Genome Repair Research Articles

PB1781: THERAPEUTIC POTENTIAL OF DNA DAMAGE RESPONSE IN ACUTE MYELOBLASTIC LEUKEMIA

Background: Genome integrity is of utmost importance for cell survival and homeostasis maintenance. However, DNA integrity is threatened by endogenous and exogenous sources. DNA lesions can block genome replication and transcription, and if unrepaired or repaired incorrectly can lead to mutations and genomic instability. Hence, cells have developed a variety of mechanisms to protect genome integrity and repair damaged DNA, collectively termed the DNA damage response (DDR). These mechanisms comprise DNA damage recognition, damage tolerance, signal transduction, transcriptional regulation, cell cycle control, and DNA repair. Key DDR elements include sensor proteins that detect damage (e.g. PARP1 and 2), mediators and signaling transducers (e.g. DNA-PK) that regulate downstream targets, and effector molecules (e.g. CHK1 and 2). Increased DNA damage and altered DDR are critical features of genetic instability presumably implicated in the pathogenesis of acute myeloblastic leukemia (AML). AML is a clonal malignant disease of hematopoietic stem and/or progenitor cells characterized by a block in myeloid differentiation and increased proliferation. This neoplasia is considered the most common acute leukemia in adults and is characterized by recurrent genetic abnormalities. Recently, specific recurrent chromosomal translocations and genetic mutations typical of AML have been associated with DNA repair defects. Aims: Our goal is to assess the potential of DDR as a therapeutic target in AML, using in vitro models, in order to identify new therapeutic approaches. Methods: Five AML cell lines of different molecular and cellular subtypes were used (HEL, HL-60, K-562, LAMA-84, and NB-4). Cells were incubated in the absence and presence of increasing concentrations of two DDR inhibitors, CCT245737 (CHK1 inhibitor) and Niraparib (PARP1/2 inhibitor). Cell density and viability were assessed, for 72 hours, by trypan blue assay. Afterward, resorting to flow cytometry, the cell cycle was assessed using propidium iodide/RNAse, and the cell death was determined using Annexin V/7-AAD double staining as well as by optic microscopy (May-Grünwald-Giemsa staining). Results were statistical analyzed, considering a significance level of 95%. Results: CCT245737 reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. NB-4 was the most sensitive cell line, with an IC50 at 48 hours of 54 µM. In contrast, LAMA-84 was the most resistant cell line with an IC50 at 48 hours of 203 µM, 3.7-fold higher than NB-4. PARP1/2 inhibition also reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. Once again NB-4 was the most sensitive cell line, with an IC50 at 48 hours of 25 µM and LAMA-84 was the most resistant with an IC50 at 48 hours of 66 µM, 2.7-fold higher than NB-4. Both inhibitors induced cell death by apoptosis since an increase in the percentage of cells in late apoptosis was observed. This result is in agreement with the observed by optic microscopy. We also detected a cytostatic effect since a cell cycle arrest in the S phase was observed after treatment with both inhibitors. Summary/Conclusion: AML cell lines have different sensibilities to DDR inhibitors, particularly CCT245737 and niraparib, with the NB-4 cell line appearing to be the most sensitive to DDR inhibition (namely CHK1 and PARP1/2 inhibition), whereas LAMA-84 appears to be the most resistant to this inhibition. In conclusion, this work may help to identify new therapeutic approaches that could eventually improve AML patients’ outcomes.

RecQ Helicase Somatic Alterations in Cancer.

Named the “caretakers” of the genome, RecQ helicases function in several pathways to maintain genomic stability and repair DNA. This highly conserved family of enzymes consist of five different proteins in humans: RECQL1, BLM, WRN, RECQL4, and RECQL5. Biallelic germline mutations in BLM, WRN, and RECQL4 have been linked to rare cancer-predisposing syndromes. Emerging research has also implicated somatic alterations in RecQ helicases in a variety of cancers, including hematological malignancies, breast cancer, osteosarcoma, amongst others. These alterations in RecQ helicases, particularly overexpression, may lead to increased resistance of cancer cells to conventional chemotherapy. Downregulation of these proteins may allow for increased sensitivity to chemotherapy, and, therefore, may be important therapeutic targets. Here we provide a comprehensive review of our current understanding of the role of RecQ DNA helicases in cancer and discuss the potential therapeutic opportunities in targeting these helicases.

