Inspiring basic and applied research in genome integrity mechanisms: Dedication to Samuel H. Wilson.

This Special Issue (SI) of Environmental and Molecular Mutagenesis (EMM), entitled "Inspiring Basic and Applied Research in Genome Integrity Mechanisms," is to update the community on recent findings and advances on genome integrity mechanisms with emphasis on their importance for basic and environmental health sciences. This SI includes two research articles, one brief research communication, and four reviews that highlight cutting edge research findings and perspectives, from both established leaders and junior trainees, on DNA repair mechanisms. In particular, the authors provided an updated understanding on several distinct enzymes (e.g., DNA polymerase beta, DNA polymerase theta, DNA glycosylase NEIL2) and the associated molecular mechanisms in base excision repair, nucleotide excision repair, and microhomology-mediated end joining of double-strand breaks. In addition, genome-wide sequencing analysis or site-specific mutational signature analysis of DNA lesions from environmental mutagens (e.g., UV light and aflatoxin) provide further characterization and sequence context impact of DNA damage and mutations. This SI is dedicated to the legacy of Dr. Samuel H. Wilson from the U.S. National Institute of Environmental Health Sciences at the National Institutes of Health.

Sequential requirements for distinct Polθ domains during theta-mediated end joining.

DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.

In Silico Design of gRNA for CRISPR/Cas9-Mediated Gene Knockout.

CRISPR/Cas9 stands as a revolutionary and versatile gene editing technology. At its core, the Cas9 DNA endonuclease is guided with precision by a specifically designed single-guide RNA (gRNA). This guidance system facilitates the introduction of double-stranded breaks (DSBs) within the DNA. Subsequent imprecise repairs, mainly through the non-homologous end-joining (NHEJ) pathway, yield insertions or deletions, resulting in frameshift mutations. These mutations are instrumental in achieving the successful knockout of the target gene. In this chapter, we describe all necessary steps to create and design a gRNA for a gene knockout to a target gene before to transfer it to a target plant.

CRISPR/Cas9 Vector Construction for Gene Knockout.

This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352 bp) and scaffold (83 bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide. The three gRNA expression fragments are then loosely assembled in a ligation-free cloning reaction and used as a template for an additional PCR step to amplify a single gRNA expression construct, ready for assembly into the transformation vector. The benefits of this design include cost efficiency, as subsequent cloning reactions only require 59 oligonucleotides and standard cloning reagents. Researchers engaged in CRISPR/Cas9-mediated genome editing in plants will find this protocol a clear and resource-efficient approach to create transformation plasmids for their experiments.

Precision Genome Editing with CRISPR-Cas9.

The CRISPR/Cas9 system is a revolutionary technology for genome editing that allows for precise and efficient modifications of DNA sequences. The system is composed of two main components, the Cas9 enzyme and a guide RNA (gRNA). The gRNA is designed to specifically target a desired DNA sequence, while the Cas9 enzyme acts as molecular scissors to cut the DNA at that specific location. The cell then repairs the digested DNA, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in either indels or precise modifications of DNA sequences with broad implications in biotechnology, agriculture, and medicine. This chapter provides a practical approach for utilizing CRISPR/Cas9 in precise genome editing, including identifying the target gene sequence, designing gRNA and protein (Cas9), and delivering the CRISPR components to target cells.

Modulating DNA damage response in uveal melanoma through embryonic stem cell microenvironment.

