DNA Replication Process Research Articles

Immune Dysfunction in Mendelian Disorders of POLA1 Deficiency.

POLA1 encodes the catalytic unit of DNA polymerase α, which together with the Primase complex launches the DNA replication process. While complete deficiency of this essential gene is presumed to be lethal, at least two conditions due to partial POLA1 deficiency have been described. The first genetic syndrome to be mapped to POLA1 was X-linked reticulate pigmentary disorder (XLPDR, MIM #301220), a rare syndrome characterized by skin hyperpigmentation, sterile multiorgan inflammation, recurrent infections, and distinct facial features. XLPDR has been shown to be accompanied by profound activation of type I interferon signaling, but unlike other interferonopathies, it is not associated with autoantibodies or classical autoimmunity. Rather, it is accompanied by marked Natural Killer (NK) cell dysfunction, which may explain the recurrent infections seen in this syndrome. To date, all XLPDR cases are caused by the same recurrent intronic mutation, which results in gene missplicing. Several hypomorphic mutations in POLA1, distinct from the XLPDR intronic mutation, have been recently reported and these mutations associate with a separate condition, van Esch-O'Driscoll syndrome (VEODS, MIM #301030). This condition results in growth retardation, microcephaly, hypogonadism, and in some cases, overlapping immunological features to those seen in XLPDR. This review summarizes our current understanding of the clinical manifestations of POLA1 gene mutations with an emphasis on its immunological consequences, as well as recent advances in understanding of its pathophysiologic basis and potential therapeutic options.

黄河口潮间带沉积物细菌群落结构特征

选取黄河口潮间带有植被覆盖和无植被覆盖两个区域采集柱状沉积物，利用实时荧光定量PCR技术和高通量测序技术分析有无植被覆盖沉积物中的细菌群落特征和功能差异，探究影响潮间带细菌群落结构的主要环境因子。结果显示，细菌丰度在有植被区域大于无植被区域。沉积物中细菌群落丰富度和多样性在有植被区域随深度的增加而增加，而在无植被区域其最高值出现在沉积物中层（14-16 cm）。两个区域在门分类水平上以变形菌（Proteobacteria）、绿弯菌（Chloroflexi）、放线菌（Actinobacteria）和酸杆菌（Acidobacteria）为主；属分类水平上的优势类群为芽孢杆菌（Bacillus）、苍白杆菌（Ochrobactrum）、拟无枝菌酸菌（Amycolatopsis）和鞘脂单胞菌（Sphingomonas）等。相关性分析发现盐度和亚硝酸盐浓度对细菌群落多样性和丰富度影响显著。功能预测分析表明，有植被区域沉积物细菌在氨基酸代谢、膜运输和碳水化合物代谢方面功能活跃，而无植被区域细菌则在核酸复制和修复、能量代谢过程方面更为活跃。;In this study, the sediment cores were collected from two areas with or without vegetation covering in the intertidal zone of the Yellow River estuary, in which the characteristics and functional differences of bacterial community were analyzed based on real-time fluorescent quantitative PCR and high-throughput sequencing. The mainly environmental influencing factors were also explored. Results showed that the bacterial abundance from the vegetation area was higher than that from the vegetation-free area. The richness and diversity of bacterial community increased with the depth in the vegetation area, while they were the highest in the sample of middle layer (14-16 cm) in the vegetation-free area. The shared dominant phyla in both areas were Proteobacteria, Chloroflexi, Actinobacteria and Acidobacteria. The dominant genera were Bacillus, Ochrobactrum, Amycolatopsis, Sphingomonas and so on. The results of correlation analysis showed that salinity and nitrite concentration were the main influencing factors of the richness and diversity of bacterial community. The functional prediction analysis showed that the processes of bacterial membrane transport, amino acid metabolism and carbohydrate metabolism in the vegetation area were more active, while the processes of bacterial DNA replication, repairing and energy metabolism in the vegetation-free area were more active.