Abstract 5484: Preclinical evaluation of CAP-0121 for multidrug resistant cancers

Abstract CAP-0121 is a second generation illudofulvene agent that produces an unusual type of DNA damage and is being developed to treat ovarian cancer by Califia Pharma. CAP-0121 DNA damage, like other illudofulvenes, can only be recognized and repaired by the Transcription-Coupled Repair pathway, and is ignored by the Global Genome Repair Pathway. The first generation illudofulvene, irofulven, displayed significant activity in clinical trials against ovarian, prostate and hepatocellular tumors. While irofulven has proven to be a potent antitumor agent against drug-resistant tumors, its efficacy is limited by hematologic toxicity that reduced the maximum dosage that can be administered to patients. CAP-0121 is as cytotoxic, or even more cytotoxic, than irofulven when tested in the NCI 60 cell line panel. Despite this increased cytotoxicity noted in vitro, CAP-0121 is less toxic to animals and the maximum tolerated dose is approximately 3 times higher than irofulven (mg/kg basis). CAP-0121 also retains activity against multidrug resistant cell lines including MRP1 and MDR1 expressing variants. As a result CAP-0121 is capable of inducing tumor remission in MDR xenograft models that are unresponsive to 35 conventional and experimental agents, and in which irofulven is only capable of slowing the rate of tumor growth. This response of CAP-0121 in drug resistant xenografts is observed at nontoxic doses. Synergistic action is observed when CAP-0121 is administered in combination with traditional drugs used for treating ovarian cancer, including taxanes, platinum agents, and PARP inhibitors. Citation Format: Michael J. Kelner, Venkata R. Kotamraju. Preclinical evaluation of CAP-0121 for multidrug resistant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5484.

Same modification, different location: the mythical role of N6-adenine methylation in plant genomes.

The present review summarizes recent advances in the understanding of 6mA in DNA as an emergent epigenetic mark with distinctive characteristics, discusses its importance in plant genomes, and highlights its chemical nature and functions. Adenine methylation is an epigenetic modification present in DNA (6mA) and RNA (m6A) that has a regulatory function in many cellular processes. This modification occurs through a reversible reaction that covalently binds a methyl group, usually at the N6 position of the purine ring. This modification carries biophysical properties that affect the stability of nucleic acids as well as their binding affinity with other molecules. DNA 6mA has been related to genome stability, gene expression, DNA replication, and repair mechanisms. Recent advances have shown that 6mA in plant genomes is related to development and stress response. In this review, we present recent advances in the understanding of 6mA in DNA as an emergent epigenetic mark with distinctive characteristics. We discuss the key elements of this modification, focusing mainly on its importance in plant genomes. Furthermore, we highlight its chemical nature and the regulatory effects that it exerts on gene expression and plant development. Finally, we emphasize the functions of 6mA in photosynthesis, stress, and flowering.

Implications of DNA Polymerase Gamma in the Repair of the Mitochondrial Genome

Mitochondrial DNA (mtDNA) encodes thirteen essential proteins of the oxidative phosphorylation system, responsible for the major production of ATP in the cell. Therefore, damages to the mitochondrial genome result in energy deprivation, which may in turn onset human diseases. Notably, due to its proximity to the electron transport chain, mtDNA remains exposed to damage by reactive oxygen species, thus the maintenance of its integrity requires a robust repair system. Until recently, DNA polymerase gamma (Pol γ) has been the only polymerase identified in mitochondria, bearing responsibility for efficient replication as well as post‐replication repair of the genome. We have previously suggested that the division of the roles of Pol γ may be controlled by the association of its catalytic subunit, Pol γA, with the accessory subunit Pol γB, such that the holoenzyme is engaged in the processive mtDNA replication, whereas, alone, Pol γA may serve the repair processes. Recently, the major repair polymerase of the nucleus, Pol β, has been discovered to also localize in mitochondria, which raises the question for its competition or cooperation with Pol γ in the mtDNA repair processes. To address this, we have tested in vitro the efficiency of DNA synthesis by the two polymerases, separately and in combination, using various DNA substrates. In agreement with previous reports, we did not observe any indication of a functional interaction between the Pol γ holoenzyme and Pol β. We did, however, observe a cooperative activity of Pol β with the Pol γA subunit. In conclusion, our results suggest that the repair of mtDNA may entail a synergistic activity of the catalytic subunit of Pol γ and Pol β.

