Primase Subunits Research Articles

The evolving genetic landscape of telomere biology disorder dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as ‘DC-like’ (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1. In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients’ blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders.

Harnessing DNA replication stress to target RBM10 deficiency in lung adenocarcinoma

The splicing factor RNA-binding motif protein 10 (RBM10) is frequently mutated in lung adenocarcinoma (LUAD) (9-25%). Most RBM10 cancer mutations are loss-of-function, correlating with increased tumorigenesis and limiting the efficacy of current LUAD targeted therapies. Remarkably, therapeutic strategies leveraging RBM10 deficiency remain unexplored. Here, we conduct a CRISPR-Cas9 synthetic lethality (SL) screen and identify ~60 RBM10 SL genes, including WEE1 kinase. WEE1 inhibition sensitizes RBM10-deficient LUAD cells in-vitro and in-vivo. Mechanistically, we identify a splicing-independent role of RBM10 in regulating DNA replication fork progression and replication stress response, which underpins RBM10-WEE1 SL. Additionally, RBM10 interacts with active DNA replication forks, relying on DNA Primase Subunit 1 (PRIM1) that synthesizes Okazaki RNA primers. Functionally, we demonstrate that RBM10 serves as an anchor for recruiting Histone Deacetylase 1 (HDAC1) to facilitate H4K16 deacetylation and R-loop homeostasis to maintain replication fork stability. Collectively, our data reveal a role of RBM10 in fine-tuning DNA replication and provide therapeutic arsenal for targeting RBM10-deficient tumors.

New Progress on DNA Primase Subunit Enzymes Research and Link to Cancer Development and Treatment Approaches.

In eukaryotic cells, primases are the key polymerase during DNA replication and DNA damage repair, which includes primase subunit 1 (PRIM1) and primase subunit 2 (PRIM2). Recent studies reported that the aberrant expression and activity of PRIM enzymes are closely associated with the carcinogenesis and development of various cancers. PRIM1 is overexpressed in hepatocellular carcinoma, breast cancer, and other cancers, while PRIM2 is highly expressed in lung cancer, gastrointestinal cancer, and other cancers. Further studies revealed that the knockdown of PRIM1 promoted the apoptosis of liver cancer cells, while Dihydroartemisinin (DHA) can inhibit PRIM2 expression, suppress lung cancer cell proliferation, and result in ferroptosis. The present review summarized the recent advancements in the research of the aberrant expression of PRIM1 and PRIM2 and their activity in DNA replication, DNA damage repair, and carcinogenesis. Furthermore, the strategies targeting PRIM1 or/and PRIM2 become potential therapeutic approaches in cancer treatment.

CryoEM insights into RNA primer synthesis by the human primosome.

Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo-electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation.

Abstract IA023: The role of RTF2 in replication and replication stress response

Abstract The replisome faithfully coordinates the essential process of genome duplication despite genomic sequences or lesions that slow or stall replication fork progression, collectively termed replication stress. Such lesions threaten the accurate transmission of the genome; as such, cells have evolved many mechanisms of replication fork protection and repair to ensure that the genome is fully replicated within the confines of S phase. Our group has previously characterized the essential protein Replication Termination Factor 2 (RTF2) as one such facilitator of replication control during both unperturbed and stalled conditions. During unperturbed replication, RTF2 localizes the Ribonuclease H2 heterotrimer (RNase H2) to replication forks, where RNase H2 facilitates the removal of ribonucleotides aberrantly incorporated into the genome to ensure replication fork speeds and promote the completion of genome duplication. When the replisome encounters fork-stalling lesions, however, RTF2-RNase H2 are removed from the replisome through the action of proteasome shuttle proteins DNA Damage Inducible 1 and 2 (DDI1/2) to promote replication restart dependent upon PRIM1, the primase subunit of DNA polymerase α. Further, we find that cells depleted of RTF2 accumulate mitotic markers, including phosphorylated FOXM1 and cyclin B1, during S phase and exhibit abnormal nuclear structures. Together, these findings suggest that RTF2-deficient cells bypass the S/G2 checkpoint, in which ATR functions to suppress CDK1 activity and mitotic progression while replication is ongoing and implicate RTF2 as a mediator of replication checkpoint control. Citation Format: Agata Smogorzewska, Cayla Broton, Nicolas J. Blobel, Brooke Conti. The role of RTF2 in replication and replication stress response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA023.

