Comparison of the Proteome of Huh7 Cells Transfected with Hepatitis B Virus Subgenotype A1, with or without G1862T.

HBeAg is a non-structural, secreted protein of hepatitis B virus (HBV). Its p25 precursor is post-translationally modified in the endoplasmic reticulum. The G1862T precore mutation leads to the accumulation of P25 in the endoplasmic reticulum and activation of unfolded protein response. Using mass spectrometry, comparative proteome profiling of Huh-7 cells transfected with wildtype (WT) or G1862T revealed significantly differentially expressed proteins resulting in 12 dysregulated pathways unique to WT-transfected cells and 7 shared between cells transfected with either WT or G1862T. Except for the p38 MAPK signalling pathway, WT showed a higher number of DEPs than G1862T-transfected cells in all remaining six shared pathways. Two signalling pathways: oxidative stress and cell cycle signalling were differentially expressed only in cells transfected with G1862T. Fifteen pathways were dysregulated in G1862T-transfected cells compared to WT. The 15 dysregulated pathways were involved in the following processes: MAPK signalling, DNA synthesis and methylation, and extracellular matrix organization. Moreover, proteins involved in DNA synthesis signalling (replication protein A (RPA) and DNA primase (PRIM2)) were significantly upregulated in G1862T compared to WT. This upregulation was confirmed by mRNA quantification of both genes and immunofluorescent confocal microscopy for RPA only. The dysregulation of the pathways involved in these processes may lead to immune evasion, persistence, and uncontrolled proliferation, which are hallmarks of cancer.

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.

Telomere C-Strand Fill-In Machinery: New Insights into the Human CST-DNA Polymerase Alpha-Primase Structures and Functions.

Telomeres at the end of eukaryotic chromosomes are extended by a specialized set of enzymes and telomere-associated proteins, collectively termed here the telomere "replisome." The telomere replisome acts on a unique replicon at each chromosomal end of the telomeres, the 3' DNA overhang. This telomere replication process is distinct from the replisome mechanism deployed to duplicate the human genome. The G-rich overhang is first extended before the complementary C-strand is filled in. This overhang is extended by telomerase, a specialized ribonucleoprotein and reverse transcriptase. The overhang extension process is terminated when telomerase is displaced by CTC1-STN1-TEN1 (CST), a single-stranded DNA-binding protein complex. CST then recruits DNA polymerase α-primase to complete the telomere replication process by filling in the complementary C-strand. In this chapter, the recent structure-function insights into the human telomere C-strand fill-in machinery (DNA polymerase α-primase and CST) will be discussed.

POT1 recruits and regulates CST-Polα/primase at human telomeres.

Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.

Primase and polymerase α tango to make an RNA-DNA hybrid primer.

Several recent cryo-electron microscopy (cryo-EM) studies about the eukaryotic primosome, including the human primosome described by Yin et al. in this issue, have uncovered the structural intricacies between the RNA primase and the DNA polymerase. These studies show that these two partners tango on DNA to synthesize a hybrid primer composed of ~ 10 nucleotide (nt) RNA and ~ 10-nt DNA. They reveal key intermediate steps involved in this process; from the self-inhibited apo state to the initiation of RNA primer synthesis, RNA primer handover to the polymerase, primer elongation by polymerase, and finally, primer termination and release. Remarkably, the polymerase domain orchestrates all major steps during primer synthesis.

Replacement of sulfonamide by sulfoximine within a helicase-primase inhibitor with restricted flexibility.

Helicase-primase is an interesting target for the therapy of herpes simplex virus (HSV) infections. Since amenamevir is already approved for varicella-zoster virus (VZV) and HSV in Japan and pritelivir has received breakthrough therapy status for the treatment of acyclovir-resistant HSV infections in immunocompromised patients, the target has sparked interest in me-too approaches. Here, we describe the attempt to improve nervous tissue penetration in Phaeno Therapeutics drug candidate HN0037 to target the latent reservoir of HSV by installing less polar moieties, mainly a difluorophenyl instead of a pyridyl group, and replacing the primary sulfonamide with a methyl sulfoximine moiety. However, all obtained stereoisomers exhibited a weaker inhibitory activity on HSV-1 and HSV-2.

Evaluation of multiple displacement amplification for metagenomic analysis of low biomass samples.

