A Simulated Shift Work Schedule Does Not Increase DNA Double-Strand Break Repair by NHEJ in the Drosophila Rr3 System

Long-term shift work is widely believed to increase the risk of certain cancers, but conflicting findings between studies render this association unclear. Evidence of interplay between the circadian clock, cell cycle regulation, and DNA damage detection machinery suggests the possibility that circadian rhythm disruption consequent to shift work could alter the DNA double-strand break (DSB) repair pathway usage to favor mutagenic non-homologous end-joining (NHEJ) repair. To test this hypothesis, we compared relative usage of NHEJ and single-strand annealing (SSA) repair of a complementary ended chromosomal double-stranded break using the Repair Reporter 3 (Rr3) system in Drosophila between flies reared on 12:12 and 8:8 (simulated shift work) light:dark schedules. Actimetric analysis showed that the 8:8 light:dark schedule effectively disrupted the rhythms in locomotor output. Inaccurate NHEJ repair was not a frequent outcome in this system overall, and no significant difference was seen in the usage of NHEJ or SSA repair between the control and simulated shift work schedules. We conclude that this circadian disruption regimen does not alter the usage of mutagenic NHEJ DSB repair in the Drosophila male pre-meiotic germline, in the context of the Rr3 system.

Dynamic configurations of meiotic DNA-break hotspot determinant proteins

Appropriate DNA double-strand-break (DSB) and crossover distributions are required for proper meiotic chromosome segregation. Schizosaccharomyces pombe linear element proteins (LinEs) determine DSB hotspots; LinE-bound hotspots form 3D clusters over ∼200 kb chromosomal regions. Here, we investigated LinE configurations and distributions in live cells using super-resolution fluorescence microscopy. We found LinEs form two chromosomal structures, dot-like and linear structures, in both zygotic and azygotic meiosis. Dot-like LinE structures appeared around the time of meiotic DNA replication, underwent dotty-to-linear-to-dotty configurational transitions, and disassembled before the first meiotic division. DSB formation and repair did not detectably influence LinE structure formation, but failure of DSB formation delayed disassembly. Recombination-deficient LinE missense mutants formed dot-like but not linear LinE structures. Our quantitative study reveals a transient form of LinE structures and suggests a novel role for LinE proteins in regulating meiotic events, such as DSB repair. We discuss the relation of LinEs and the synaptonemal complex in other species.

Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice

A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5 end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5 resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330- 338) is important for two events that drive NHEJ, stimulating ligation and inhibiting resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly through inhibiting Dna2-mediated resection.

DNA Double Strand Break Repair and Its Control by Nucleosome Remodeling

DNA double strand breaks (DSBs) are repaired in eukaryotes by one of several cellular mechanisms. The decision-making process controlling DSB repair takes place at the step of DNA end resection, the nucleolytic processing of DNA ends, which generates single-stranded DNA overhangs. Dependent on the length of the overhang, a corresponding DSB repair mechanism is engaged. Interestingly, nucleosomes—the fundamental unit of chromatin—influence the activity of resection nucleases and nucleosome remodelers have emerged as key regulators of DSB repair. Nucleosome remodelers share a common enzymatic mechanism, but for global genome organization specific remodelers have been shown to exert distinct activities. Specifically, different remodelers have been found to slide and evict, position or edit nucleosomes. It is an open question whether the same remodelers exert the same function also in the context of DSBs. Here, we will review recent advances in our understanding of nucleosome remodelers at DSBs: to what extent nucleosome sliding, eviction, positioning and editing can be observed at DSBs and how these activities affect the DSB repair decision.

HP1β Chromo Shadow Domain facilitates H2A ubiquitination for BRCA1 recruitment at DNA double-strand breaks

Efficient DNA double strand break (DSB) repair by homologous recombination (HR), as orchestrated by histone and non-histone proteins, is critical to genome stability, replication, transcription, and cancer avoidance. Here we report that Heterochromatin Protein1 beta (HP1β) acts as a key component of the HR DNA resection step by regulating BRCA1 enrichment at DNA damage sites, a function largely dependent on the HP1β chromo shadow domain (CSD). HP1β itself is enriched at DSBs within gene-rich regions through a CSD interaction with Chromatin Assembly Factor 1 (CAF1) and HP1β depletion impairs subsequent BRCA1 enrichment. An added interaction of the HP1β CSD with the Polycomb Repressor Complex 1 ubiquitinase component RING1A facilitates BRCA1 recruitment by increasing H2A lysine 118-119 ubiquitination, a marker for BRCA1 recruitment. Our findings reveal that HP1β interactions, mediated through its CSD with RING1A, promote H2A ubiquitination and facilitate BRCA1 recruitment at DNA damage sites, a critical step in DSB repair by the HR pathway. These collective results unveil how HP1β is recruited to DSBs in gene-rich regions and how HP1β subsequently promotes BRCA1 recruitment to further HR DNA damage repair by stimulating CtIP-dependent resection.