Pan‐Cancer Analysis Reveals PRIM2 as a Potential Biomarker for Diagnosis, Prognosis, and Immunomodulatory

Purpose: DNA primase subunit 2 (PRIM2) is a component of the DNA polymerase α‐primase complex involved in DNA replication. Previous studies have implicated PRIM2 in cancer progression, but a comprehensive pan‐cancer analysis is lacking. Here, we systematically analyzed PRIM2 across various cancer types to elucidate its potential role in cancer biology.Methods: The expression data of PRIM2 was obtained from The Cancer Genome Atlas (TCGA) and Genotype‐Tissue Expression Project (GTEx) databases. A range of bioinformatics tools were employed to analyze the expression of PRIM2 with the correlation of clinical data, diagnosis, prognosis, subtypes, immunomodulatory, and drug sensitivity. Further validation experiments were performed in HT‐29 and HepG2 cell lines.Results: PRIM2 expression was elevated in most tumor tissues, indicating its potential diagnostic value. Elevated PRIM2 levels affected prognosis and survival and varied with clinical status. Mutations and methylation events drove the aberrant expression of PRIM2 in various cancers. Furthermore, PRIM2 was linked to key immunoregulatory genes such as PD‐L1 and infiltration of Th2 cells in tumor tissues. Our findings suggested that PRIM2 is correlated to immunotherapy and may be sensitive to specific small molecule PLK1 inhibitors. Knocking down PRIM2 in HT‐29 and HepG2 cell lines reduced their proliferation, migration, invasion, and epithelial‐mesenchymal transition.Conclusion: This research highlights the potential of PRIM2 as a biomarker for cancer diagnosis and prognosis, and as a target for immunomodulatory therapy, offering valuable insights for clinical applications.

DNA primase large subunit is an essential plant gene for geminiviruses, putatively priming viral ss-DNA replication.

The family of Geminiviridae consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their genome in the nucleus of a plant cell, taking advantage of the host's DNA replication machinery. For converting their DNA into double-stranded DNA, and subsequent replication, these viruses rely on host DNA polymerases. However, the priming of the very first step of this process, i.e. the conversion of incoming circular ssDNA into a dsDNA molecule, has remained elusive for almost 30 years. In this study, sequencing of melon (Cucumis melo) accession K18 carrying the Tomato leaf curl New Delhi virus (ToLCNDV) recessive resistance quantitative trait locus (QTL) in chromosome 11, and analyses of DNA sequence data from 100 melon genomes, showed a conservation of a shared mutation in the DNA Primase Large subunit (PRiL) of all accessions that exhibited resistance upon a challenge with ToLCNDV. Silencing of (native) Nicotiana benthamiana PriL and subsequent challenging with three different geminiviruses showed a severe reduction in titers of all three viruses, altogether emphasizing an important role of PRiL in geminiviral replication. A model is presented explaining the role of PriL during initiation of geminiviral DNA replication, i.e. as a regulatory subunit of primase that generates an RNA primer at the onset of DNA replication in analogy to DNA Primase-mediated initiation of DNA replication in all living organisms.

Identification of potential biomarkers for colorectal cancer by clinical database analysis and Kaplan-Meier curves analysis.