BACKGROUND: Uveal melanoma (UVM) is the most common primary intraocular tumor in adults, with a median survival of 4-5 months following metastasis. DNA damage response (DDR) upregulation in UVM, which could be linked to its frequent activation of the PI3K/AKT pathway, contributes to its treatment resistance. We have reported that embryonic stem cell microenvironments (ESCMe) can revert cancer cells to less aggressive states through downregulation of the PI3K signaling, showing promise in modulating the DDR of UVM. METHODS: Since nonhomologous end joining (NHEJ) is the main DNA repair mechanism in UVM, this study utilized gene expression analysis and survival prognosis analysis to investigate the role of NHEJ-related genes in UVM based on public databases. Xenograft mouse models were established to assess the therapeutic potential of ESC transplantation and exposure to ESC-conditioned medium (ESC-CM) on key DNA repair pathways in UVM. Quantitative PCR and immunohistochemistry were used to analyze NHEJ pathway-related gene expression in UVM and surrounding normal tissues. Apoptosis in UVM tissues was evaluated using the TUNEL assay. RESULTS: PRKDC, KU70, XRCC5, LIG4 and PARP1 showed significant correlations with UM progression. High expression of PRKDC and XRCC5 predicted poorer overall survival, while low PARP1 and XRCC6 expression predicted better disease-free survival in UVM patients. ESCMe treatment significantly inhibited the NHEJ pathway transcriptionally and translationally and promoted apoptosis in tumor tissues in mice bearing UVM. Furthermore, ESC transplantation enhanced DDR activities in surrounding normal cells, potentially mitigating the side effects of cancer therapy. Notably, direct cell-to-cell contact with ESCs was more effective than their secreted factors in regulating the NHEJ pathway. CONCLUSIONS: Our results suggest that NHEJ-related genes might serve as prognostic markers and therapeutic targets in UVM. These findings support the therapeutic potential of ESC-based therapy in enhancing UVM sensitivity to radiochemotherapy and improving treatment outcomes while minimizing damage to healthy cells.

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient's death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.

Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair.

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.

Developmental progression of DNA double-strand break repair deciphered by a single-allele resolution mutation classifier.

DNA double-strand breaks (DSBs) are repaired by a hierarchically regulated network of pathways. Factors influencing the choice of particular repair pathways, however remain poorly characterized. Here we develop an Integrated Classification Pipeline (ICP) to decompose and categorize CRISPR/Cas9 generated mutations on genomic target sites in complex multicellular insects. The ICP outputs graphic rank ordered classifications of mutant alleles to visualize discriminating DSB repair fingerprints generated from different target sites and alternative inheritance patterns of CRISPR components. We uncover highly reproducible lineage-specific mutation fingerprints in individual organisms and a developmental progression wherein Microhomology-Mediated End-Joining (MMEJ) or Insertion events predominate during early rapid mitotic cell cycles, switching to distinct subsets of Non-Homologous End-Joining (NHEJ) alleles, and then to Homology-Directed Repair (HDR)-based gene conversion. These repair signatures enable marker-free tracking of specific mutations in dynamic populations, including NHEJ and HDR events within the same samples, for in-depth analysis of diverse gene editing events.

DNA polymerase λ Loop1 variant yields unexpected gain-of-function capabilities in nonhomologous end-joining.

DNA polymerases lambda (Polλ) and mu (Polµ) are X-Family polymerases that participate in DNA double-strand break (DSB) repair by the nonhomologous end-joining pathway (NHEJ). Both polymerases direct synthesis from one DSB end, using template derived from a second DSB end. In this way, they promote the NHEJ ligation step and minimize the sequence loss normally associated with this pathway. The two polymerases differ in cognate substrate, as Polλ is preferred when synthesis must be primed from a base-paired DSB end, while Polµ is required when synthesis must be primed from an unpaired DSB end. We generated a Polλ variant (PolλKGET) that retained canonical Polλ activity on a paired end-albeit with reduced incorporation fidelity. We recently discovered that the variant had unexpectedly acquired the activity previously unique to Polµ-synthesis from an unpaired primer terminus. Though the sidechains of the Loop1 region make no contact with the DNA substrate, PolλKGET Loop1 amino acid sequence is surprisingly essential for its unique activity during NHEJ. Taken together, these results underscore that the Loop1 region plays distinct roles in different Family X polymerases.

DNA Double Strand Break and Response Fluorescent Assays: Choices and Interpretation.

Accurately characterizing DNA double-stranded breaks (DSBs) and understanding the DNA damage response (DDR) is crucial for assessing cellular genotoxicity, maintaining genomic integrity, and advancing gene editing technologies. Immunofluorescence-based techniques have proven to be invaluable for quantifying and visualizing DSB repair, providing valuable insights into cellular repair processes. However, the selection of appropriate markers for analysis can be challenging due to the intricate nature of DSB repair mechanisms, often leading to ambiguous interpretations. This comprehensively summarizes the significance of immunofluorescence-based techniques, with their capacity for spatiotemporal visualization, in elucidating complex DDR processes. By evaluating the strengths and limitations of different markers, we identify where they are most relevant chronologically from DSB detection to repair, better contextualizing what each assay represents at a molecular level. This is valuable for identifying biases associated with each assay and facilitates accurate data interpretation. This review aims to improve the precision of DSB quantification, deepen the understanding of DDR processes, assay biases, and pathway choices, and provide practical guidance on marker selection. Each assay offers a unique perspective of the underlying processes, underscoring the need to select markers that are best suited to specific research objectives.