Unlocking the PIP-box: A peptide library reveals interactions that drive high-affinity binding to human PCNA

The human sliding clamp, Proliferating Cell Nuclear Antigen (hPCNA), interacts with over 200 proteins through a conserved binding motif, the PIP-box, to orchestrate DNA replication and repair. It is not clear how changes to the features of a PIP-box modulate protein binding and thus how they fine-tune downstream processes. Here, we present a systematic study of each position within the PIP-box to reveal how hPCNA-interacting peptides bind with drastically varied affinities. We synthesized a series of 27 peptides derived from the native protein p21 with small PIP-box modifications and another series of 19 peptides containing PIP-box binding motifs from other proteins. The hPCNA-binding affinity of all peptides, characterized as KD values determined by surface plasmon resonance, spanned a 4000-fold range, from 1.83 nM to 7.59 μM. The hPCNA-bound peptide structures determined by X-ray crystallography and modeled computationally revealed intermolecular and intramolecular interaction networks that correlate with high hPCNA affinity. These data informed rational design of three new PIP-box sequences, testing of which revealed the highest affinity hPCNA-binding partner to date, with a KD value of 1.12 nM, from a peptide with PIP-box QTRITEYF. This work showcases the sequence-specific nuances within the PIP-box that are responsible for high-affinity hPCNA binding, which underpins our understanding of how nature tunes hPCNA affinity to regulate DNA replication and repair processes. In addition, these insights will be useful to future design of hPCNA inhibitors.

BTF3 confers oncogenic activity in prostate cancer through transcriptional upregulation of Replication Factor C

High levels of Basic Transcription Factor 3 (BTF3) have been associated with prostate cancer. However, the mechanisms underlying the role of BTF3 as an oncogenic transcription factor in prostate tumorigenesis have not been explored. Herein, we report that BTF3 confers oncogenic activity in prostate cancer cells. Mechanistically, while both BTF3 splicing isoforms (BTF3a and BTF3b) promote cell growth, BTF3b, but not BTF3a, regulates the transcriptional expression of the genes encoding the subunits of Replication Factor C (RFC) family that is involved in DNA replication and damage repair processes. BTF3 knockdown results in decreased expression of RFC genes, and consequently attenuated DNA replication, deficient DNA damage repair, and increased G2/M arrest. Furthermore, knockdown of the RFC3 subunit diminishes the growth advantage and DNA damage repair capability conferred by ectopic overexpression of BTF3b. Importantly, we show that enforced BTF3 overexpression in prostate cancer cells induces substantial accumulation of cisplatin-DNA adducts and render the cells more sensitive to cisplatin treatment both in vitro and in vivo. These findings provide novel insights into the role of BTF3 as an oncogenic transcription factor in prostate cancer and suggest that BTF3 expression levels may serve as a potential biomarker to predict cisplatin treatment response.

Correlation between Charge Transport and Base Excision Repair in the MutY-DNA Glycosylase.

Experimental evidence suggests that DNA-mediated redox signaling between high-potential [Fe4S4] proteins is relevant to DNA replication and repair processes, and protein-mediated charge transfer (CT) between [Fe4S4] clusters and nucleic acids is a fundamental process of the signaling and repair mechanisms. We analyzed the dominant CT pathways in the base excision repair glycosylase MutY using molecular dynamics simulations and hole hopping pathway analysis. We find that the adenine nucleobase of the mismatched A·oxoG DNA base pair facilitates [Fe4S4]-DNA CT prior to adenine excision by MutY. We also find that the R153L mutation in MutY (linked to colorectal adenomatous polyposis) influences the dominant [Fe4S4]-DNA CT pathways and appreciably decreases their effective CT rates.

Comparative genomic analysis reveals evolutionary and structural attributes of MCM gene family in Arabidopsis thaliana and Oryza sativa

The mini-chromosome maintenance (MCM) family, a large and functionally diverse protein family belonging to the AAA+ superfamily, is essential for DNA replication in all eukaryotic organisms. The MCM 2-7 form a hetero-hexameric complex which serves as licensing factor necessary to ensure the proper genomic DNA replication during the S phase of cell cycle. MCM 8-10 are also associated with the DNA replication process though their roles are particularly unclear. In this study, we report an extensive in silico analysis of MCM gene family (MCM 2-10) in Arabidopsis and rice. Comparative analysis of genomic distribution across eukaryotes revealed conservation of core MCMs 2-7 while MCMs 8-10 are absent in some taxa. Domain architecture analysis underlined MCM 2-10 subfamily specific features. Phylogenetic analyses clustered MCMs into 9 clades as per their subfamily. Duplication events are prominent in plant MCM family, however no duplications are observed in Arabidopsis and rice MCMs. Synteny analysis among Arabidopsis thaliana, Oryza sativa, Glycine max and Zea mays MCMs demonstrated orthologous relationships and duplication events. Further, estimation of synonymous and non-synonymous substitution rates illustrated evolution of MCM family under strong constraints. Expression profiling using available microarray data and qRT-PCR revealed differential expression under various stress conditions, hinting at their potential use to develop stress resilient crops. Homology modeling of Arabidopsis and rice MCM 2-7 and detailed comparison with yeast MCMs identified conservation of eukaryotic specific insertions and extensions as compared to archeal MCMs. Protein-protein interaction analysis revealed an extensive network of putative interacting partners mainly involved in DNA replication and repair. The present study provides novel insights into the MCM family in Arabidopsis and rice and identifies unique features, thus opening new perspectives for further targeted analyses.

