Thwarting endogenous stress: BRCA protects against aldehyde toxicity

Homologous recombination (HR) and the Fanconi Anemia (FA) pathways constitute essential repair pathways for DNA damage, which includes DNA double-stranded breaks (DSB) and inter-strand cross-links (ICL), respectively. Germline mutations affecting a single copy of the HR factors BRCA1 and BRCA2 predispose individuals to cancers of the breast, ovary, prostate, and pancreas. Cells deficient for BRCA proteins display high levels of genome instability due to defective repair of endogenous DSBs and are also exquisitely sensitive to DNA-damaging agents. In addition to their roles in repair of DSBs and ICLs, HR and FA proteins have a genetically separable function in the protection of stalled DNA replication forks from nuclease-mediated degradation (Schlacher et al, ). Although it has been hypothesized that loss of functional HR and ICL repair is the primary cause of cancer in BRCA- and FA-deficient patients (Prakash et al, ), the contribution of replication fork instability associated with the degradation of nascent DNA remains unclear. Two recent papers explain how endogenous toxins render cells vulnerable to genomic instability, which explains how the BRCA/FA pathway suppresses tumorigenesis (Tacconi et al, ; Tan et al, )

Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8

53BP1 is a well-known mediator of the cellular response to DNA damage. Two alternative mechanisms have been proposed to explain 53BP1’s interaction with DNA double-strand breaks (DSBs), one by binding to methylated histones and the other via an RNF8 E3 ligase–dependent ubiquitylation pathway. The formation of RNF8 and 53BP1 irradiation-induced foci are both dependent on histone H2AX. To evaluate the contribution of the RNF8-dependent pathway to 53BP1 function, we generated RNF8 knockout mice. We report that RNF8 deficiency results in defective class switch recombination (CSR) and accumulation of unresolved immunoglobulin heavy chain–associated DSBs. The CSR DSB repair defect is milder than that observed in the absence of 53BP1 but similar to that found in H2AX−/− mice. Moreover, similar to H2AX but different from 53BP1 deficiency, RNF8−/− males are sterile, and this is associated with defective ubiquitylation of the XY chromatin. Combined loss of H2AX and RNF8 does not cause further impairment in CSR, demonstrating that the two genes function epistatically. Importantly, although 53BP1 foci formation is RNF8 dependent, its binding to chromatin is preserved in the absence of RNF8. This suggests a two-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage–inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin

Loss of ATM kinase activity leads to embryonic lethality in mice

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis

Replication Fork Stability Confers Chemoresistance in BRCA-deficient Cells

Brca1- and Brca2-deficient cells have reduced capacity to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) and consequently are hypersensitive to DNA damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore HR activity at DSBs. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARPi and cisplatin resistance is associated with replication fork (RF) protection in Brca2-deficient tumor cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of RF protection, highlighting the complexities by which tumor cells evade chemotherapeutic interventions and acquire drug resistance

The AID-induced DNA damage response in chromatin

Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity with somatic hypermutation and class switch recombination, chromatin must be made accessible for Activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways, but if not handled properly, can lead to formation of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment for which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles of histone post-translational modifications and the significance of AID function outside of antibody diversity

Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals.

Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities

ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm–/– spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing

Oocytes in the Tc1 accessory chromosome mouse model do not exhibit diplotene elimination.

<p>(<b>A</b>) Pachytene Tc1 oocyte with an asynapsed h21 chromosome (arrow, inset), labelled with γH2AFX (red, arrow) and HORMAD1 (magenta, inset). (<b>B</b>) Pachytene Tc1 oocyte with self-synapsed h21 chromosome (arrow, inset), which is negative for γH2AFX and HORMAD1. Scale bar represents 10μm. (<b>C</b>) Mean percentage (± s.e.m.) of Tc1 oocytes with a γH2AFX-positive or γH2AFX-negative h21 chromosome between pachynema and late diplonema, demonstrating no significant elimination of Tc1 oocytes with asynapsed h21 chromosomes. n is the number of oocytes counted from three non-littermate mice at 18.5 d<i>pc</i>. (<b>D</b>) Comparison of γH2AFX domain integrated intensity between XO and Tc1 oocytes at diplonema, the stage when oocyte losses are observed in XO but not Tc1 females. n refers to the number of oocytes analyzed. (<b>E</b>) Schematic showing that the asynapsed h21 chromosome does not trigger oocyte losses at diplonema.</p

<i>H2afx</i> deletion reverses perinatal oocyte losses in XO females.

<p>(<b>A</b>) Pachytene XO <i>H2afx-/-</i> oocyte with an asynapsed X chromosome (arrow; marked by HORMAD1, green, and HORMAD2, red and inset). (<b>B</b>) Early diplotene XO <i>H2afx</i>-/- oocyte, showing intermediate levels of desynapsis (HORMAD1-only positive axes, green) and an asynapsed X chromosome (arrow; marked with both HORMAD1 and HORMAD2, inset). (<b>C</b>) Late diplotene XO <i>H2afx</i>-/- oocyte, showing extensive desynapsis and an asynapsed X chromosome (arrow). Scale bar represents 10μm. (<b>D</b>) Mean percentage (± s.e.m.) of XO <i>H2afx+/-</i> oocytes with a HORMAD1/2 double-positive X chromosome between pachynema and late diplonema at 19.5 d<i>pc</i>. Statistics were performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005462#pgen.1005462.g001" target="_blank">Fig 1</a>. (<b>E</b>) Mean percentage (± s.e.m.) of XO <i>H2afx-/-</i> oocytes with a HORMAD1/2 double-positive X chromosome between pachynema and late diplonema at 19.5 d<i>pc</i>. No statistically significant difference was found between means at pachynema and late diplonema (Tukey’s test). (<b>F</b>) Enrichment of oocytes with an asynapsed X chromosome in XO <i>H2afx-/-</i> compared to XO <i>H2afx+/-</i> between pachynema and late diplonema. Enrichment was defined as the ratio of the mean percentage of oocytes with an asynapsed X in XO <i>H2afx-/-</i> versus XO <i>H2afx+/-</i> control, using data from part (D) and (E). (<b>G</b>) Mean number (± s.e.m.) of oocytes in XO and XX, ovaries with varying doses of <i>H2afx</i>, XO <i>H2afx-/-</i>,XX <i>H2afx+/-</i> and XX <i>H2afx+/+</i> at 20.5 d<i>pc</i>. n is the number of non-littermate ovaries analyzed. Tukey multiple comparison tests. (<b>H</b>) Summary showing the importance of H2AFX in XO oocyte elimination.</p