Nuclear NAD+ homeostasis is essential for naive and chemoresistant BRCA1/2-deficient tumor survival

Resistance to PARP inhibitors (PARPi) is emerging as the major obstacle to their effectiveness for the treatment of BRCA1/2-mutated, also referred as homologous recombination (HR)-deficient, tumors (HRD). Over the years, mechanistic studies gained insights on effectors acting downstream of PARP1, lagging behind the understanding of earlier events upstream - and thus independent - of PARP1. Here, we investigated the role of nuclear NAD+, an essential cofactor for the activity of key DNA repair proteins, including PARP1 and sirtuins. We show that NMNAT1- the enzyme synthesizing nuclear NAD+ - is synthetically lethal with BRCA1/2 in a PARP1-independent but SIRT6-dependent manner. Consequently, inhibition of NMNAT1/SIRT6 axis not only kills naive but also PARPi-resistant HRD cancer cells. Our results unravel a unique vulnerability of HRD tumors, therapeutically exploitable even upon PARPi resistance development.

Nucleotide excision repair leaves a mark on chromatin: DNA damage detection in nucleosomes

Global genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair proteins to access DNA lesions buried in nucleosomal DNA. The DNA-damage sensors XPC and DDB2 recognize DNA lesions in nucleosomal DNA and initiate repair. The emerging view is that a tight interplay between XPC and DDB2 is regulated by post-translational modifications on the damage sensors themselves as well as on chromatin containing DNA lesions. The choreography between XPC and DDB2, their interconnection with post-translational modifications such as ubiquitylation, SUMOylation, methylation, poly(ADP-ribos)ylation, acetylation, and the functional links with chromatin remodelling activities regulate not only the initial recognition of DNA lesions in nucleosomes, but also the downstream recruitment and necessary displacement of GG-NER factors as repair progresses. In this review, we highlight how nucleotide excision repair leaves a mark on chromatin to enable DNA damage detection in nucleosomes.

SLX4 dampens MutSα-dependent mismatch repair

ABSTRACTThe tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSβ (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSβ and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.

Beyond Base Excision Repair: An evolving picture of mitochondrial DNA repair

Mitochondria are highly specialised organelles required for cellular processes including ATP-production through cellular respiration and controlling apoptosis. Mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function – deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular aging and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear encoded DNA repair proteins that are translocated into the mitochondria. Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.

Beyond the Double-Strand Breaks: The Role of DNA Repair Proteins in Cancer Stem-Cell Regulation

Cancer stem cells (CSCs) are pluripotent and highly tumorigenic cells that can re-populate a tumor and cause relapses even after initially successful therapy. As with tissue stem cells, CSCs possess enhanced DNA repair mechanisms. An active DNA damage response alleviates the increased oxidative and replicative stress and leads to therapy resistance. On the other hand, mutations in DNA repair genes cause genomic instability, therefore driving tumor evolution and developing highly aggressive CSC phenotypes. However, the role of DNA repair proteins in CSCs extends beyond the level of DNA damage. In recent years, more and more studies have reported the unexpected role of DNA repair proteins in the regulation of transcription, CSC signaling pathways, intracellular levels of reactive oxygen species (ROS), and epithelial–mesenchymal transition (EMT). Moreover, DNA damage signaling plays an essential role in the immune response towards tumor cells. Due to its high importance for the CSC phenotype and treatment resistance, the DNA damage response is a promising target for individualized therapies. Furthermore, understanding the dependence of CSC on DNA repair pathways can be therapeutically exploited to induce synthetic lethality and sensitize CSCs to anti-cancer therapies. This review discusses the different roles of DNA repair proteins in CSC maintenance and their potential as therapeutic targets.

DNArepairK: An Interactive Database for Exploring the Impact of Anticancer Drugs onto the Dynamics of DNA Repair Proteins

Cells are constantly exposed to numerous mutagens that produce diverse types of DNA lesions. Eukaryotic cells have evolved an impressive array of DNA repair mechanisms that are able to detect and repair these lesions, thus preventing genomic instability. The DNA repair process is subjected to precise spatiotemporal coordination, and repair proteins are recruited to lesions in an orderly fashion, depending on their function. Here, we present DNArepairK, a unique open-access database that contains the kinetics of recruitment and removal of 70 fluorescently tagged DNA repair proteins to complex DNA damage sites in living HeLa Kyoto cells. An interactive graphical representation of the data complemented with live cell imaging movies facilitates straightforward comparisons between the dynamics of proteins contributing to different DNA repair pathways. Notably, most of the proteins included in DNArepairK are represented by their kinetics in both nontreated and PARP1/2 inhibitor-treated (talazoparib) cells, thereby providing an unprecedented overview of the effects of anticancer drugs on the regular dynamics of the DNA damage response. We believe that the exclusive dataset available in DNArepairK will be of value to scientists exploring the DNA damage response but, also, to inform and guide the development and evaluation of novel DNA repair-targeting anticancer drugs.

H3K36me3 and PSIP1/LEDGF associate with several DNA repair proteins, suggesting their role in efficient DNA repair at actively transcribing loci

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.