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Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m 5 C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m 5 C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m 5 C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m 5 C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair. Post-translational modifications of proteins at DNA damage sites can facilitate the recruitment of DNA repair factors. Here, the authors show that mRNA is locally modified with m 5 C at sites of DNA damage by the RNA methyltransferase TRDMT1 to promote homologous recombination repair.

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle 1 – 3 . Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci 4 – 10 , but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA–RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA–RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term ‘DR-loops’, which contain both DNA–DNA and DNA–RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops. RNA transcripts stimulate homologous recombination through the formation of DR-loops, intermediate structures that contain both DNA–DNA and DNA–RNA hybrids.

Actively transcribed regions of the genome are protected by transcription-coupled DNA repair mechanisms, including transcription-coupled homologous recombination (TC-HR). Here we used reactive oxygen species (ROS) to induce and characterize TC-HR at a transcribed locus in human cells. As canonical HR, TC-HR requires RAD51. However, the localization of RAD51 to damage sites during TC-HR does not require BRCA1 and BRCA2, but relies on RAD52 and Cockayne Syndrome Protein B (CSB). During TC-HR, RAD52 is recruited by CSB through an acidic domain. CSB in turn is recruited by R loops, which are strongly induced by ROS in transcribed regions. Notably, CSB displays a strong affinity for DNA:RNA hybrids in vitro, suggesting that it is a sensor of ROS-induced R loops. Thus, TC-HR is triggered by R loops, initiated by CSB, and carried out by the CSB-RAD52-RAD51 axis, establishing a BRCA1/2-independent alternative HR pathway protecting the transcribed genome. Transcription-coupled homologous recombination (TC-HR) is activated by reactive oxygen species-induced DNA damage to maintain transcribed genome stability. The authors demonstrate that R loops are induced by ROS at the transcribed genome, triggering a CSB-RAD52- dependent but BRCA1/2-independent RAD51 loading for repair.

RNA modifications regulate a variety of cellular processes including DNA repair. The RNA methyltransferase TRDMT1 generates methyl-5-cytosine (m5C) on messenger RNA (mRNA) at DNA double-strand breaks (DSBs) in transcribed regions, promoting transcription-coupled homologous recombination (HR). Here, we identified that Fragile X mental retardation protein (FMRP) promotes transcription-coupled HR via its interaction with both the m5C writer TRDMT1 and the m5C eraser ten-eleven translocation protein 1 (TET1). TRDMT1, FMRP, and TET1 function in a temporal order at the transcriptionally active sites of DSBs. FMRP displays a higher affinity for DNA:RNA hybrids containing m5C-modified RNA than for hybrids without modification and facilitates demethylation of m5C by TET1 in vitro. Loss of either the chromatin- or RNA-binding domain of FMRP compromises demethylation of damage-induced m5C in cells. Importantly, FMRP is required for R-loop resolving in cells. Due to unresolved R-loop and m5C preventing completion of DSB repair, FMRP depletion or low expression leads to delayed repair of DSBs at transcriptionally active sites and sensitizes cancer cells to radiation in a BRCA-independent manner. Together, our findings present an m5C reader, FMRP, which acts as a coordinator between the m5C writer and eraser to promote mRNA-dependent repair and cell survival in cancer.

Abstract In all living organisms, the response to double-strand breaks (DSBs) is critical for the maintenance of chromosome integrity. Homologous recombination (HR), which utilizes a homologous template to prime DNA synthesis and to restore genetic information lost at the DNA break site, is a complex multistep response. In Bacillus subtilis, this response can be subdivided into five general acts: (1) recognition of the break site(s) and formation of a repair center (RC), which enables cells to commit to HR; (2) end-processing of the broken end(s) by different avenues to generate a 3′-tailed duplex and RecN-mediated DSB ‘coordination’; (3) loading of RecA onto single-strand DNA at the RecN-induced RC and concomitant DNA strand exchange; (4) branch migration and resolution, or dissolution, of the recombination intermediates, and replication restart, followed by (5) disassembly of the recombination apparatus formed at the dynamic RC and segregation of sister chromosomes. When HR is impaired or an intact homologous template is not available, error-prone nonhomologous end-joining directly rejoins the two broken ends by ligation. In this review, we examine the functions that are known to contribute to DNA DSB repair in B. subtilis, and compare their properties with those of other bacterial phyla. Double strand break repair in bacteria

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.