This study aimed to explore critical genes as potential biomarkers for the diagnosis and prognosis of colorectal cancer (CRC) for clinical utility. To identify and screen candidate genes involved in CRC carcinogenesis and disease progression, we downloaded microarray datasets GSE89076, GSE73360, and GSE32323 from the GEO database identified differentially expressed genes (DEGs), and performed a functional enrichment analysis. A protein-protein interaction network was constructed, and correlated module analysis was performed using STRING and Cytoscape. The Kaplan-Meier survival curve shows the survival of the hub genes. The expression of cyclin-dependent kinase (CDK1), cyclin B1 (CCNB1), and PCNA in tissues and changes in tumor grade were analyzed. A total of 329 DEGs were identified, including 264 upregulated and 65 downregulated genes. The functions and pathways of DEGs include the mitotic cell cycle, poly(A) RNA binding replication, ATP binding, DNA replication, ribosome biogenesis in eukaryotes, and RNA transport. Forty-seven Hub genes were identified, and biological process analysis showed that these genes were mainly enriched in cell cycle and DNA replication. Patients with mutations in CDK1, PCNA, and CCNB1 had poorer survival rates. CDK1, PCNA, and CCNB1 were significantly overexpressed in the tumor tissues. The expression of CDK1 and CCNB1 gradually decreased with increasing tumor grade. CDK1, CCNB1, and PCNA can be used as potential markers for the diagnosis and prognosis of CRC. These genes are overexpressed in colon cancer tissues and are associated with low survival rates in CRC patients.

The primase subunits of DNA polymerase α, PRIM1 and PRIM2, are required for the replication of the geminivirus tomato yellow leaf curl virus in the host plant.

Geminiviruses are causal agents of devastating diseases in crops. Geminiviral genomes are single-stranded (ss) circular DNA molecules that replicate in the nucleus of the infected cell through double-stranded (ds) intermediates by co-opting the plant DNA replication machinery. However, the identity of the plant DNA polymerases enabling geminiviral replication has remained largely elusive. Recently, we showed that DNA polymerase α mediates the ss-to-ds conversion of tomato yellow leaf curl virus (TYLCV), and is therefore essential for the infection. Here, we provide data indicating that the primase subunits of DNA polymerase α, PRIM1 and PRIM2, are also required for TYLCV replication.

Leveraging pleiotropy to discover and interpret GWAS results for sleep-associated traits.

Genetic association studies of many heritable traits resulting from physiological testing often have modest sample sizes due to the cost and burden of the required phenotyping. This reduces statistical power and limits discovery of multiple genetic associations. We present a strategy to leverage pleiotropy between traits to both discover new loci and to provide mechanistic hypotheses of the underlying pathophysiology. Specifically, we combine a colocalization test with a locus-level test of pleiotropy. In simulations, we show that this approach is highly selective for identifying true pleiotropy driven by the same causative variant, thereby improves the chance to replicate the associations in underpowered validation cohorts and leads to higher interpretability. Here, as an exemplar, we use Obstructive Sleep Apnea (OSA), a common disorder diagnosed using overnight multi-channel physiological testing. We leverage pleiotropy with relevant cellular and cardio-metabolic phenotypes and gene expression traits to map new risk loci in an underpowered OSA GWAS. We identify several pleiotropic loci harboring suggestive associations to OSA and genome-wide significant associations to other traits, and show that their OSA association replicates in independent cohorts of diverse ancestries. By investigating pleiotropic loci, our strategy allows proposing new hypotheses about OSA pathobiology across many physiological layers. For example, we identify and replicate the pleiotropy across the plateletcrit, OSA and an eQTL of DNA primase subunit 1 (PRIM1) in immune cells. We find suggestive links between OSA, a measure of lung function (FEV1/FVC), and an eQTL of matrix metallopeptidase 15 (MMP15) in lung tissue. We also link a previously known genome-wide significant peak for OSA in the hexokinase 1 (HK1) locus to hematocrit and other red blood cell related traits. Thus, the analysis of pleiotropic associations has the potential to assemble diverse phenotypes into a chain of mechanistic hypotheses that provide insight into the pathogenesis of complex human diseases.

