Transcript-RNA-templated DNA recombination and repair

[INTRODUCTION]

If you are interested in nucleic acids, such as DNA and RNA, you must have heard that nucleic acids can be damaged and repaired in most of the organisms, including humans, even though RNA repair mechanisms have not yet been explored much. For example, in human cells, DNA damages are commonly caused by environmental factors like UV radiation that causes thymine-thymine dimers, consisting of covalently-linked, adjacent thymine residues, result in frame-shift mutations [1], [2]. However, cells themselves have abilities to repair mutations without foreign aids. Among several DNA repair mechanisms, it is well known that homologous recombination is widely used for double strand breaks (DSBs). The two videos below would help you understand how homologous recombination occurs in repairing damaged DNA.

Video 1. Double stranded break repair by homologous recombination (2D version).

 

Video 2. Homology-dependent double strand break repair by transposon (3D version).

 

As shown, the DNA repair of homologous recombination utilizes DNA strands having complementary sequences. However, would you imagine that if there is insufficient amount of DNA for a substrate of repair or what if no complementary DNA strand exists in cells? It is known that RNA can act as a template for DNA synthesis in the reverse transcription of retroviruses and retrotransposons and in the elongation of telomeres [3]. 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 DSB, which are repaired in most organisms by homologous recombination or by non-homologous end joining. In 2007, Francesca Storici discovered that DSB can be repaired by RNA-containing oligonucleotides in Saccharomyces cerevisiae (Figure 1) [4].

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Figure 1. Repair of a DSB in LEU2 by RNA-containing oligonucleotides.
Comparison of  the broken leu2 gene repair frequencies using ssDNA oligonucleotides, RNA oligonucleotides, or RNA-DNA combined oligonucleotides. DNA (D; blue), and RNA (R; red).

 

This figure shows how effectively DNA repair occurs with served nucleic acid oligonucleotides into the yeast cells. As shown in the figure, the broken leu2 gene is repaired by either ssDNA oligonucleotides (blue-marked), RNA oligonucleotides (red-marked), or DNA-RNA combined oligonucleotides in leu- solid media (lack of leucine) after replica-plating [Video 3], [Video 4]. In the leu- solid media, cells are not able to survive unless they produce the amino acid leucine. Therefore, colonies shown in the media represent the leu2 gene is repaired, and then the numbers of colonies were counted to indicate the repair frequencies.

With in-frame 12-base insert containing 0, 4, 6 or 12 RNA bases (a, b, c and d, respectively), or a 6-base insert with 0 or 6 RNA bases (e and f) as a template, DSB repair was accomplished and a functional LEU2 gene was restored. Remarkably, repair by b (containing 4 ribonucleotides) was only a factor of 3 lower than repair by the DNA-only control (a). It was insisted from this paper that even though the frequencies of DSB repair is decreased with increasing RNA-tract length (b, c, d, Figure 1), RNA-containing molecules participate directly in the repair.

Based on the fact that RNA would be another substrate to be used for DSB repair, an interesting idea came up that complementary DNA (cDNA), resulted from reverse-transcription of mRNA, and/or transcript RNA itself may aid DSB repair via homologous recombination [5].

 

[EXPERIMENTAL SYSTEMS]

In order to investigate the capacity of transcript RNA to recombine with genomic DNA, two experimental systems were designed in S. cerevisiae (Figure 2) [5].

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Figure 2. Repair of a chromosomal DSB by transcript of RNA. (a) and (b) Schemes of trans and cis cell systems used to detect DSB repair by transcript RNA. AI, artificial intron; HO, homothallic switching endonuclease; pGAL1, galactose-inducible promoter; RT, reverse transctiptase. Yellow triangles, cleavage activity by HO; red question marks, hypothesis for transcript-RNA-templated DSB repair mechanism.

 

In trans system, a his3 gene, disrupted by a HO (homothallic switching endonuclease) site, is present on chromosome XV and another his3 gene, disrupted by an artificial intron (AI) is shown on chromosome III (Figure 2a). Once HO is added into the cell media, HO will recognize its site and cut it, which causes a DSB in the middle of the his3 gene. On chromosome III, the disrupted his3 is located under GAL1 promoter, which can be induced by galactose so that addition of galactose would accelerate the expression of the his3. The expressed RNA of his3 undergoes splicing to be a complete messenger RNA (mRNA). Then, the AI is spliced out, and his3 mRNA is formed. Afterward, the his3 mRNA  initiates its cDNA (HIS3 cDNA) synthesis by reverse transcriptase (RT). Lastly, the synthesized cDNA is used as a substrate to repair the DSB on chromosome XV via recombination. In contrast to the trans system, cis system has the HO site and the AI in the same position on chromosome III (Figure 2b). In both systems, the spliced antisense his3 RNA transcript can serve as a homologous template to repair the broken his3 DNA and restore its function. However, given the abundance of Ty retrotransposons, which can copy themselves to RNA and then synthesize DNA that may integrate back to the genome, in yeast cells, the spliced antisense his3 RNA could potentially be reverse- transcribed to cDNA by the Ty reverse transcriptase in the cytoplasm. Thus, the newly synthesized his3 cDNA could then recombine with the homologous broken his3 sequence and then repair of the his3 gene is initiated by non-homologous end joining at the HO site. As a result, HIS+ colonies are shown in his- solid media.

