Senataxin Ortholog Sen1 Limits DNA:RNA Hybrid Accumulation at DNA Double-Strand Breaks to Control End Resection and Repair Fidelity

Summary: An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity

An Expanding Toolkit for Heterochromatin Repair Studies

Pericentromeric heterochromatin is mostly composed of repetitive DNA sequences prone to aberrant recombination. Cells have developed highly specialized mechanisms to enable ‘safe’ homologous recombination (HR) repair while preventing aberrant recombination in this domain. Understanding heterochromatin repair responses is essential to understanding the critical mechanisms responsible for genome integrity and tumor suppression. Here, we review the tools, approaches, and methods currently available to investigate double-strand break (DSB) repair in pericentromeric regions, and also suggest how technologies recently developed for euchromatin repair studies can be adapted to characterize responses in heterochromatin. With this ever-growing toolkit, we are witnessing exciting progress in our understanding of how the ‘dark matter’ of the genome is repaired, greatly improving our understanding of genome stability mechanisms

Rad9/53BP1 promotes DNA repair via crossover recombination by limiting the Sgs1 and Mph1 helicases

In budding yeast, the 53BP1 ortholog Rad9 limits the resection nucleolytic processing of DNA double strand breaks. Here the authors reveal that Rad9 promotes long tract gene conversions, BIR and CO, during the HR repair of a DSB via modulation of Sgs1 and Mph1 helicases

Reduced kinase activity of polo kinase Cdc5 affects chromosome stability and DNA damage response in <i>S. cerevisiae</i>

<p>Polo-like kinases (PLKs) control several aspects of eukaryotic cell division and DNA damage response. Remarkably, PLKs are overexpressed in several types of cancer, being therefore a marker of bad prognosis. As such, specific PLK kinase activity inhibitors are already used in clinical trials and the regulation of PLK activation is a relevant topic of cancer research. Phosphorylation of threonine residues in the T-loop of the kinase domain is pivotal for PLKs activation. Here, we show that T238A substitution in the T-loop reduces the kinase activity of Cdc5, the only PLK in <i>Saccharomyces cerevisiae</i>, with minor effect on cell growth in unperturbed conditions. However, the <i>cdc5</i>-T238A cells have increased rate of chromosome loss and gross chromosomal rearrangements, indicating altered genome stability. Moreover, the T238A mutation affects timely localization of Cdc5 to the spindle pole bodies and blocks cell cycle restart after one irreparable double-strand break. In cells responding to alkylating agent metylmethane sulfonate (MMS), the <i>cdc5</i>-T238A mutation reduces the phosphorylation of Mus81-Mms4 resolvase and exacerbates the MMS sensitivity of <i>sgs1</i>Δ cells that accumulate Holliday junctions. Of importance, the previously described checkpoint adaptation defective allele, <i>cdc5</i>-ad does not show reduced kinase activity, defective Mms4 phosphorylation and genetic interaction with <i>sgs1</i>Δ. Our data define the importance of regulating Cdc5 activity through T-loop phosphorylation to preserve genome integrity and respond to DNA damage.</p

Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection, End-Tethering and Repair of a Double-Strand Break

<div><p>The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog <i>RAD9</i> reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of <i>RAD9</i> restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.</p></div

Rad9 oligomers affect cell viability following a DSB, in the absence of Sae2, mainly through the interaction with Dpb11.

<p>(A and D) Viability of the wild type YMV80 strain and the indicated derivatives, plated on YEP+raf+gal. For each strain, the number of colonies grown after 3 days at 28°C in YEP+raf+gal was normalized respect YEP+raf. Plotted values are the mean values ± SD from three independent experiments. (B) Cells of the wild type JKM139 strain and the indicated derivatives, expressing a Rad9-3HA fusion protein, were grown in YEP+raf and synchronized in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. Relative fold enrichment of Rad9-3HA at 0.1 kb from the HO cleavage site was evaluated after ChIP with anti-HA antibodies and qPCR analysis. Plotted values are the mean values ± SEM from three independent experiments. (C) Schematic representation of Rad9 functional domains and sites phosphorylated by CDK1, Mec1 and Tel1. (E) Exponentially growing cell cultures of the wild type YMV80 strain and the indicated derivatives were incubated for 2 hours with or without the dimerization-inducing molecule AP20187, before plating in YEP+Raf or YEP+Raf+Gal, with/without AP20187. For each strain, the number of colonies grown after 3 days at 28°C in YEP+raf+gal was normalized with respect to YEP+raf. Plotted values are the mean values ± SD from three independent experiments. Expression level of Rad9-2A, Rad9-7xA and Rad9-ΔBRCT-FKBP protein variants, described in this Figure, were determined by western blotting in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen.1004928.s006" target="_blank">S6 Fig</a>.</p

Model to explain the interplay between Mre11 complex and Rad9 at a DSB in G2/M phase.

<p>Ku and Mre11 complexes, together with Rad9, are recruited soon after a DSB formation and limit the action of Exo1 and Dna2-Sgs1 pathways. The order of appearance of the various factors was based on both literature and our results. See details in the text.</p

Rad9 limits an Sgs1- and Sae2- dependent initial step of DSB resection.

<p>(A) Scheme of the <i>MAT</i> locus. The figure shows the positions of the HO-cut site, and the probe used in experiments shown in (B and C) and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen.1004928.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen.1004928.s004" target="_blank">S4 Figs</a>. (B, C) Exponentially growing YEP+raf cell cultures of the wild type JKM139 strain and the indicated derivatives, carrying a unique HO cut site at <i>MAT</i> locus and expressing the HO nuclease under GAL1 promoter, were synchronized and kept in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. SspI-digested genomic DNA, extracted from samples taken at the indicated times, was analysed by Southern blotting to test 3′ filament formation. (C) The mean values ± SEM corresponding to the resection products of two independent experiments were determined by densitometry. (D) Schematic representation of the quantitative PCR method used to monitor HO-induced DSB resection. (E–F) Plots showing the ratio of resected DNA among HO cut DNAs at each time points by qPCR analysis. The mean values from three independent experiments are shown with SEM. Significance was calculated by one-tailed paired Student's <i>t</i> test (* for P<0.05; ** for P<0.01; where not indicated, the P value was higher than 0.05) (G) JKM139 derivatives were nocodazole-arrested in G2/M and 2% galactose was added to induce HO cut. After 2 hours of HO induction, cells were plated on YEP+raf and YEP+raf+gal, and incubated at 28°C for three days. Viability results were obtained from the ratio between number of colonies on YEP+raf+gal and YEP+raf. The mean values from three independent experiments are shown with SD.</p