding to them (that is, complementary to retrotranscribed sequences) are in light colors (orange, grey and light blue) while the DNA sequences derived from retrotranscription of the RNA are in dark colors (red, black and dark blue). (a) Sequence of the mouse telomerase RNA component. The nucleotides of the canonical telomerase template, located near the 5' end, are shown in orange (nucleotides 3-10). Nucleotides adjacent to the template that are retrotranscribed together with the first inserted hexamer are grey underlined. The nucleotides of the 3' domain of TERC involved in the TERC-ITS loci are indicated in light blue. The 17 nt core sequence, present in all TERC-ITSs, has a blue background. In the 3' domain of the RNA, the mouse TERC sequence homologous to the human TERC sequence interacting with Ku is underlined. (b) Example of a mouse specific TERC-ITS locus (MMU9qA5). The top row shows the 5' domain of TERC containing the canonical template (orange) and the adjacent sequence (grey underlined). The second row shows the sequence of the mouse locus: telomeric repeats are in red; the nucleotides complementary to those adjacent to the hexameric template are black underlined; the light blue nucleotides indicate the region derived from the 3' domain of TERC. The third row reports, in light blue, the sequence of the 3' domain of TERC from nucleotides 314 to nucleotides 373. The bottom row shows the sequence of the orthologous empty rat locus RNO8q23. The CG dinucleotide (yellow) is present both in the ancestral rat sequence, at the 3' end of the break, and in the region of the TERC RNA immediately preceding the retrotranscribed 3' domain. (c) Overall organization of TERC-ITS loci. At the top is the structure of TERC: orange oval, canonical template; grey square, adjacent nucleotides; light blue strip, 3' domain. At the bottom is the organization of the double-stranded DNA at TERC-ITS loci: light blue strip, sequence corresponding to the 3' domain of TERC; blue strip, complementary sequence; black square, sequence complementary to the nucleotides adjacent to the canonical template; grey square, sequence corresponding to the nucleotides adjacent to the canonical template; red ovals, TTAGGG repeats; orange ovals, complementary repeats.

Figure 4

Model for TERC-ITS insertion. (a) TERC interaction at DNA double strand break. (b) Retrotranscription of TERC 3' domain. (c) Second strand synthesis. (d) Retrotranscription of TERC 5' domain. (e) Digestion of DNA/RNA junction and addition of canonical telomeric repeats. (f) Gap filling. Curved thin lines represent the telomerase RNA (TERC) in which the orange oval corresponds to the canonical telomeric template, the grey line to the nucleotides immediately adjacent to the 3' side of the template, the yellow line to nucleotides homologous to the last nucleotides of the break site; the light blue line represents the retrotranscribed 3' region of TERC. Straight thick lines represent DNA strands. The DNA involved in the double-strand break is in black, the yellow boxes correspond to nucleotides homologous to the region of TERC preceding the sequence retrotranscribed from the 3' end, the dark blue line represents the DNA strand retrotranscribed from the 3' end of TERC and the light blue line is the complementary strand. Red and orange ovals represent TTAGGG and CCCTAA repeats, respectively. Black and grey lines correspond to the sequence homologous to the nucleotides immediately adjacent to the telomeric template.