 

[RESULTS]

In order to observe colonies containing restored HIS3 gene , grown cells were transferred into his- solid media by replica-plating, and then incubated at 30 degree Celsius (Figure 3).

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Figure 3. Comparison of DNA repair frequency in his- media.

 

The wild-type (WT) cells grown in his- media (lack of histidine) prove the the DSB was repaired, and HIS3 gene was restored in the both systems by HIS3 cDNA originated from its mRNA transcript. To distinguish DSB repair mediated by the transcript RNA template from repair mediated by the cDNA template, SPT3 gene, a key enzyme required for Ty transcription, was knocked out (spt3), and  no HIS+ colony is shown. It is assumed that DNA-RNA duplex hybrids were not formed during reverse transcription. Ribonuclease H (RNAse H) plays a role in digestion of the RNA strand of RNA-DNA hybrid. The two types of null-RNAse Hs(RNH1 and RNH201)  were also tested to confirm if RNAse Hs affect the frequency of DSB repair. While rnh1 cells has a little of impact on the DSB repair frequency, rnh201 cells show significant difference when knocked out alone and knocked out with rnh1 together. In the absence of RNAse Hs, DSB repair using RNA-DNA hybrids occurs more active than that occurs in the wild-type in both systems. More information of the experiments are indicated in Table 1. The DSB repair frequency of each yeast strain is displayed with the number of colonies found in his- solid media. The most abundant HIS3+ colonies are found in the double knock-out mutant rnh1 rnh201 strain in both trans and cis systems. This finding suggests the both RNAse Hs act as an inhibitor in  DSB repair mediated by cDNA.

table1

 Table 1. Frequencies of cDNA and trasnscript-RNA-templated DSB repair in trans and cis systems.

 

Additionally, RAD52, important for homologous recombination both via single-strand annealing and via strand invasion, was knocked out. Also, by deletion of RAD51, promoting RNA-DNA hybrids in yeast, the effect of RAD51 in DSB repair was confirmed (Table 1b). When rad52 was deleted, the restored HIS+ colonies were not found. Here, it is shown transcript-RNA-directed chromosomal DNA repair is stimulated by the function of Rad52 but not Rad51 recombination protein. DSB repair by transcript RNA was reduced over 14 fold in cis system rnh1 rnh201 spt3 rad51 compated to rnh1 rnh201 spt3 cells. All things considered,  in the null-RNAse H cells, it can be assumed that RNA-DNA hybrids are directly used as template for DSB repair with RAD51 and RAD52.

Overall, transcript-RNA-templated DNA repair are taken placed by Ty retrotransposons (reverse transcription) and homologous recombination together. RNAse Hs and SPT3 are key enzymes to aid DNA repair by reverse transcription, and RAD51 and RAD52 are essential for the repair by homologous recombination.

 

[PROPOSED MODEL]

modelFigure 4. Models of transcipt-RNA-templated DSB repair in cis.

This research proposes a model that in the absence of RNAse H function, transcript RNA mediates DSB repair preferentially in cis systems via a Rad52-facilitated annealing mechanism. In this mechanism, the transcript may provide a template that either bridges-broken DNA ends to facilitate precise re-ligation or initiate single-strand annealing via a reverse-transcriptase-dependent extension of the broken DNA ends (Figure 4). The reverse transcriptase activity could be provided by a replicative DNA poloymerase, minimal Ty reverse transcriptase, or both. The current view in the field is that RNA-DNA hybrids formed by the annealing of transcript RNA with complementary chromosomal DNA either in cis or in trans systems are mainly a cause of DNA breaks, DNA damage, and genome instability.

 

[CITATIONS]

[1] Wacker, A., Progr. Nucl. Acid Res., 1, 369 (1963).

[2] Smith, K.C., Photophysiology, ed. A. C. Giese (New York: Academic Press, 1964), vol. 2, pp. 329-388.

[3] Autexier, C. & Lue, N. F. The structure and function of telomerase reverse
transcriptase. Annu. Rev. Biochem. 75, 493–517 (2006).

[4] Storici, F.,Bebenek, K.,Kunkel, T. A.,Gordenin, D. A.&Resnick, M.A. RNA-templated
DNA repair. Nature 447, 338–341 (2007).

[5] Keskin, H., Shen, Y., Huang, Fei., Patel, M., Yang, T., Ashley K., Mazin, AV., Storici, F. Transcript-RNA-templated DNA recombination and repair. Nature 515, 436-439 (2014).

[6] Dombroski BA, Feng Q, Mathias SL et al. An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Saccharomyces cerevisiae. Mol. Cell. Biol. 14 (7): 4485–92. PMC 358820 (1994).

[Video 1] Lo J.H. and Engelward P., Double Holiday Junction model of homology directed repair for a two-ended double strand break, https://www.youtube.com/watch?v=y_OWCU5HwMU.

[Video 2] Oxford Academic (Oxford University Press), Animation 13: Homology-dependent double strand break repair, Molecular Biology: Principles of Genome Function 2nd, https://www.youtube.com/watch?v=86JCMM5kb2A.

[Video 3] https://www.youtube.com/watch?v=-6uBKK5_ckg.

[Video 4] https://www.youtube.com/watch?v=zDm9RaOUHcI.

 

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