Remodeler Catalyzed Nucleosome Repositioning: Influence of Structure and Stability.

The packaging of the eukaryotic genome into chromatin regulates the storage of genetic information, including the access of the cell’s DNA metabolism machinery. Indeed, since the processes of DNA replication, translation, and repair require access to the underlying DNA, several mechanisms, both active and passive, have evolved by which chromatin structure can be regulated and modified. One mechanism relies upon the function of chromatin remodeling enzymes which couple the free energy obtained from the binding and hydrolysis of ATP to the mechanical work of repositioning and rearranging nucleosomes. Here, we review recent work on the nucleosome mobilization activity of this essential family of molecular machines.

KIF14 and KIF23 Promote Cell Proliferation and Chemoresistance in HCC Cells, and Predict Worse Prognosis of Patients with HCC.

BackgroundHepatocellular carcinoma (HCC) is one of the most common human malignant tumors. The prognosis of HCC patients is still unsatisfying. In this study, we performed the integrated bioinformatics analysis to identify potential biomarkers and biological pathways in HCC.MethodsGene expression profiles were obtained from the Gene Expression Omnibus database (GSE55048, GSE55758, and GSE56545) for the screening of the common differentially expressed genes (DEGs) between HCC tissues and matched non-tumor tissues. DEGs were subjected to Gene Ontology, KEGG pathway, and Reactome pathway analysis. The hub genes were identified by using protein–protein interaction (PPI) network analysis. The hub genes in HCC were further subjected to overall survival analysis of HCC patients. The hub genes were further validated by in vitro functional assays.ResultsA total of 544 common differentially expressed genes were screened from three datasets. Gene Ontology, KEGG and Reactome analysis results showed that DEGs are significantly associated with the biological process of cell cycle, cell division, and DNA replication. PPI network analysis identified 20 hub genes from the DEGs. These hub genes except CENPE were all significantly up-regulated in the HCC tissues when compared to non-tumor tissues. The Kaplan–Meier survival analysis results showed that the high expression of the 20 hub genes was associated with shorter survival of the HCC patients. Further validation studies showed that knockdown of KIF14 and KIF23 both suppressed the proliferative potential, increased the caspase-3/-7 activity, up-regulated Bax expression, and promoted the invasive and migratory abilities in the HCC cells. In addition, knockdown of KIF14 and KIF23 enhanced chemosensitivity to cisplatin and sorafenib in the HCC cells. Finally, the high expression of KIF14 and KIF23 was associated with shorter progression-free survival, recurrence-free survival, and disease-specific survival of patients with HCC.ConclusionIn conclusion, the present study performed the integrated bioinformatics analysis and showed that KIF14 and KIF23 silence attenuated cell proliferation, invasion, and migration, and promoted chemosensitivity of HCC cells. KIF14 and KIF23 may serve as potential biomarkers for predicting the worse prognosis of patients with HCC.

Computational prediction and interpretation of cell-specific replication origin sites from multiple eukaryotes by exploiting stacking framework.

Origins of replication sites (ORIs), which refers to the initiative locations of genomic DNA replication, play essential roles in DNA replication process. Detection of ORIs' distribution in genome scale is one of key steps to in-depth understanding their regulation mechanisms. In this study, we presented a novel machine learning-based approach called Stack-ORI encompassing 10 cell-specific prediction models for identifying ORIs from four different eukaryotic species (Homo sapiens, Mus musculus, Drosophila melanogaster and Arabidopsis thaliana). For each cell-specific model, we employed 12 feature encoding schemes that cover nucleic acid composition, position-specific and physicochemical properties information. The optimal feature set was identified from each encoding individually and developed their respective baseline models using the eXtreme Gradient Boosting (XGBoost) classifier. Subsequently, the predicted scores of 12 baseline models are integrated as a novel feature vector to train XGBoost and develop the final model. Extensive experimental results show that Stack-ORI achieves significantly better performance as compared with their baseline models on both training and independent datasets. Interestingly, Stack-ORI consistently outperforms existing predictor in all cell-specific models, not only on training but also on independent test. Moreover, our novel approach provides necessary interpretations that help understanding model success by leveraging the powerful SHapley Additive exPlanation algorithm, thus underlining the most important feature encoding schemes significant for predicting cell-specific ORIs.