Combining multiple displacement amplification (MDA) with metagenomics enables the analysis of samples with extremely low DNA concentrations, making them suitable for high-throughput sequencing. Although amplification bias and nonspecific amplification have been reported from MDA-amplified samples, the impact of MDA on metagenomic datasets is not well understood. We compared three MDA methods (i.e. bulk MDA, emulsion MDA, and primase MDA) for metagenomic analysis of two DNA template concentrations (approx. 1 and 100 pg) derived from a microbial community standard "mock community" and two low biomass environmental samples (i.e. borehole fluid and groundwater). We assessed the impact of MDA on metagenome-based community composition, assembly quality, functional profiles, and binning. We found amplification bias against high GC content genomes but relatively low nonspecific amplification such as chimeras, artifacts, or contamination for all MDA methods. We observed MDA-associated representational bias for microbial community profiles, especially for low-input DNA and with the primase MDA method. Nevertheless, similar taxa were represented in MDA-amplified libraries to those of unamplified samples. The MDA libraries were highly fragmented, but similar functional profiles to the unamplified libraries were obtained for bulk MDA and emulsion MDA at higher DNA input and across these MDA libraries for the groundwater sample. Medium to low-quality bins were possible for the high input bulk MDA metagenomes for the most simple microbial communities, borehole fluid, and mock community. Although MDA-based amplification should be avoided, it can still reveal meaningful taxonomic and functional information from samples with extremely low DNA concentration where direct metagenomics is otherwise impossible.

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.

The unfolded protein response of the endoplasmic reticulum protects Caenorhabditis elegans against DNA damage caused by stalled replication forks.

All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis, while DNA damage responses (DDRs) maintain genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but this remains poorly understood. Here, we demonstrate that depletion of the DNA primases pri-1 or pri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) in Caenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like endoplasmic reticulum kinase (pek-1) branches of the (UPR-ER). Interestingly, activation of the (UPR-ER) output gene heat shock protein 4 (hsp-4) was partially independent of its canonical activators, ire-1 and X-box binding protein (xbp-1), and instead required the third branch of the (UPR-ER), activating transcription factor 6 (atf-6), suggesting functional redundancy. We further found that primase depletion specifically induces the (UPR-ER), but not the distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss of ire-1 or pek-1 sensitizes animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis of pri-1 embryos revealed several deregulated processes that could cause (UPR-ER) activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the (UPR-ER), but not other UPRs, responds to replication fork stress and that the (UPR-ER) is required to alleviate this stress.

Mass Balance and Metabolite Profile after Single and Multiple Oral Doses of Pritelivir in Healthy Subjects.

Pritelivir is a helicase-primase inhibitor active against HSV. Two human mass balance trials (a multiple-dose trial and a single-dose trial) were performed to characterize the absorption, distribution, metabolism, and excretion of 100 mg oral pritelivir combined with a single microdose of 14C-pritelivir. Blood, urine, and feces samples were collected up to 26 days postdose. The plasma half-life of pritelivir was 63-67 hours. Overall, 92% and 66% of the administered dose was recovered in the multiple and single dose trials, respectively. The low recovery after the single dose (66%) was most likely related to the formulation used. The major metabolic pathway was amide hydrolysis leading to amino thiazole sulfonamide (ATS) and pyridinyl phenyl acetic acid (PPA). In plasma, pritelivir, ATS, PPA, and PPA-acyl glucuronide accounted for 40.6%, 9.4%, 5.1%, and 0.2% of total radioactivity. More than 90% of drug-related material was eliminated 624 hours postdose. The majority was excreted in urine (75% and 77%), followed by feces (16% and 23%). The main components in urine were PPA-acyl glucuronide (and its isomers), ATS, and its N-demethylated isomers. Only minor metabolites were observed in feces. In conclusion, the major metabolic pathways of pritelivir have been identified with the primary excretion route being renal.

Response of PRIMPOL-Knockout Human Lung Adenocarcinoma A549 Cells to Genotoxic Stress.

Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.

Coordination of Primer Initiation Within the Catalytic Domain of Human PrimPol.

To facilitate the eukaryotic repriming pathway of DNA damage tolerance, PrimPol synthesises de novo oligonucleotide primers downstream of polymerase-stalling obstacles. These primers enable replicative polymerases to resume synthesis and ensure the timely completion of DNA replication. Initiating synthesis de novo requires the coordination of single-stranded DNA, initiating nucleotides, and metal ions within PrimPol's active site to catalyze the formation of the first phosphodiester bond. Here we examine the interactions between human PrimPol's catalytic domain, nucleotides, and DNA template during each of the various catalytic steps to determine the 'choreography' of primer synthesis, where substrates bind in an ordered manner. Our findings show that the ability of PrimPol to conduct de novo primer synthesis is underpinned by a network of stabilising interactions between the enzyme, template, and nucleotides, as we previously observed for related primase CRISPR-Associated Prim-Pol (CAPP). Together, these findings establish a detailed model for the initiation of DNA synthesis by human PrimPol, which appears highly conserved.

Intermittent therapy with helicase-primase inhibitor IM-250 efficiently controls recurrent herpes disease and reduces reactivation of latent HSV.

