CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway

The RNA world is changing our views about sensing and resolution of DNA damage. Here, we developed single-molecule DNA/RNA analysis approaches to visualize how nascent RNA facilitates the repair of DNA double-strand breaks (DSBs). RNA polymerase II (RNAPII) is crucial for DSB resolution in human cells. DSB-flanking, RNAPII-generated nascent RNA forms RNA:DNA hybrids, guiding the upstream DNA repair steps towards favouring the error-free Homologous Recombination (HR) pathway over Non-Homologous End Joining. Specific RNAPII inhibitor, THZ1, impairs recruitment of essential HR proteins to DSBs, implicating nascent RNA in DNA end resection, initiation and execution of HR repair. We further propose that resection factor CtIP interacts with and re-activates RNAPII when paused by the RNA:DNA hybrids, collectively promoting faithful repair of chromosome breaks to maintain genomic integrity.

Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control

p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, we find that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B are both required for the normal repair of meiotic DNA breaks, an activity that is more crucial when meiotic recombination is defective. We find that in oocytes with persistent DNA breaks p53A is also required to activate a meiotic pachytene checkpoint. Our findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control.

XRCC4 and MRE11 Roles and Transcriptional Response to Repair of TALEN-Induced Double-Strand DNA Breaks

Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered.

Dysregulation of X-Ray Repair Cross Complementing 4 Expression in the Eutopic Endometrium of Women with Endometriosis

Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of double-stranded DNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Towards this, we focused on the X-Ray Repair Cross-Complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (p<0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the GEO database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in-vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AFX and 8-hydroxy-2’-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (ThESCs) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AFX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.

Effects of tamoxifen inducible MerCreMer on gene expression in cardiac myocytes in mice

The Cre-LoxP technology, including the tamoxifen (TAM) inducible MerCreMer (MCM), is increasingly used to delineate gene function, understand the disease mechanisms, and test therapeutic interventions. We set to determine the effects of TAM-MCM on cardiac myocyte transcriptome. Expression of the MCM was induced specifically in cardiac myocytes upon injection of TAM to myosin heavy chain 6-MCM (Myh6-Mcm) mice for 5 consecutive days. Cardiac function, myocardial histology, and gene expression (RNA-sequencing) were analyzed 2 weeks after TAM injection. A total of 346 protein coding genes (168 up- and 178 down-regulated) were differentially expressed. Transcript levels of 85 genes, analyzed by a reverse transcription-polymerase chain reaction in independent samples, correlated with changes in the RNA-sequencing data. The differentially expressed genes were modestly enriched for genes involved in the interferon response and the tumor protein 53 (TP53) pathways. The changes in gene expression were relatively small and mostly transient and had no discernible effects on cardiac function, myocardial fibrosis, and apoptosis or induction of double-stranded DNA breaks. Thus, TAM-inducible activation of MCM alters cardiac myocytes gene expression, provoking modest and transient interferon and DNA damage responses without exerting other discernible phenotypic effects. Thus, the effects of TAM-MCM on gene expression should be considered in discerning the bona fide changes that result from the targeting of the gene of interest.

PAR recognition by multiple reader domains of PARP1 allosterically regulates the DNA-dependent activities and independently stimulates the catalytic activity of PARP1

Poly(ADP-ribosyl)ation is a post translational modification, predominantly catalyzed by Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage, mediating the DNA repair process to maintain genomic integrity. Single strand (SSB) and double strand (DSB) DNA breaks are bonafide stimulators of PARP1 activity. We identified that, in addition, single strand (ss) DNA also binds and stimulates the PARP1 activity. Poly(ADP-ribose) (PAR) is chemically similar to ssDNA. However, PAR mediated PARP1 regulation remains unexplored. Here, we report ZnF3, BRCT and WGR, hitherto uncharacterized, as PAR-specific reader domains of PARP1. Surprisingly, these domains recognize PARylated protein with a higher affinity compared to PAR, but do not bind to DNA. Conversely, N-terminal domains, ZnF1 and ZnF2, of PARP1 recognize DNA but not PAR. Further competition binding studies suggest that PAR binding, allosterically releases DNA from PARP1. Unexpectedly, PAR showed catalytic stimulation of PARP1 but hampers the DNA dependent stimulation. Altogether, our work discovers dedicated PAR and DNA reader domains of the PARP1, and uncovers a novel mechanism of allosteric stimulation of the catalytic activity of PARP1 but retardation of DNA-dependent activities of PARP1 by its catalytic product PAR.