Lysine acetylation regulates the activity of nuclear Pif1

In Saccharomyces cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase, ATPase, and DNA-binding activities, whereas maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity.

Discovery of Novel Substituted N-(4-Amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide Analogues as Potent Human Adenovirus Inhibitors.

An effective therapy for human adenovirus (HAdV) infections in immunocompromised patients and healthy individuals with community-acquired pneumonia remains an unmet medical need. We herein reported a series of novel substituted N-(4-amino-2-chlorophenyl)-5-chloro-2-hydroxybenzamide analogues as potent HAdV inhibitors. Compounds 6, 15, 29, 40, 43, 46, 47, and 54 exhibited increased selectivity indexes (SI > 100) compared to the lead compound niclosamide, while maintaining sub-micromolar to low micromolar potency against HAdV. The preliminary mechanistic studies indicated that compounds 6 and 43 possibly target the HAdV DNA replication process, while compounds 46 and 47 suppress later steps of HAdV life cycle. Notably, among these derivatives, compound 15 showed improved anti-HAdV activity (IC50 = 0.27 μM), significantly decreased cytotoxicity (CC50 = 156.8 μM), and low in vivo toxicity (maximum tolerated dose = 150 mg/kg in hamster) as compared with niclosamide, supporting its further in vivo efficacy studies for the treatment of HAdV infections.

Molecular dynamics simulations of sulfone derivatives in complex with DNA topoisomerase IIα ATPase domain

Human topoisomerase II alpha (TopoIIα) is a crucial enzyme involved in maintaining genomic integrity during the process of DNA replication and mitotic division. It is a vital therapeutic target for designing novel anticancer agents in targeted cancer therapy. Sulfones, members of organosulfur compounds, have been reported to possess various biological activities such as antimicrobial, anti-inflammatory, anti-HIV, anticancer, and antimalarial properties. In the present study, a series of sulfones was selected to evaluate their inhibitory activity against TopoIIα using computational approaches. Molecular docking results revealed that several sulfone analogs bind efficiently to the ATPase domain of TopoIIα. Among them, sulfones 18a, 60a, *4 b, *8 b, *3c, and 8c exhibit higher binding affinity than the known TopoII inhibitor, salvicine. Molecular dynamics simulations and free energy calculations based on MM/PB(GB)SA method demonstrated that sulfone *8 b strongly interacts with amino acid residues in the ATP-binding pocket (E87, N91, D94, I125, I141, F142, S149, G161, and A167), driven mainly by an electrostatic attraction and a strong H-bond formation at G161 residue. Altogether, the obtained results predicted that sulfones could have a high potential to be a lead molecule for targeting TopoIIα. Communicated by Ramaswamy H. Sarma

Serinol-Based Benzoic Acid Esters as New Scaffolds for the Development of Adenovirus Infection Inhibitors: Design, Synthesis, and In Vitro Biological Evaluation.

Over the years, human adenovirus (HAdV) has progressively been recognized as a significant viral pathogen. Traditionally associated with self-limited respiratory, gastrointestinal, and conjunctival infections, mainly in immunocompromised patients, HAdV is currently considered to be a pathogen presenting significant morbidity and mortality in both immunosuppressed and otherwise healthy individuals. Currently available therapeutic options are limited because of their lack of effectivity and related side effects. In this context, there is an urgent need to develop effective anti-HAdV drugs with suitable therapeutic indexes. In this work, we identified new serinol-derived benzoic acid esters as novel scaffolds for the inhibition of HAdV infections. A set of 38 compounds were designed and synthesized, and their antiviral activity and cytotoxicity were evaluated. Four compounds (13, 14, 27, and 32) inhibited HAdV infection at low micromolar concentrations (2.82-5.35 μM). Their half maximal inhibitory concentration (IC50) values were lower compared to that of cidofovir, the current drug of choice. All compounds significantly reduced the HAdV DNA replication process, while they did not block any step of the viral entry. Our results showed that compounds 13, 14, and 32 seem to be targeting the expression of the E1A early gene. Moreover, all four derivatives demonstrated a significant inhibition of human cytomegalovirus (HCMV) DNA replication. This new scaffold may represent a potential tool useful for the development of effective anti-HAdV drugs.

