Genomes undergo different types of alterations, including DNA damage, point mutations, and genome rearrangements, that can trigger genome instability, a hallmark of cancer, ageing and genetic diseases. Cells possess a powerful system, called DNA damage response (DDR) that senses and repair DNA damage to warrant proper cell proliferation, senescence or apoptosis.
Transcription constitutes an important natural source of DNA metabolic errors that can compromise genome integrity. Transcription can create the conditions for high levels of mutations and recombination by both its ability to become a barrier to DNA replication and to open the DNA structure and remodel chromatin. Furthermore, nascent RNAs can reinvade the DNA double helix to form a structure termed the R loop, where a single-stranded DNA (ssDNA) is accompanied by formation of a DNA:RNA hybrid, which can impede transcription and replication processes and lead to genomic instability.
However, it is now emerging that RNA and its potential to form hybrids with single-stranded DNA are important for maintaining genome stability. In fact, DNA:RNA hybrids can form also under physiological conditions, such as the result of RNA polymerase backtracking at negatively supercoiled regions, or at telomeres, where a long noncoding telomeric repeat-containing RNA (TERRA) is transcribed and forms R loops structures in both yeast and humans. Interestingly, these DNA:RNA hybrids at Saccharomyces cerevisiae telomeres appear to be important to induce telomere elongation through an homologous recombination-based pathway, named alternative lengthening of telomeres (ALT), that in human cells allows telomere elongation in a small but significant number of cancers. Furthermore, we have shown that long non-coding RNAs (dilncRNAs) are generated at DNA double-strand breaks (DSBs) in mammalian cells and are processed to generate DDRNAs that are important to trigger a full DDR and to repair DSBs. DDRNAs and dilncRNAs are generated also at mammalian dysfunctional telomeres, suggesting that they could form DNA:RNA hybrids and control HR-based telomere lengthening in ALT cancer cells.
It is thus important to appreciate how RNA, and its potential to form a hybrid with single-stranded DNA, although initially studied only as a threat to genome stability is now emerging as key regulator of the pathways maintaining genome stability. Equally importantly it is also to highlight how DDR pathways, so far considered only the result of interacting proteins governed by their post-translational modifications, in fact rely on the synthesis and processing of RNA molecules generated in situ at sites of DNA damage. Therefore, the study of the emerging role of RNA both as threat and a guardian of genome stability is both topical and likely ripe for many important discoveries.
In this proposal, we plan to integrate different set of competences, spanning from genetics to cell and molecular biology, in order to investigate the mechanisms leading to DNA:RNA hybrid formation and those preventing their formation, under physiological and pathological conditions, using the yeast S. cerevisiae and mammalian cells as model systems. In particular, our specific aims are designed to (1) investigate how DNA super helical tension and higher order chromatin organization influence DNA:RNA hybrid formation within the genome, in yeast ALT cells and at telomeres; 2) determine the mechanisms regulating DNA:RNA hybrid formation by studying the contribution of proteins involved in topological tension, telomere metabolism, and arrest of DNA replication or transcription; 3) determine the role of dilncRNA and DDRNA in DNA:RNA hybrid formation and ALT in cancer cells; 4) determine the role of TERRA and DNA:RNA hybrids in promoting ALT at yeast type II survivors; 5) determine the crosstalks between DNA:RNA hybrids formation, topological feature and ALT mechanisms at yeast telomeres.
These research aims will be developed by using a combinat