PRIM2 Promotes Cell Cycle and Tumor Progression in p53-Mutant Lung Cancer.

Simple SummaryThe mutation or inactivation of tumor suppressor genes is a key driving force during tumorigenesis, among which, p53 mutation is a common feature of human cancer. Therefore, exploring the potential role of p53 mutation in the occurrence and development of tumors is a powerful support for tumor diagnosis and treatment. In this study, we found that PRIM2 expression was abnormally elevated in p53-mutated lung cancer patients, and the elevated PRIM2 promoted DNA replication, enhanced mismatch repair, activated cell cycle, and promoted lung cancer progression. Here, we first report that the expression of PRIM2 is regulated by p53, and is identified as a biomarker of lung cancer malignancy and survival prognosis.p53 is a common tumor suppressor, and its mutation drives tumorigenesis. What is more, p53 mutations have also been reported to be indicative of poor prognosis in lung cancer, but the detailed mechanism has not been elucidated. In this study, we found that DNA primase subunit 2 (PRIM2) had a high expression level and associated with poor prognosis in lung cancer. Furthermore, we found that PRIM2 expression was abnormally increased in lung cancer cells with p53 mutation or altered the p53/RB pathway based on database. We also verified that PRIM2 expression was elevated by mutation or deletion of p53 in lung cancer cell lines. Lastly, silence p53 increased the expression of RPIM2. Thus, these data suggest that PRIM2 is a cancer-promoting factor which is regulated by the p53/RB pathway. The p53 tumor-suppressor gene integrates numerous signals that control cell proliferation, cell cycle, and cell death; and the p53/RB pathway determines the cellular localization of transcription factor E2F, which regulates the expression of downstream targets. Next, we explored the role of PRIM2 in lung cancer and found that knockdown of PRIM2 induced cell cycle arrest, increased DNA damage, and increased cell senescence, leading to decreased lung cancer cell proliferation. Lastly, the positive correlation between PRIM2 and E2F/CDK also indicated that PRIM2 was involved in promoting cell cycle mediated by p53/RB pathway. These results confirmed that the expression of PRIM2 is regulated by the p53/RB pathway in lung cancer cells, promotes DNA replication and mismatch repair, and activates the cell cycle. Overall, we found that frequent p53 mutations increased PRIM2 expression, activated the cell cycle, and promoted lung cancer progression.

Insights into Binding of Single-Stranded Viral RNA Template to the Replication-Transcription Complex of SARS-CoV-2 for the Priming Reaction from Molecular Dynamics Simulations.

A minimal replication–transcription complex (RTC) of SARS-CoV-2 for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase and two nsp8 RNA primase subunits for de novo primer synthesis, one nsp8 in complex with its accessory nsp7 subunit and the other without it. The RTC is responsible for faithfully copying the entire (+) sense viral genome from its first 5′-end to the last 3′-end nucleotides through a replication-intermediate (RI) template. The single-stranded (ss) RNA template for the RI is its 33-nucleotide 3′-poly(A) tail adjacent to a well-characterized secondary structure. The ssRNA template for viral transcription is a 5′-UUUAU-3′ next to stem-loop (SL) 1′. We analyze the electrostatic potential distribution of the nsp8 subunit within the RTC around the template strand of the primer/template (P/T) RNA duplex in recently published cryo-EM structures to address the priming reaction using the viral poly(A) template. We carried out molecular dynamics (MD) simulations with a P/T RNA duplex, the viral poly(A) template, or a generic ssRNA template. We find evidence that the viral poly(A) template binds similarly to the template strand of the P/T RNA duplex within the RTC, mainly through electrostatic interactions, providing new insights into the priming reaction by the nsp8 subunit within the RTC, which differs significantly from the existing proposal of the nsp7/nsp8 oligomer formed outside the RTC. High-order oligomerization of nsp8 and nsp7 for SARS-CoV observed outside the RTC of SARS-CoV-2 is not found in the RTC and not likely to be relevant to the priming reaction.

