RESUMO
DNA double-strand breaks (DSBs) are the most severe DNA lesions and need to be removed immediately to prevent loss of genomic information. Recently, it has been revealed that DSBs induce novel transcription from the cleavage sites in various species, resulting in RNAs being referred to as damage-induced RNAs (diRNAs). While diRNA synthesis is an early event in the DNA damage response and plays an essential role in DSB repair activation, the location where diRNAs are newly generated in plants remains unclear, as does their transcriptional mechanism. Here, we performed the sequencing of polyadenylated (polyA) diRNAs that emerged around all DSB loci in Arabidopsis thaliana under the expression of the exogenous restriction enzyme Sbf I and observed 88 diRNAs transcribed via RNA polymerase II in 360 DSB loci. Most of the detected diRNAs originated within active genes and were transcribed from DSBs in a bidirectional manner. Furthermore, we found that diRNA elongation tends to terminate at the boundary of an endogenous gene located near DSB loci. Our results provide reliable evidence for understanding the importance of new transcription at DSBs and show that diRNA is a crucial factor for successful DSB repair.
Assuntos
Arabidopsis , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Transcrição Gênica , Arabidopsis/genética , Arabidopsis/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , RNA Polimerase II/metabolismo , RNA Polimerase II/genéticaRESUMO
In modern molecular biology, RNA has emerged as a versatile macromolecule capable of mediating an astonishing number of biological functions beyond its role as a transient messenger of genetic information. The recent discovery and functional analyses of new classes of noncoding RNAs (ncRNAs) have revealed their widespread use in many pathways, including several in the nucleus. This Review focuses on the mechanisms by which nuclear ncRNAs directly contribute to the maintenance of genome stability. We discuss how ncRNAs inhibit spurious recombination among repetitive DNA elements, repress mobilization of transposable elements (TEs), template or bridge DNA double-strand breaks (DSBs) during repair, and direct developmentally regulated genome rearrangements in some ciliates. These studies reveal an unexpected repertoire of mechanisms by which ncRNAs contribute to genome stability and even potentially fuel evolution by acting as templates for genome modification.
Assuntos
Núcleo Celular/metabolismo , Instabilidade Genômica , RNA não Traduzido/genética , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Dosagem de Genes , Inativação Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Conformação de Ácido Nucleico , RNA não Traduzido/química , RNA não Traduzido/classificação , RNA não Traduzido/metabolismo , Relação Estrutura-Atividade , Telômero/genética , Telômero/metabolismoRESUMO
Repair of DNA double-strand break (DSB) is critical for the maintenance of genome integrity. A class of DSB-induced small RNAs (diRNAs) has been shown to play an important role in DSB repair. In humans, diRNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination (HR). Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions. However, the role of Ago2 phosphorylation in DNA damage repair is unexplored. Here, we show that S672, S828, T830, and S831 of human Ago2 are phosphorylated in response to ionizing radiation (IR). S672A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency. We further show that defective association of Ago2 S672A variant with DSB sites, instead of defects in diRNA and Rad51 binding, may account for decreased Rad51 foci formation and HR efficiency. Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.
Assuntos
Proteínas Argonautas/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Rad51 Recombinase/genética , Aminoácidos/genética , Aminoácidos/efeitos da radiação , DNA/genética , DNA/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Genoma/genética , Genoma/efeitos da radiação , Recombinação Homóloga/efeitos da radiação , Humanos , Fosforilação/efeitos da radiação , Ligação Proteica/genética , RNA/genética , RNA/efeitos da radiação , Radiação IonizanteRESUMO
DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Regulação da Expressão Gênica , RNA/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , DNA/química , Instabilidade Genômica , Humanos , RNA/química , RNA/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética , Transdução de SinaisRESUMO
Historically, the role of cellular RNA has been subordinate and ancillary to DNA. Protein-coding mRNA conveys the information content of DNA, and transfer RNAs and ribosomal RNAs allow the polymerization of amino acids into proteins. The discovery of non-protein-coding RNAs (ncRNAs) provided an additional role for RNA in finely tuning DNA expression. However, it has recently become apparent that the safeguard of DNA integrity depends on small ncRNAs acting at the site of DNA lesions to signal the presence of DNA damage in the cell, and on the genes involved in their biogenesis to achieve accurate DNA repair. I review here evidence supporting a role for small ncRNAs, termed DNA damage-response RNAs (DDRNAs) or double-strand break (DSB)-induced RNAs (diRNAs), that are generated at sites of DNA damage and control the DNA damage response (DDR). I also discuss their biogenesis, potential mechanisms of action, and their relevance in cancer.