A Multidrug Approach to Modulate the Mitochondrial Metabolism Impairment and Relative Oxidative Stress in Fanconi Anemia Complementation Group A

Fanconi Anemia (FA) is a rare recessive genetic disorder characterized by aplastic anemia due to a defective DNA repair system. In addition, dysfunctional energy metabolism, lipid droplets accumulation, and unbalanced oxidative stress are involved in FA pathogenesis. Thus, to modulate the altered metabolism, Fanc-A lymphoblast cell lines were treated with quercetin, a flavonoid compound, C75 (4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid), a fatty acid synthesis inhibitor, and rapamycin, an mTOR inhibitor, alone or in combination. As a control, isogenic FA cell lines corrected with the functional Fanc-A gene were used. Results showed that: (i) quercetin recovered the energy metabolism efficiency, reducing oxidative stress; (ii) C75 caused the lipid accumulation decrement and a slight oxidative stress reduction, without improving the energy metabolism; (iii) rapamycin reduced the aerobic metabolism and the oxidative stress, without increasing the energy status. In addition, all molecules reduce the accumulation of DNA double-strand breaks. Two-by-two combinations of the three drugs showed an additive effect compared with the action of the single molecule. Specifically, the quercetin/C75 combination appeared the most efficient in the mitochondrial and lipid metabolism improvement and in oxidative stress production reduction, while the quercetin/rapamycin combination seemed the most efficient in the DNA breaks decrement. Thus, data reported herein suggest that FA is a complex and multifactorial disease, and a multidrug strategy is necessary to correct the metabolic alterations.

Structural Insight into the Mechanism of PALB2 Interaction with MRG15

The tumor suppressor protein partner and localizer of BRCA2 (PALB2) orchestrates the interactions between breast cancer susceptibility proteins 1 and 2 (BRCA1, -2) that are critical for genome stability, homologous recombination (HR) and DNA repair. PALB2 mutations predispose patients to a spectrum of cancers, including breast and ovarian cancers. PALB2 localizes HR machinery to chromatin and links it with transcription through multiple DNA and protein interactions. This includes its interaction with MRG15 (Morf-related gene on chromosome 15), which is part of many transcription complexes, including the HAT-associated and the HDAC-associated complexes. This interaction is critical for PALB2 localization in actively transcribed genes, where transcription/replication conflicts lead to frequent replication stress and DNA breaks. We solved the crystal structure of the MRG15 MRG domain bound to the PALB2 peptide and investigated the effect of several PALB2 mutations, including patient-derived variants. PALB2 interacts with an extended surface of the MRG that is known to interact with other proteins. This, together with a nanomolar affinity, suggests that the binding of MRG15 partners, including PALB2, to this region is mutually exclusive. Breast cancer-related mutations of PALB2 cause only minor attenuation of the binding affinity. New data reveal the mechanism of PALB2-MRG15 binding, advancing our understanding of PALB2 function in chromosome maintenance and tumorigenesis.

Replication origins drive genetic and phenotypic variation in humans

DNA replication starts with the activation of the replicative helicases, polymerases and associated factors at thousands of origins per S-phase. Due to local torsional constraints generated during licensing and the switch between polymerases of distinct fidelity and proofreading ability following firing, origin activation has the potential to induce DNA damage and mutagenesis. However, whether sites of replication initiation exhibit a specific mutational footprint has not yet been established. Here we demonstrate that mutagenesis is increased at early and highly efficient origins. The elevated mutation rate observed at these sites is caused by two distinct mutational processes consistent with formation of DNA breaks at the origin itself and local error-prone DNA synthesis in the immediate vicinity of the origin. We demonstrate that these replication-dependent mutational processes create the skew in base composition observed at human replication origins. Further, we show that mutagenesis associated with replication initiation exerts an influence on phenotypic diversity in human populations disproportionate to the origins genomic footprint: by increasing mutational loads at gene promoters and splice junctions the presence of an origin influences both gene expression and mRNA isoform usage. These findings have important implications for our understanding of the mutational processes that sculpt the human genome.