Biological and clinical significance of flap endonuclease‑1 in triple‑negative breast cancer: Support of metastasis and a poor prognosis.

Flap endonuclease-1 (FEN1), a structure-specific nuclease participating in DNA replication and repair processes, has been confirmed to promote the proliferation and drug resistance of tumor cells. However, the biological functions of FEN1 in cancer cell migration and invasion have not been defined. In the present study, using online database analysis and immunohistochemistry of the specimens, it was found that FEN1 expression was associated with a highly invasive triple-negative breast cancer (TNBC) subtype in both breast cancer samples from the Oncomine database and from patients recruited into the study. Furthermore, FEN1 was an important biomarker of lymph node metastasis and poor prognosis in patients with TNBC. FEN1 promoted migration of TNBC cell lines and FEN1 knockdown reduced the number of spontaneous lung metastasis in vivo. Ingenuity Pathway Analysis of FEN1-related transcripts in 198 patients with TNBC demonstrated that the polo-like kinase family may be the downstream target of FEN1. PLK4 was further identified as a critical target of FEN1 mediating TNBC cell migration, by regulating actin cytoskeleton rearrangement. The results of the present study validate FEN1 as a therapeutic target in patients with TNBC and revealed a new role for FEN1 in regulating TNBC invasion and metastasis.

Hydroxy Piperlongumines: Synthesis, Antioxidant, Cytotoxic Effect on Human Cancer Cell Lines, Inhibitory Action and ADMET Studies

AbstractPiperlongumine, an anticancer natural product, was sequentially demethylated to obtain mono, di and trihydroxy piperlongumines. The hydroxy piperlongumines were evaluated for cytotoxic effect on five human cancer cell lines (kidney, skin, cervical, breast, and lung) in vitro. Results from cytotoxic and cell growth inhibition studies showed monohydroxy piperlongumine (MHPL) was the most active of the series, exhibited potent cytotoxic effect on all the tested cell lines. Flow cytometry analysis shows MHPL inhibited cell cycle progression of HeLa cells in the S phase, indicated that it inhibits the DNA replication process. Fluorescence staining indicated MHPL treated cells were apoptotic. MHPL upregulated the apoptotic genes (caspase‐3, −8, Bax, and p53) and downregulated anti‐apoptotic gene (Bcl‐2). The antioxidant study revealed that hydroxy piperlongumines are effective antioxidants. For MHPL, all the molecular descriptors were within the range of 95 % of approved drugs suggesting high druglikeliness. Our results suggest that MHPL could be developed as a potential lead molecule for cancer therapy.

MCM10 Acts as a Potential Prognostic Biomarker and Promotes Cell Proliferation in Hepatocellular Carcinoma: Integrated Bioinformatics Analysis and Experimental Validation.

BackgroundHepatocellular carcinoma (HCC) is one of the most common human malignant tumors. The prognosis of HCC patients is still unsatisfying. Thus, it is of great importance to identify novel molecules and functional pathways associated with the pathophysiology of HCC. In this study, we performed the integrated bioinformatics analysis and experiment validation to identify novel biomarkers in the prognosis and progression of HCC.Materials and MethodsGene expression profiles were obtained from Gene Expression Omnibus database (GSE33294) for the screening of the differentially expressed genes (DEGs) between HCC tissues and matched non-tumor tissues. The DEGs were subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Set Enrichment Analysis (GSEA). The key genes in HCC were further subjected to overall survival analysis of HCC patients. The in vitro functional studies were performed to validate the biological functions of the key gene in HCC cell progression.ResultsA total of 2,334 DEGs were screened from GSE33294 dataset, including 1,120 up-regulated and 1,214 down-regulated genes. GO, KEGG and GSEA results showed that DEGs are significantly associated with the biological process of cell cycle, cell division and DNA replication. The Kaplan–Meier survival analysis results showed that the key genes from the minichromosome maintenance protein complex (MCM) family including MCM8, MCM10, MCM2, MCM3, MCM4, MCM6 and MCM7 were significantly correlated with the overall survival of the HCC patients. Further validation studies showed that MCM10 was significantly up-regulated in the HCC cell lines, and knockdown of MCM10 significantly suppressed cell proliferation as determined by the cell counting kit-8 and BrdU incorporation assays and increased the caspase-3 activity of HCC cells.ConclusionThe comprehensive bioinformatics analysis identified several key genes that were associated with the prognosis of HCC patients. The validation study results indicated that MCM10 may be an important predictor for poorer prognosis of HCC patients and may act as an oncogene to promote HCC cell progression.