Nucleotide excision repair: a versatile and smart toolkit.

Nucleotide excision repair (NER) is a major pathway to deal with bulky adducts induced by various environmental toxins in all cellular organisms. The two sub-pathways of NER, global genome repair (GGR) and transcription-coupled repair (TCR), differ in the damage recognition modes. In this review, we describe the molecular mechanism of NER in mammalian cells, especially the details of damage recognition steps in both sub-pathways. We also introduce new sequencing methods for genome-wide mapping of NER, as well as recent advances about NER in chromatin by these methods. Finally, the roles of NER factors in repairing oxidative damages and resolving R-loops are discussed.

Transcription of ncRNAs promotes repair of UV induced DNA lesions in Saccharomyces cerevisiae subtelomeres.

Ultraviolet light causes DNA lesions that are removed by nucleotide excision repair (NER). The efficiency of NER is conditional to transcription and chromatin structure. UV induced photoproducts are repaired faster in the gene transcribed strands than in the non-transcribed strands or in transcriptionally inactive regions of the genome. This specificity of NER is known as transcription-coupled repair (TCR). The discovery of pervasive non-coding RNA transcription (ncRNA) advocates for ubiquitous contribution of TCR to the repair of UV photoproducts, beyond the repair of active gene-transcribed strands. Chromatin rules transcription, and telomeres form a complex structure of proteins that silences nearby engineered ectopic genes. The essential protective function of telomeres also includes preventing unwanted repair of double-strand breaks. Thus, telomeres were thought to be transcriptionally inert, but more recently, ncRNA transcription was found to initiate in subtelomeric regions. On the other hand, induced DNA lesions like the UV photoproducts must be recognized and repaired also at the ends of chromosomes. In this study, repair of UV induced DNA lesions was analyzed in the subtelomeric regions of budding yeast. The T4-endonuclease V nicking-activity at cyclobutene pyrimidine dimer (CPD) sites was exploited to monitor CPD formation and repair. The presence of two photoproducts, CPDs and pyrimidine (6,4)-pyrimidones (6-4PPs), was verified by the effective and precise blockage of Taq DNA polymerase at these sites. The results indicate that UV photoproducts in silenced heterochromatin are slowly repaired, but that ncRNA transcription enhances NER throughout one subtelomeric element, called Y', and in distinct short segments of the second, more conserved element, called X. Therefore, ncRNA-transcription dependent TCR assists global genome repair to remove CPDs and 6-4PPs from subtelomeric DNA.

X-ray crystal structure of the Escherichia coli RadD DNA repair protein bound to ADP reveals a novel zinc ribbon domain.

Genome maintenance is an essential process in all cells. In prokaryotes, the RadD protein is important for survival under conditions that include DNA-damaging radiation. Precisely how RadD participates in genome maintenance remains unclear. Here we present a high-resolution X-ray crystal structure of ADP-bound Escherichia coli RadD, revealing a zinc-ribbon element that was not modelled in a previous RadD crystal structure. Insights into the mode of nucleotide binding and additional structure refinement afforded by the new RadD model will help to drive investigations into the activity of RadD as a genome stability and repair factor.

