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, 447 (7142), 338-41

RNA-templated DNA Repair

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RNA-templated DNA Repair

Francesca Storici et al. Nature.

Erratum in

  • Nature. 2007 Aug 30;448(7157):1076

Abstract

RNA can act as a template for DNA synthesis in the reverse transcription of retroviruses and retrotransposons and in the elongation of telomeres. Despite its abundance in the nucleus, there has been no evidence for a direct role of RNA as a template in the repair of any chromosomal DNA lesions, including DNA double-strand breaks (DSBs), which are repaired in most organisms by homologous recombination or by non-homologous end joining. An indirect role for RNA in DNA repair, following reverse transcription and formation of a complementary DNA, has been observed in the non-homologous joining of DSB ends. In the yeast Saccharomyces cerevisiae, in which homologous recombination is efficient, RNA was shown to mediate recombination, but only indirectly through a cDNA intermediate generated by the reverse transcriptase function of Ty retrotransposons in Ty particles in the cytoplasm. Although pairing between duplex DNA and single-strand (ss)RNA can occur in vitro and in vivo, direct homologous exchange of genetic information between RNA and DNA molecules has not been observed. We show here that RNA can serve as a template for DNA synthesis during repair of a chromosomal DSB in yeast. The repair was accomplished with RNA oligonucleotides complementary to the broken ends. This and the observation that even yeast replicative DNA polymerases such as alpha and delta can copy short RNA template tracts in vitro demonstrate that RNA can transfer genetic information in vivo through direct homologous interaction with chromosomal DNA.

Figures

Figure 1
Figure 1. Repair of a DSB by RNA-containing oligonucleotides
The diagram shows the broken LEU2 chromosomal DNA along with the oligonucleotides containing DNA (D; blue) or RNA (R; red) sequences that were used to repair the DSB, and corresponding frequencies of LEU2 repair. The HO cutting site (124 bp) is split in two halves shown as thicker short black lines (not to scale). Oligonucleotides are shown as lines with arrows at the 3′ end; nucleotide inserts are shown as thick lines; dotted lines indicate non-homologous tails. Potential for homologous pairing is presented as short, thin parallel vertical lines. Numbers of nucleotides homologous to the LEU2 sequence are indicated in square brackets. Insertions are indicated by ‘ins::’; a comma separates DNA from RNA bases within the insertions; a hyphen separates the different parts of the oligonucleotides. Oligonucleotide sequences are given in Supplementary Table 1. Presented are numbers of Leu+ transformants per 107 viable cells resulting from targeting by 1 or 5* (adjacent column) nmoles of oligonucleotides a to s. Targeting frequencies with a pair of oligonucleotides (connected by braces) are shown in parentheses. Confidence intervals, as well as results of sequence verification are provided in Supplementary Table 2a.
Figure 2
Figure 2. Strand bias of oligonucleotide targeting to sites distant from the DSB
a, System to detect oligonucleotide strand bias in yeast diploid strains. One copy of chromosome VII with the TRP5 locus inactivated by a 31 bp frameshift insertion plus an I-SceI-induced DSB either 10 kb upstream or downstream. The Trp+ phenotype can be restored by the oligonucleotides R.w or R.c (corresponding to the ‘Watson’ or ‘Crick’ strand in the TRP5 coding sequence), containing six central bases of RNA, or only DNA (D.w and D.c), while the intact copy of chromosome VII, in which TRP5 is replaced by LEU2, provides a template for repair of the DSB. A restriction site created by the oligonucleotides is indicated by an asterisk b, Number of Trp+ transformants per 107 viable cells resulting from targeting 1 nmole of R.w, R.c, D.w or D.c following DSB induction. Presented are mean + s.d. from six independent experiments.
Figure 3
Figure 3. Synthesis by Pol α and Pol δ across RNA templates
a, Substrates consist of DNA (D; blue) and/or RNA (R; red) and include primer P (same in all substrates). b, Lanes 1, 7, 13, template I, no enzyme; lanes 2–6 and 8–12, Pol α products; lanes 14–18, Pol δ products. Reactions use templates: I in lanes 2, 8, 14; II in lanes 3, 9, 15; III in lanes 4, 10 and 16; IV in lanes 5, 11, and 17; and V in lanes 6, 12, and 18. Red asterisks, ribonucleotide positions in template II and first ribonucleotide position in templates III and IV. Red arrows, RNA tracts in templates III, IV and V. The black asterisk in lane 15 marks where Pol δ was impeded when the RNA–DNA duplex is upstream of the polymerase active site. c, Synthesis on a gapped substrate. Lane 1, template with no enzyme V; lanes 2 and 4, products with template V and indicated enzyme; lanes 3 and 5, products with gapped substrate (V-Dw). Extension of the primer by one nucleotide is shown as +1.

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