Suchergebnisse

Endogenously-arising DNA double-strand breaks (DSBs) rarely harbor canonical 5′-phosphate, 3′-hydroxyl moieties at the ends, which are, regardless of the pathway used, ultimately required for their repair. Cells are therefore endowed with a wide variety of enzymes that can deal with these chemical and structural variations and guarantee the formation of ligatable termini. An important distinction is whether the ends are directly "unblocked" by specific enzymatic activities without affecting the integrity of the DNA molecule and its sequence, or whether they are "processed" by unspecific nucleases that remove nucleotides from the termini. DNA end structure and configuration, therefore, shape the repair process, its requirements, and, importantly, its final outcome. Thus, the molecular mechanisms that coordinate and integrate the cellular response to blocked DSBs, although still largely unexplored, can be particularly relevant for maintaining genome integrity and avoiding malignant transformation and cancer. ; Work in the FC-L laboratory was funded with grants from the Spanish and Andalusian Government (SAF2017-89619-R, CVI-7948, European Regional Development Fund), and the European Research Council (ERC-CoG-2014-647359); and with an individual fellowship for AS-B (Beca Predoctoral AEFAT, Asociación Española Familia Ataxia Telangiectasia). CABIMER was supported by the Andalusian Government. ; Peer reviewed

DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr540 amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells. © 2013 Ruiz et al. ; Spanish Ministry of Science and Innovation (BFU2009-10085, BFU2010-16370 and Consolider Ingenio 2010 CSD2007-015); European Union (FEDER); Spanish Council for Scientific Research (CSIC). ; Peer Reviewed

R loops are an important source of genome instability, largely due to their negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess causes lethality and genome instability. Here, we show that, in addition to ssDNA and ssRNA, Yra1 binds RNA–DNA hybrids in vitro and, when artificially overexpressed, can be recruited to chromatin in an RNA–DNA hybrid-dependent manner, stabilizing R loops and converting them into replication obstacles in vivo. Importantly, an excess of Yra1 increases R-loop-mediated genome instability caused by transcription–replication collisions regardless of whether they are codirectional or head-on. It also induces telomere shortening in telomerase-negative cells and accelerates senescence, consistent with a defect in telomere replication. Our results indicate that RNA–DNA hybrids form transiently in cells regardless of replication and, after stabilization by excess Yra1, compromise genome integrity, in agreement with a two-step model of R-loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells ; Research was funded by grants from the European Research Council (ERC2014 AdG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2016-75058-P), the Junta de Andalucía (PA12-BIO1238), and the European Union Regional Funds (FEDER) to A.A., and the Ligue Nationale Contre le Cancer (Équipe Labellisée) to V.G. P.A. was the recipient of Région Provence- Alpes-Côte d'Azur and Association pour la Recherche Contre le Cancer (ARC) predoctoral training grants. ; Peer reviewed