The yeast Dbf4 Zn2+ finger domain suppresses single-stranded DNA at replication forks initiated from a subset of origins

Dbf4 is the cyclin-like subunit for the Dbf4-dependent protein kinase (DDK), required for activating the replicative helicase at DNA replication origin that fire during S phase. Dbf4 also functions as an adaptor, targeting the DDK to different groups of origins and substrates. Here we report a genome-wide analysis of origin firing in a budding yeast mutant, dbf4-zn, lacking the Zn2+ finger domain within the C-terminus of Dbf4. At one group of origins, which we call dromedaries, we observe an unanticipated DNA replication phenotype: accumulation of single-stranded DNA spanning ± 5kbp from the center of the origins. A similar accumulation of single-stranded DNA at origins occurs more globally in pri1-m4 mutants defective for the catalytic subunit of DNA primase and rad53 mutants defective for the S phase checkpoint following DNA replication stress. We propose the Dbf4 Zn2+ finger suppresses single-stranded gaps at replication forks emanating from dromedary origins. Certain origins may impose an elevated requirement for the DDK to fully initiate DNA synthesis following origin activation. Alternatively, dbf4-zn may be defective for stabilizing/restarting replication forks emanating from dromedary origins during replication stress.

Select DNA Replication And Repair Genes Are Transcriptionally Modified By Acute Exercise In Hypoxia

DNA replication and repair in eukaryote cells are critical processes that ensures the correct transfer of DNA material through mitosis. Errors in the replication and repair of DNA during and following cell division may lead to genome instability. PURPOSE: To investigate the regulation of select gene transcripts aligned to DNA replication and repair following exercise in hypoxia. METHODS: Twenty-four (n = 24) apparently healthy male participants were recruited (age 28 ± 5 years; mass 74 ± 8 kg; stature 177 ± 6 cm; VO2max hypoxia 45 ± 2 ml·kg-1·min-1: normoxia 60 ± 9 ml·kg-1·min-1), and completed 1 hr of exercise at a workload corresponding to 75% of pre-determined VO2max in hypoxia (Fio2 = 0.16%), and repeated in normoxia (Fio2 = 0.21%). RNA was extracted from whole blood. Two-colour oligonucleotide microarrays [for DNA primase subunit 1 - PRIM1; Chromatin assembly factor 1 subunit B - CHAF1B; and Werner syndrome RecQ like helicase - WRN] were processed on an Agilent DNA microarray scanner, while mRNA analysis was conducted using GeneSpring software incorporating checks for data normalisation, quality control, filtering grouping (by experimental condition) and statistical analysis (paired t-test with Benjamini & Hochberg False Discovery Rate corrections for multiple comparisons). RESULTS: Exercise performed in hypoxia per se downregulated PRIM1 and CHAF1B (p = 0.03 and p = 0.01 respectively vs. pre-exercise), with no change observed in normoxia (p > 0.05). Compared with normoxia, CHAF1B and WRN were differentially downregulated following exercise in hypoxia (p = 0.05 and p = 0.05 respectively vs. pre-exercise). CONCLUSION: Acute exercise in moderate hypoxia interferes with gene transcription aligned to blood cell DNA replication and repair. Further work is necessary to ascertain the consequence of these select modifications on genome stability.