Genotoxicity-Suppressing Effect of Sophora japonica L. Aqueous Extract

The dry flower buds of Sophora japonica L. are used as a hemostatic agent in traditional Chinese medicine. In the comet assay, aqueous extracts of S. japonica decreased and increased the tail length significantly in cultured human lymphoblastoid WTK1 cells exposed to UV in the absence and presence of DNA repair inhibitors, respectively. The extract did not affect the tail length in methyl methanesulfonate-exposed cells. In the present study, the aqueous extract of the flower buds of S. japonica was separated by repeated column chromatography, yielding four types of flavonoid glycosides. Among them, only rutin, similar to the extract, decreased and increased the tail length significantly in WTK1 cells exposed to UV in the absence and presence of DNA repair inhibitors hydroxyurea (10 mM) and cytosine-1-β-D-arabinofuranoside (1.8 mM), respectively. The genotoxicity-suppressing effect of rutin was further studied using the micronucleus test. Rutin significantly decreased the frequency of micronucleated binucleate cells in UV-exposed WTK1 cells but did not affect this frequency in UV-exposed XPL3KA (Xeroderma pigmentosum group C) cells. These results suggest that the anti-genotoxic potential of rutin is due to an enhanced incision step of global genome repair (GGR) sub-pathways in nucleotide excision repair (NER). Herein, we show that S. japonica exhibits heretofore unknown anti-genotoxic potential against UV by enhancing the incision of GGR sub-pathways in NER, and that its anti-genotoxic component is rutin.

UV Irradiation of Vaccinia Virus-Infected Cells Impairs Cellular Functions, Introduces Lesions into the Viral Genome, and Uncovers Repair Capabilities for the Viral Replication Machinery.

Vaccinia virus (VV), the prototypic poxvirus, encodes a repertoire of proteins responsible for the metabolism of its large dsDNA genome. Previous work has furthered our understanding of how poxviruses replicate and recombine their genomes, but little is known about whether the poxvirus genome undergoes DNA repair. Our studies here are aimed at understanding how VV responds to exogenous DNA damage introduced by UV irradiation. Irradiation of cells prior to infection decreased protein synthesis and led to an ∼12-fold reduction in viral yield. On top of these cell-specific insults, irradiation of VV infections at 4 h postinfection (hpi) introduced both cyclobutene pyrimidine dimer (CPD) and 6,4-photoproduct (6,4-PP) lesions into the viral genome led to a nearly complete halt to further DNA synthesis and to a further reduction in viral yield (∼35-fold). DNA lesions persisted throughout infection and were indeed present in the genomes encapsidated into nascent virions. Depletion of several cellular proteins that mediate nucleotide excision repair (XP-A, -F, and -G) did not render viral infections hypersensitive to UV. We next investigated whether viral proteins were involved in combatting DNA damage. Infections performed with a virus lacking the A50 DNA ligase were moderately hypersensitive to UV irradiation (∼3-fold). More strikingly, when the DNA polymerase inhibitor cytosine arabinoside (araC) was added to wild-type infections at the time of UV irradiation (4 hpi), an even greater hypersensitivity to UV irradiation was seen (∼11-fold). Virions produced under the latter condition contained elevated levels of CPD adducts, strongly suggesting that the viral polymerase contributes to the repair of UV lesions introduced into the viral genome. IMPORTANCE Poxviruses remain of significant interest because of their continuing clinical relevance, their utility for the development of vaccines and oncolytic therapies, and their illustration of fundamental principles of viral replication and virus/cell interactions. These viruses are unique in that they replicate exclusively in the cytoplasm of infected mammalian cells, providing novel challenges for DNA viruses. How poxviruses replicate, recombine, and possibly repair their genomes is still only partially understood. Using UV irradiation as a form of exogenous DNA damage, we have examined how vaccinia virus metabolizes its genome following insult. We show that even UV irradiation of cells prior to infection diminishes viral yield, while UV irradiation during infection damages the genome, causes a halt in DNA accumulation, and reduces the viral yield more severely. Furthermore, we show that viral proteins, but not the cellular machinery, contribute to a partial repair of the viral genome following UV irradiation.