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.

Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.

The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK.

BRCA1-associated protein 1 (BAP1) has emerged as a major tumor suppressor gene in diverse cancer types, notably in malignant pleural mesothelioma (DPM), and has also been identified as a germline cancer predisposition gene for DPM and other select cancers. However, its role in the response to DNA damage has remained unclear. Here, we show that BAP1 inactivation is associated with increased DNA damage both in Met-5A human mesothelial cells and human DPM cell lines. Through proteomic analyses, we identified PRKDC as an interaction partner of BAP1 protein complexes in DPM cells and 293 T human embryonic kidney cells. PRKDC encodes the catalytic subunit of DNA protein kinase (DNA-PKcs) which functions in the nonhomologous end-joining (NHEJ) pathway of DNA repair. Double-stranded DNA damage resulted in prominent nuclear expression of BAP1 in DPM cells and phosphorylation of BAP1 at serine 395. A plasmid-based NHEJ assay confirmed a significant effect of BAP1 knockdown on cellular NHEJ activity. Combination treatment with X-ray irradiation and gemcitabine (as a radiosensitizer) strongly suppressed the growth of BAP1-deficient cells. Our results suggest reciprocal positive interactions between BAP1 and DNA-PKcs, based on phosphorylation of BAP1 by the latter and deubiquitination of DNA-PKcs by BAP1. Thus, functional interaction of BAP1 with DNA-PKcs supports a role for BAP1 in NHEJ DNA repair and may provide the basis for new therapeutic strategies and new insights into its role as a tumor suppressor.

A di-acetyl-decorated chromatin signature couples liquid condensation to suppress DNA end synapsis.

Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.

The role of RNF138 in DNA end resection is regulated by ubiquitylation and CDK phosphorylation.

Double-strand breaks (DSBs) are DNA lesions that pose a significant threat to genomic stability. The repair of DSBs by the homologous recombination (HR) pathway is preceded by DNA end resection, the 5' to 3' nucleolytic degradation of DNA away from the DSB. We and others previously identified a role for RNF138, a really interesting new gene finger E3 ubiquitin ligase, in stimulating DNA end resection and HR. Yet, little is known about how RNF138's function is regulated in the context of DSB repair. Here, we show that RNF138 is phosphorylated at residue T27 by cyclin-dependent kinase (CDK) activity during the S and G2 phases of the cell cycle. We also observe that RNF138 is ubiquitylated constitutively, with ubiquitylation occurring in part on residue K158 and rising during the S/G2 phases. Interestingly, RNF138 ubiquitylation decreases upon genotoxic stress. By mutating RNF138 at residues T27, K158, and the previously identified S124 ataxia telangiectasia mutated phosphorylation site (Han et al., 2016, ref. 22), we find that post-translational modifications at all three positions mediate DSB repair. Cells expressing the T27A, K158R, and S124A variants of RNF138 are impaired in DNA end resection, HR activity, and are more sensitive to ionizing radiation compared to those expressing wildtype RNF138. Our findings shed more light on how RNF138 activity is controlled by the cell during HR.

Loss of Dna2 fidelity results in decreased Exo1-mediated resection at DNA double-strand breaks.

A DNA double-strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination or nonhomologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of homologous recombination, and it is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harboring nuclease-dead dna2-1 compared with dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harboring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater resection defect compared with cells where DNA2 was deleted. Both the resection defect and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change; however, Nej1 increased as did the frequency of alt-end joining/microhomology-mediated end-joining repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.

New Discoveries on Protein Recruitment and Regulation during the Early Stages of the DNA Damage Response Pathways.

Maintaining genomic stability and properly repairing damaged DNA is essential to staying healthy and preserving cellular homeostasis. The five major pathways involved in repairing eukaryotic DNA include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), and homologous recombination (HR). When these pathways do not properly repair damaged DNA, genomic stability is compromised and can contribute to diseases such as cancer. It is essential that the causes of DNA damage and the consequent repair pathways are fully understood, yet the initial recruitment and regulation of DNA damage response proteins remains unclear. In this review, the causes of DNA damage, the various mechanisms of DNA damage repair, and the current research regarding the early steps of each major pathway were investigated.

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells using GFP Based Reporter Systems.

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining.

Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.