Herpes is a contagious life-long infection with persistently high incidence and prevalence, causing significant disease worldwide. Current therapies have efficacy against active HSV infections but no impact on the latent viral reservoir in neurons. Thus, despite treatment, disease recurs from latency and the infectious potential remains unaffected within patients. Here, efficacy of the helicase-primase inhibitor (HPI) IM-250 against chronic neuronal HSV infections utilizing two classic herpes in vivo latency/reactivation animal models (intravaginal guinea pig HSV-2 infection model and ocular mouse HSV-1 infection model) is presented. Intermittent therapy of infected animals with 4-7 cycles of IM-250 during latency silences subsequent recurrences analyzed up to 6 months. In contrast to common experience, our studies show that the latent reservoir is indeed accessible to antiviral therapy altering the latent viral reservoir such that reactivation frequency can be reduced significantly by prior IM-250 treatment. We provide evidence that antiviral treatment during HSV latency can reduce future reactivation from the latent reservoir, supporting a conceptual shift in the antiviral field, and reframing what is achievable with respect to therapy of latent neuronal HSV infections.

Interaction and assembly of the DNA replication core proteins of Kaposi's sarcoma-associated herpesvirus.

IMPORTANCE: Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell's DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi's sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation.

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain.

BACKGROUND: The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD). RESULTS: Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase. CONCLUSIONS: We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.

Regulation of Human DNA Primase-Polymerase PrimPol.

Transmission of genetic information depends on successful completion of DNA replication. Genomic DNA is subjected to damage on a daily basis. DNA lesions create obstacles for DNA polymerases and can lead to the replication blockage, formation of DNA breaks, cell cycle arrest, and apoptosis. Cells have evolutionary adapted to DNA damage by developing mechanisms allowing elimination of lesions prior to DNA replication (DNA repair) and helping to bypass lesions during DNA synthesis (DNA damage tolerance). The second group of mechanisms includes the restart of DNA synthesis at the sites of DNA damage by DNA primase-polymerase PrimPol. Human PrimPol was described in 2013. The properties and functions of this enzyme have been extensively studied in recent years, but very little is known about the regulation of PrimPol and association between the enzyme dysfunction and diseases. In this review, we described the mechanisms of human PrimPol regulation in the context of DNA replication, discussed in detail interactions of PrimPol with other proteins, and proposed possible pathways for the regulation of human PrimPol activity. The article also addresses the association of PrimPol dysfunction with human diseases.

Models for human telomere C-strand fill-in by CST-Polα-primase.

Telomere maintenance is essential for the genome integrity of eukaryotes, and this function is underpinned by the two-step telomeric DNA synthesis process: telomere G-overhang extension by telomerase and complementary strand fill-in by DNA polymerase alpha-primase (polα-primase). Compared to the telomerase step, the telomere C-strand fill-in mechanism is less understood. Recent studies have provided new insights into how telomeric single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) and polα-primase coordinate to synthesize the telomeric C-strand for telomere overhang fill-in. Cryogenic electron microscopy (cryo-EM) structures of CST-polα-primase complexes have provided additional insights into how they assemble at telomeric templates and de novo synthesize the telomere C-strand. In this review, we discuss how these latest findings coalesce with existing understanding to develop a human telomere C-strand fill-in mechanism model.

How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication.

During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.

Primase-polymerases: how to make a primer from scratch.

To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicative primases (eukaryotes and archaea) belong to a functionally diverse enzyme superfamily known as Primase-Polymerases (Prim-Pols), with orthologues present throughout all domains of life. Characterised by a conserved catalytic Prim-Pol domain, these enzymes have evolved various roles in DNA metabolism, including DNA replication, repair, and damage tolerance. Many of these biological roles are fundamentally underpinned by the ability of Prim-Pols to generate primers de novo. This review examines our current understanding of the catalytic mechanisms utilised by Prim-Pols to initiate primer synthesis.

Association of Mutations in Replicative DNA Polymerase Genes with Human Disease: Possible Application of Drosophila Models for Studies.

Replicative DNA polymerases, such as DNA polymerase α-primase, δ and ε, are multi-subunit complexes that are responsible for the bulk of nuclear DNA replication during the S phase. Over the last decade, extensive genome-wide association studies and expression profiling studies of the replicative DNA polymerase genes in human patients have revealed a link between the replicative DNA polymerase genes and various human diseases and disorders including cancer, intellectual disability, microcephalic primordial dwarfism and immunodeficiency. These studies suggest the importance of dissecting the mechanisms involved in the functioning of replicative DNA polymerases in understanding and treating a range of human diseases. Previous studies in Drosophila have established this organism as a useful model to understand a variety of human diseases. Here, we review the studies on Drosophila that explored the link between DNA polymerases and human disease. First, we summarize the recent studies linking replicative DNA polymerases to various human diseases and disorders. We then review studies on replicative DNA polymerases in Drosophila. Finally, we suggest the possible use of Drosophila models to study human diseases and disorders associated with replicative DNA polymerases.