Модели митохондриальной дисфункции с индуцируемой экспрессией мутантного варианта гена Polg

Mitochondrial dysfunctions, which underlie many systemic diseases in animals and humans, may arise from accumulation of mutations in the mitochondrial genome. PolG-alpha enzyme encoded by Polg gene is crucial for replication and repair of the mitochondrial genome. The aim of this study was to assess the possible role of Polg mutations in mitochondrial dysfunctions using in vitro and in vivo animal models. The experiments involved transgenic mice with inducible expression of Polg mutant variant; the methods included cell culture, real time PCR assay, fluorescence flow cytometry, and skeletal muscle functional tests. The results indicate that mouse embryonic fibroblasts (MEFs) expressing Polg pathogenic mutant variant have decreased mitochondrial membrane potential and increased expression of mitophagy markers compared with control cultures. Transgenic animals with systemic expression of the pathogenic variant develop mitochondrial dysfunction which significantly affects muscular performance. In addition, systemic expression of mutated Polg in transgenic animals significantly inhibits expression of TCR subunit α and CD3 coreceptor complex subunits δ and ε in total splenocyte populations and significantly affects cellularity of the thymus without altering its CD4/CD8 subpopulation ratio. Thus, inducible expression of mutated Polg in transgenic animals provides a relevant model for studying mitochondrial dysfunction and its treatment in vitro and in vivo.

Pervasive Transcription-coupled DNA repair in E. coli

Global Genomic Repair (GGR) and Transcription-Coupled Repair (TCR) have been viewed, respectively, as major and minor sub-pathways of the nucleotide excision repair (NER) process that removes bulky lesions from the genome. Here we applied a next generation sequencing assay, CPD-seq, in E. coli to measure the levels of cyclobutane pyrimidine dimer (CPD) lesions before, during, and after UV-induced genotoxic stress, and, therefore, to determine the rate of genomic recovery by NER at a single nucleotide resolution. We find that active transcription is necessary for the repair of not only the template strand (TS), but also the non-template strand (NTS), and that the bulk of TCR is independent of Mfd – a DNA translocase that is thought to be necessary and sufficient for TCR in bacteria. We further show that repair of both TS and NTS is enhanced by increased readthrough past Rho-dependent terminators. We demonstrate that UV-induced genotoxic stress promotes global antitermination so that TCR is more accessible to the antisense, intergenic, and other low transcribed regions. Overall, our data suggest that GGR and TCR are essentially the same process required for complete repair of the bacterial genome.

Crucial role and mechanism of transcription-coupled DNA repair in bacteria.

Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd-a DNA translocase of a marginal NER phenotype1-3. Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo. We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD-RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.

Removal of an Internal Translational Start Site from mRNA While Retaining Expression of the Full-Length Protein.

The CRISPR-Cas9 gene-editing system, based on genome repair mechanisms, enables the generation of gene-modified mouse models more quickly and easily relative to traditional homologous recombination. The CRISPR-Cas9 system is particularly attractive when a single-point mutation is desired. The gap junction protein, Connexin 43 (Cx43), is encoded by gene Gja1, which has a single coding exon and cannot be spliced. However, Gja1 produces not only full-length Cx43 protein but up to six N-terminus truncated isoforms by a process known as internal translation, the result of ribosomal translation initiation at internal AUG (Methionine) start sites. GJA1-20k is the most commonly generated truncated isoform of Cx43 initiated at the AUG codon at position 213 of Gja1 mRNA. Because residue 213 occurs at the end of the last transmembrane domain of Cx43, GJA1-20k is effectively the 20 kDa C-terminus tail of Cx43 as an independent protein. Previous investigators identified, in cells, that a critical role of GJA1-20k is to facilitate trafficking of full-length Cx43 gap junction hemichannels to the plasma membrane. To examine this phenomenon in vivo, a mutant mouse with a Gja1 point-mutation was generated that replaces the ATG (Methionine) at residue 213 with TTA (Leucine, M213L mutation). The result of M213L is that Gja1 mRNA and full-length Cx43 are still generated, yet the translation of Gja1-20k is significantly reduced. This report focuses on choosing the restriction enzyme site to develop a one amino acid mutated (Gja1M213L/M213L) mouse model. This protocol describes genetically modified mice by the CRISPR-Cas9 system and rapid genotyping by combining PCR and restriction enzyme treatments.