DNA primase subunit 1 deteriorated progression of hepatocellular carcinoma by activating AKT/mTOR signaling and UBE2C-mediated P53 ubiquitination

BackgroundDNA primase subunit 1 (PRIM1) has been reported as a novel oncogene in several cancer types. However, its roles in hepatocellular carcinoma (HCC) remain unclear. This study aimed to investigate underlying mechanisms of PRIM1 and identify it as a potential molecular target for HCC.MethodsHub genes were screened between HCC tissues and normal liver tissues in 3 gene expression omnibus (GEO) datasets and the cancer genome atlas (TCGA). The expression features and prognostic value of one of the hub genes PRIM1 were analyzed by bioinformatic analyses and immunohistochemistry. Loss-of-function and gain-of-function studies were used to investigate the regulatory role of PRIM1 in HCC cells. Real-time (RT)-qPCR, western blotting, and ubiquitin immunoprecipitation assays were performed to explore the underlying mechanisms. The xenograft model was employed to detect the roles of PRIM1 in tumor growth in vivo. Finally, the 3D spheroid model was conducted to validate the role of PRIM1 in tumor growth and sorafenib resistance.ResultsThe hub genes of HCC were screened in multiple bioinformatic datasets. PRIM1, as one of the hub genes, was significantly overexpressed in HCC tissues in mRNA and protein levels. In addition, high expression of PRIM1 indicated poor prognosis of HCC patients in TCGA, ICGC, and Nantong cohorts. Overexpression of PRIM1 promoted the proliferation, migration/invasion, and sorafenib resistance of HCC cells, with the decrease in apoptosis and cell cycle arrest. Mechanically, PRIM1 facilitated epithelial-mesenchymal transition (EMT) process and the activity of PI3K/AKT/mTOR signaling of HCC cells. Additionally, PRIM1 could cause the ubiquitination and degradation of P53 by upregulating Ubiquitin Conjugating Enzyme E2 C (UBE2C). Furthermore, knockdown of PRIM1 significantly inhibited the growth of xenograft tumors and HCC cells-derived spheroids with enhanced sorafenib resistance.ConclusionThis study implies that PRIM1 may play a key role in the progression of HCC and may serve as a potential target for HCC treatment.

PRIM1 deficiency causes a distinctive primordial dwarfism syndrome.

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.

DNA Primase Subunit 1 Expression in Hepatocellular Carcinoma and Its Clinical Implication.

DNA Primase Subunit 1 (PRIM1) is crucial for cancer development and progression. However, there remains a lack of comprehension concerning the clinical implication of PRIM1 in HCC. Here, aberrant expression of PRIM1 was identified in HCC according to available databases. The prognostic value of PRIM1 in patients presenting with HCC was further assessed based on TCGA data. Gene set enrichment analysis (GSEA) was subsequently conducted to investigate the potential function of PRIM1. Additionally, the correlations between tumor-infiltrating immune cells (TIICs) and PRIM1 expression were evaluated. The data from TCGA, GEO, ONCOMINE, and HCCDB databases illustrated that PRIM1 was overexpressed in HCC tissues, compared to normal liver tissues (all p < 0.05). Kaplan-Meier analysis revealed that high PRIM1 expression in HCC was closely correlated with worse overall survival (p < 0.05). The univariate and multivariate analyses illustrated that PRIM1 expression was an independent novel prognostic indicator in HCC. Additionally, the area under the receiver operating characteristic (AUROC) curve for PRIM1 reached 0.8651, indicating the diagnostic significance of PRIM1 in patients with HCC. GSEA showed that PRIM1 overexpression was significantly enriched in several tumor-related signaling pathways. Besides, TIIC analysis clarified the association between PRIM1 expression and TIICs in HCC. The findings disclose that PRIM1 profoundly implicated in promoting tumorigenesis might work as a desirable biomarker for HCC.