Mechanism of Dynamic Binding of Replication Protein A to ssDNA.

Replication protein A (RPA) serves as a hub protein inside eukaryotic cells, where it coordinates crucial DNA metabolic processes and activates the DNA-damage response system. A characteristic feature of its action is to associate with single-stranded DNA (ssDNA) intermediates before handing them over to downstream proteins. The length of ssDNA intermediates differs for different pathways. This means that RPA must have mechanisms for selective processing of ssDNA intermediates based on their length, the knowledge of which is fundamental to elucidate when and how DNA repair and replication processes are symphonized. By employing extensive molecular dynamics simulations, we investigated the mechanism of binding of RPA to ssDNA of different lengths. We show that the binding involves dynamic equilibrium with a stable intermediate, the population of which increases with the length of ssDNA. The vital underlying factors are decoded through collective variable principal component analysis. It suggests a differently orchestrated set of interactions that define the action of RPA based on the length of ssDNA intermediates. We further estimated the association kinetics that matches excellently well with previous experimental studies and probed the diffusion mechanism of RPA to ssDNA. RPA diffuses on short ssDNA through progressive "bulge" formation. With long ssDNA, we observed a conformational change in ssDNA coupled with its binding to RPA in a cooperative fashion. This unanticipated binding mechanism successfully explains how the "short-lived", long ssDNA intermediates are processed quickly in vivo. This study thus reveals the molecular basis of several recent experimental observations related to RPA binding to ssDNA and provides novel insights into the RPA functioning in DNA repair and replication.

The Analysis of Recombination-Dependent Processing of Blocked Replication Forks by Bidimensional Gel Electrophoresis.

The perturbation of the DNA replication process is a threat to genome stability and is an underlying cause of cancer development and numerous human diseases. It has become central to understanding how stressed replication forks are processed to avoid their conversion into fragile and pathological DNA structures. The engineering of replication fork barriers (RFBs) to conditionally induce the arrest of a single replisome at a defined locus has made a tremendous impact in our understanding of replication fork processing. Applying the bidimensional gel electrophoresis (2DGE) technique to those site-specific RFBs allows the visualization of replication intermediates formed in response to replication fork arrest to investigate the mechanisms ensuring replication fork integrity. Here, we describe the 2DGE technique applied to the site-specific RTS1-RFB in Schizosaccharomyces pombe and explain how this approach allows the detection of arrested forks undergoing nascent strands resection.

Rapid progressive ALS in a patient with a DNAJC7 loss-of-function mutation.

ABSTRACT In S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.

Abstract 500: Impact of Oxidative Stress and Fancj on Heart Health

G-quadruplexes (or G4s) are structures formed by guanine-rich nucleic acid sequences. G4s must be promptly unfolded in cells; otherwise, they can interfere with DNA replication, RNA transcription, and other essential processes. Guanines bases are susceptible to oxidative stress forming 8-oxoguanines (8oxoG). Although 8oxoG-modified DNA sequences can still fold into stable G4s, it is not known how 8oxoG4s are repaired in human cells. In our previous study, we have shown that the FANCJ DNA helicase targets G4s using an AKKQ amino acid motif. Here, we have examined the interactions of FANCJ with various 8oxoG4s using biolayer interferometry and fluorescence spectroscopy. We show that a FANCJ AKKQ peptide alone can recognize G4s with an affinity of 3.7uM. Moreover, this motif binds to 8oxoG4s with greater affinities of 1.3 to 2.3uM. A detailed description of the mechanisms by which 8oxoG4s are repaired is essential for understanding how human hearts respond to oxidative stress. To test the importance of FANCJ AKKQ-G4 interactions in cells, we measured the total extent of oxidative DNA damage in human cardiac cells by single-cell electrophoresis. Cells that overexpress FANCJ can readily overcome the chemical stress induced by hydrogen peroxide treatment and the G4-stabilizing compound telomestatin. On the contrary, cells that produce a FANCJ AAAQ mutant, which cannot interact with G4s, resulted in an accumulation of 8oxoG4s. Based on this evidence, FANCJ plays an important role to alleviate the damage caused by oxidative stress. In future experiments, we plan to further examine the cardiovascular risks of DNA damage caused by FANCJ malfunctions.