Oligomeric XPD helicase exhibits enhanced unwinding activity by pushing in unstable regions of DNA

Helicases are a class of motor proteins that utilize ATP hydrolysis to translocate and unwind double-stranded DNA (dsDNA), and play key roles in genome maintenance processes. The mechanisms by which the unwinding activity of helicases is regulated remain poorly understood. XPD serves has a model for understanding a family of biomedically important helicases involved in genome repair. XPD as a monomer is an inefficient unwinder of dsDNA, able to unwind only short DNA duplexes due to difficulties melting stable (i.e.

The Mechanism and Prognostic Value of DNA Polymerase δ Subunits in Hepatocellular Carcinoma: Implications for Precision Therapy.

PurposePolymerase δ (POLD) proteins is a pivotal B-family DNA polymerase in the process of genome replication and repair and are comprised of POLD1-4. The predictive value of POLDs in hepatocellular carcinoma (HCC) has not been evaluated until now.Patients and MethodsA total of 369 hepatocellular carcinoma samples and 50 adjacent normal samples were enrolled from the TCGA-LIHC database, and the GSE10186 database was also used. Transcription, methylation and genetic alteration status of HCC patients were evaluated by GEPIA, Kaplan–Meier plotter, cBioPortal, MethHC, MethSurv. SurvExpress was employed to generate the overall prognosis prediction signature of POLDs. POLDs coexpressed genes were explored and enriched in potential pathways. K-M curves were generated to compare the different survival results in different groups, while ROC curves were used to validate the efficiency of the POLD signature.ResultsAll four POLD subunits were highly expressed in HCC tumor tissues. POLD1-3 and increased mRNA levels were also positively associated with advanced tumor stage and OS prognosis. Methylation in the promoter of POLDs affects mRNA expression and OS, especially for some specific CpG sites. Meanwhile, POLDs could preferably predict the prognosis for patients who suffered from a high gene mutation burden. We evaluated the combined prognostic predictive value of four POLD subunits in both the TCGA-LIHC and GSE10186 databases and recognized the statistically significant HR of the high-risk group, along with the reliable predictive value. The coexpressed gene sets and annotation results showed that the POLD coexpressed genes were mostly associated with DNA repair and cell cycle regulation pathways.ConclusionPOLD is an essential predictive factor for the prognosis of HCC. The united signature could precisely identify unfavorable clinical outcome of HCC.

Activated or Impaired: An Overview of DNA Repair in Neurodegenerative Diseases.

As the population ages, age-related neurodegenerative diseases have become a major challenge in health science. Currently, the pathology of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, is still not fully understood. Remarkably, emerging evidence indicates a role of genomic DNA damage and repair in various neurodegenerative disorders. Here, we summarized the current understanding of the function of DNA damage repair, especially base excision repair and double strand break repair pathways, in a variety of neurodegenerative diseases. We concluded that exacerbation of DNA lesions is found in almost all types of neurodegenerative diseases, whereas the activities of different DNA repair pathways demonstrate distinct trends, depending on disease type and even brain region. Specifically, key enzymes involved in base excision repair are likely impaired in Alzheimer's disease and amyotrophic lateral sclerosis but activated in Parkinson's disease, while nonhomologous end joining is likely downregulated in most types of neurodegenerative diseases. Hence, impairment of nonhomologous end joining is likely a common etiology for most neurodegenerative diseases, while defects in base excision repair are likely involved in the pathology of Alzheimer's disease and amyotrophic lateral sclerosis but are Parkinson's disease, based on current findings. Although there are still discrepancies and further studies are required to completely elucidate the exact roles of DNA repair in neurodegeneration, the current studies summarized here provide crucial insights into the pathology of neurodegenerative diseases and may reveal novel drug targets for corresponding neurodegenerative diseases.