Evaluation of potential drugs against leishmaniasis targeting catalytic subunit of Leishmania donovani nuclear DNA primase using ligand based virtual screening, docking and molecular dynamics approaches

Leishmania donovani, causes leishmaniasis, a global health trouble with around 89 different countries and its population under its risk. Replication initiation events have been instrumental in regulating the DNA duplication and as the small subunit of L. donovani nuclear DNA primase (Ld-PriS) inherits the catalytic site, it plays a vital role in DNA replication. In this study we have aimed Ld-PriS for the first time as a prospective target for the application of drug against Leishmania parasite. 3-D structures of Ld-PriS were built and ligand-based virtual screening was performed using hybrid similarity recognition techniques. Ligands from the ZINC database were used for the screening purposes based on known DNA primase inhibitor Sphingosine as a query. Top 150 ligands were taken into consideration for molecular docking against the query protein (Ld-PriS) using PyRx and iGEMDOCK softwares. Top five compounds with the best docking score were selected for pharmacokinetic investigation and molecular dynamic simulation. These top five screened inhibitors showed very poor binding affinity toward the catalytic subunit of human primase indicating their safety toward the host normal replication mechanism. The top five compounds showed good pharmacokinetic profiles and ADMET predictions revealed good absorption, solubility, permeability, uniform distribution, proper metabolism, minimal toxicity and good bioavailability. Simulation studies upto 50 ns revealed the three leads ZINC000009219046, ZINC000025998119 and ZINC000004677901 bind with Ld-PriS throughout the simulation and there were no huge variations in their backbone suggesting that these three may play as potential lead compounds for developing new drug against leishmaniasis. Communicated by Ramaswamy H. Sarma

Mutation effects on charge transport through the p58c iron-sulfur protein.

Growing experimental evidence indicates that iron–sulfur proteins play key roles in DNA repair and replication. In particular, charge transport between [Fe4S4] clusters, mediated by proteins and DNA, may convey signals to coordinate enzyme action. Human primase is a well studied [Fe4S4] protein, and its p58c domain (which contains an [Fe4S4] cluster) plays a role in the initiation of DNA replication. The Y345C mutation in p58c is linked to gastric tumors and may influence the protein-mediated charge transport. The complexity of protein–DNA systems, and the intricate electronic structure of [Fe4S4] clusters, have impeded progress into understanding functional charge transport in these systems. In this study, we built force fields to describe the high potential [Fe4S4] cluster in both oxidation states. The parameterization is compatible with AMBER force fields and enabled well-balanced molecular dynamics simulations of the p58c–RNA/DNA complex relevant to the initiation of DNA replication. Using the molecular mechanics Poisson–Boltzmann and surface area solvation method on the molecular dynamics trajectories, we find that the p58c mutation induces a modest change in the p58c–duplex binding free energy in agreement with recent experiments. Through kinetic modeling and analysis, we identify key features of the main charge transport pathways in p58c. In particular, we find that the Y345C mutation partially changes the composition and frequency of the most efficient (and potentially relevant to the biological function) charge transport pathways between the [Fe4S4] cluster and the duplex. Moreover, our approach sets the stage for a deeper understanding of functional charge transfer in [Fe4S4] protein–DNA complexes.

Role of Cdc23/Mcm10 in generating the ribonucleotide imprint at the mat1 locus in fission yeast.

The developmental asymmetry of fission yeast daughter cells derives from inheriting ‘older Watson’ versus ‘older Crick’ DNA strand from the parental cell, strands that are complementary but not identical with each other. A novel DNA strand-specific ‘imprint’, installed during DNA replication at the mating-type locus (mat1), imparts competence for cell type inter-conversion to one of the two chromosome replicas. The catalytic subunit of DNA Polymerase α (Polα) has been implicated in the imprinting process. Based on its known biochemical function, Polα might install the mat1 imprint during lagging strand synthesis. The nature of the imprint is not clear: it is either a nick or a ribonucleotide insertion. Our investigations do not support a direct role of Polα in nicking through putative endonuclease domains but confirm its indirect role in installing an alkali-labile moiety as the imprint. While ruling out the role of the primase subunit of Polα holoenzyme, we find that mutations in the Polα-recruitment and putative primase homology domain in Mcm10/Cdc23 abrogate the ribonucleotide imprint formation. These results, while confirming the ribonucleotide nature of the imprint suggest the possibility of a direct role of Mcm10/Cdc23 in installing it in cooperation with Polα and Swi1.