Identification and Characterization of an Exonic Duplication in PALB2 in a Man with Synchronous Breast and Prostate Cancer

PALB2 (partner and localizer of BRCA2), as indicated by its name, is a BRCA2-interacting protein that plays an important role in homologous recombination (HR) and DNA double-strand break (DSB) repair. While pathogenic variants of PALB2 have been well proven to confer an increased risk of breast cancer, data on its involvement in prostate cancer (PrC) have not been clearly demonstrated. We investigated, using targeted next generation sequencing (NGS), a 59-year-old Caucasian man who developed synchronous breast and prostate cancers. This genetic investigation allowed to identify an intragenic germline heterozygous duplication in PALB2, implicating intronic repetitive sequences spanning exon 11. This variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints have been identified and characterized at the nucleotide level (c.3114-811_3202-1756dup) using an approach based on walking PCR, long range PCR, and Sanger sequencing. RT-PCR using mRNA extracted from lymphocytes and followed by Sanger sequencing revealed a tandem duplication r.3114_3201dup; p.(Gly1068Glufs * 14). This duplication results in the synthesis of a truncated, and most-likely, non-functional protein. These findings expand the phenotypic spectrum of PALB2 variants and may improve the yield of genetic diagnoses in this field.

The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

Auger Emitter Conjugated PARP Inhibitor for Therapy in Triple Negative Breast Cancers: A Comparative In-Vitro Study

PARP1 inhibitors (PARPi) are currently approved for BRCAmut metastatic breast cancer, but they have shown limited response in triple negative breast cancer (TNBC) patients. Combination of an Auger emitter with PARPis enables PARP inhibition and DNA strand break induction simultaneously. This will enhance cytotoxicity and additionally allow a theranostic approach. This study presents the radiosynthesis of the Auger emitter [125I] coupled olaparib derivative: [125I]-PARPi-01, and its therapeutic evaluation in a panel of TNBC cell lines. Specificity was tested by a blocking assay. DNA strand break induction was analysed by γH2AX immunofluorescence staining. Cell cycle analysis and apoptosis assays were studied using flow cytometry in TNBC cell lines (BRCAwt/mut). Anchorage independent growth potential was evaluated using soft agar assay. [125I]-PARPi-01 showed PARP1-specificity and higher cytotoxicity than olaparib in TNBC cell lines irrespective of BRCA their status. Cell lines harbouring DNA repair deficiency showed response to [125I]-PARPi-01 monotherapy. Combined treatment with Dox-NP further enhanced therapeutic efficiency in metastatic resistant BRCAwt cell lines. The clonogenic survival was significantly reduced after treatment with [125I]-PARPi-01 in all TNBC lines investigated. Therapeutic efficacy was further enhanced after combined treatment with chemotherapeutics. [125I]-PARPi-01 is a promising radiotherapeutic agent for low radiation dosages, and mono/combined therapies of TNBC.

INO80 requires a polycomb subunit to regulate the establishment of poised chromatin in murine spermatocytes

ABSTRACT INO80 is the catalytic subunit of the INO80-chromatin remodeling complex that is involved in DNA replication, repair and transcription regulation. Ino80 deficiency in murine spermatocytes (Ino80cKO) results in pachytene arrest of spermatocytes due to incomplete synapsis and aberrant DNA double-strand break repair, which leads to apoptosis. RNA-seq on Ino80cKO spermatocytes revealed major changes in transcription, indicating that an aberrant transcription program arises upon INO80 depletion. In Ino80WT spermatocytes, genome-wide analysis showed that INO80-binding sites were mostly promoter proximal and necessary for the regulation of spermatogenic gene expression, primarily of premeiotic and meiotic genes. Furthermore, most of the genes poised for activity, as well as those genes that are active, shared INO80 binding. In Ino80cKO spermatocytes, most poised genes demonstrated de-repression due to reduced H3K27me3 enrichment and, in turn, showed increased expression levels. INO80 interacts with the core PRC2 complex member SUZ12 and promotes its recruitment. Furthermore, INO80 mediates H2A.Z incorporation at the poised promoters, which was reduced in Ino80cKO spermatocytes. Taken together, INO80 is emerging as a major regulator of the meiotic transcription program by mediating poised chromatin establishment through SUZ12 binding.