Targeted Cancer Therapy Based on Acetylation and Deacetylation of Key Proteins Involved in Double-Strand Break Repair.

DNA double-strand breaks (DSBs) play an important role in promoting genomic instability and cell death. The precise repair of DSBs is essential for maintaining genome integrity during cancer progression, and inducing genomic instability or blocking DNA repair is an important mechanism through which chemo/radiotherapies exert killing effects on cancer cells. The two main pathways that facilitate the repair of DSBs in cancer cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). Accumulating data suggest that the acetylation and deacetylation of DSB repair proteins regulate the initiation and progression of the cellular response to DNA DSBs, which may further affect the chemosensitivity or radiosensitivity of cancer cells. Here, we focus on the role of acetylation/deacetylation in the regulation of ataxia-telangiectasia mutated, Rad51, and 53BP1 in the HR pathway, as well as the relevant roles of PARP1 and Ku70 in NHEJ. Notably, several histone deacetylase (HDAC) inhibitors targeting HR or NHEJ have been demonstrated to enhance chemo/radiosensitivity in preclinical studies. This review highlights the essential role of acetylation/deacetylation in the regulation of DSB repair proteins, suggesting that HDAC inhibitors targeting the HR or NHEJ pathways that downregulate DNA DSB repair genes may be worthwhile cancer therapeutic agents.

Cryptomphalus aspersa Eggs Extract Potentiates Human Epidermal Stem Cell Regeneration and Amplification

Modern life and extended life expectancy have prompted the search for natural compounds alleviating skin aging. Evidence supports the beneficial effects on skin integrity and health from the topical administration of preparations of the mollusc Cryptomphalus aspersa eggs extract (IFC-CAF®) and suggests these effects are partly derived from an impact on skin renewal and repair mechanisms. The objective was to dissect in vitro the specific impact of IFC-CAF® on different parameters related to the regenerative potential, differentiation phenotype and exhaustion of skin stem cells. A prominent impact of IFC-CAF® was the induction of stratification and differentiated phenotypes from skin stem cells. IFC-CAF® slowed down the cell cycle at the keratinocyte DNA repair phase and, decelerated proliferation. However, it preserved the proliferative potential of the stem cells. IFC-CAF® reduced the DNA damage marker, γH2AX, and induced the expression of the transcription factor p53. These features correlated with significant protection in telomere shortening upon replicative exhaustion. Thus, IFC-CAF® helps maintain orderly cell cycling and differentiation, thus potentiating DNA repair and integrity. Our observations support the regenerative and repair capacity of IFC-CAF® on skin, through the improved mobilization and ordered differentiation of keratinocyte precursors and the enhancement of genome surveillance and repair mechanisms that counteract aging.

A J Domain Protein Functions as a Histone Chaperone to Maintain Genome Integrity and the Response to DNA Damage in a Human Fungal Pathogen.

ABSTRACTHistone chaperoning ensures genomic integrity during routine processes such as DNA replication and transcription as well as DNA repair upon damage. Here, we identify a nuclear J domain protein, Dnj4, in the fungal pathogen Cryptococcus neoformans and demonstrate that it interacts with histones 3 and 4, suggesting a role as a histone chaperone. In support of this idea, a dnj4Δ deletion mutant had elevated levels of DNA damage and was hypersensitive to DNA-damaging agents. The transcriptional response to DNA damage was also impaired in the dnj4Δ mutant. Genes related to DNA damage and iron homeostasis were upregulated in the wild-type strain in response to hydroxyurea treatment; however, their upregulation was either absent from or reduced in the dnj4Δ mutant. Accordingly, excess iron rescued the mutant’s growth in response to DNA-damaging agents. Iron homeostasis is crucial for virulence in C. neoformans; however, Dnj4 was found to be dispensable for disease in a mouse model of cryptococcosis. Finally, we confirmed a conserved role for Dnj4 as a histone chaperone by expressing it in Saccharomyces cerevisiae and showing that it disrupted endogenous histone chaperoning. Altogether, this study highlights the importance of a JDP cochaperone in maintaining genome integrity in C. neoformans.