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Journal of Virology May 2022Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes high mortality in piglets. Interferon (IFN) responses are the primary...
Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes high mortality in piglets. Interferon (IFN) responses are the primary defense mechanism against viral infection; however, viruses always evolve elaborate strategies to antagonize the antiviral action of IFN. Previous study showed that PEDV nonstructural protein 7 (nsp7), a component of the viral replicase polyprotein, can antagonize ploy(I:C)-induced type I IFN production. Here, we found that PEDV nsp7 also antagonized IFN-α-induced JAK-STAT signaling and the production of IFN-stimulated genes. PEDV nsp7 did not affect the protein and phosphorylation levels of JAK1, Tyk2, STAT1, and STAT2 or the formation of the interferon-stimulated gene factor 3 (ISGF3) complex. However, PEDV nsp7 prevented the nuclear translocation of STAT1 and STAT2. Mechanistically, PEDV nsp7 interacted with the DNA binding domain of STAT1/STAT2, which sequestered the interaction between karyopherin α1 (KPNA1) and STAT1, thereby blocking the nuclear transport of ISGF3. Collectively, these data reveal a new mechanism developed by PEDV to inhibit type I IFN signaling pathway. In recent years, an emerging porcine epidemic diarrhea virus (PEDV) variant has gained attention because of serious outbreaks of piglet diarrhea in China and the United States. Coronavirus nonstructural protein 7 (nsp7) has been proposed to act with nsp8 as part of an RNA primase to generate RNA primers for viral RNA synthesis. However, accumulating evidence indicates that coronavirus nsp7 can also antagonize type I IFN production. Our present study extends previous findings and demonstrates that PEDV nsp7 also antagonizes IFN-α-induced IFN signaling by competing with KPNA1 for binding to STAT1, thereby enriching the immune regulation function of coronavirus nsp7.
Topics: Animals; Cell Line; Interferons; Janus Kinase 1; Porcine epidemic diarrhea virus; STAT1 Transcription Factor; Signal Transduction; Swine; Viral Nonstructural Proteins; alpha Karyopherins
PubMed: 35442061
DOI: 10.1128/jvi.00400-22 -
PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy.Biomolecules Feb 2022DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One... (Review)
Review
DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with conventional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a DNA primase, elongating primers that PrimPol itself sythesizes, or as translesion synthesis (TLS) DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only acts as a DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer.
Topics: DNA Primase; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Multifunctional Enzymes; Neoplasms
PubMed: 35204749
DOI: 10.3390/biom12020248 -
Current Genetics Apr 2022Dbf4 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...
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 Zn 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 Zn 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.
Topics: Cell Cycle Proteins; DNA Replication; DNA, Single-Stranded; Protein Serine-Threonine Kinases; Replication Origin; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Zinc
PubMed: 35147742
DOI: 10.1007/s00294-022-01230-6 -
Frontiers in Microbiology 2021[This corrects the article DOI: 10.3389/fmicb.2021.766612.].
[This corrects the article DOI: 10.3389/fmicb.2021.766612.].
PubMed: 35087509
DOI: 10.3389/fmicb.2021.838050 -
FEBS Open Bio Mar 2022The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function...
The initiation of Okazaki fragment synthesis during cellular DNA replication is a crucial step for lagging strand synthesis, which is carried out by the primase function of DNA polymerase α-primase (Pol-prim). Since cellular replication protein A (RPA) prevents primase from starting RNA synthesis on single-stranded DNA (ssDNA), primase requires auxiliary factors, such as the simian virus 40 (SV40) T antigen (Tag), for the initiation reaction on RPA-bound ssDNA. Here, we investigated the ability of Tag variants and Tag protein complexes to bind to ssDNA and their resulting effects on the stimulation of Pol-prim on free and RPA-bound ssDNA. Atomic force microscopy imaging showed that while Tag (V350E/P417D) and Tag (L286D/R567E) (abbreviated as M1 and M2, respectively) could bind to ssDNA as monomers, these monomeric Tags could come together and bind to ssDNA as dimers as well. In a model assay for the initiation of Okazaki fragment synthesis, full-length Tag SV40 Tag and monomeric M2 stimulated DNA synthesis of Pol-prim on ssDNA and on RPA-bound ssDNA. In contrast, neither monomeric M1 nor M1-M2 dimers could stimulate Pol-prim, on ssDNA or on RPA-bound ssDNA. Overall, we show that a lack of stimulatory activity of monomeric M1 and M1-M2 dimers suggests that residues V350 and P417 are not only important for interactions between Tag molecules but also for protein-protein interactions within Okazaki fragment initiation complexes. Thus, we highlight that mutations in M1 are dominant negative with regard to Okazaki fragment initiation.
Topics: Antigens, Viral, Tumor; DNA; Replication Protein A; Simian virus 40
PubMed: 35073603
DOI: 10.1002/2211-5463.13373 -
Nature Communications Jan 2022Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then...
Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then extended by the DNA polymerase. In diverse archaeal species, the primase has long been known to have the ability to synthesize both RNA and DNA. However, the relevance of these dual nucleic acid synthetic modes for productive primer synthesis has remained enigmatic. In the current work, we reveal that the ability of primase to polymerize DNA serves dual roles in promoting the hand-off of the primer to the replicative DNA polymerase holoenzyme. First, it creates a 5'-RNA-DNA-3' hybrid primer which serves as an optimal substrate for elongation by the replicative DNA polymerase. Second, it promotes primer release by primase. Furthermore, modeling and experimental data indicate that primase incorporates a deoxyribonucleotide stochastically during elongation and that this switches the primase into a dedicated DNA synthetic mode polymerase.
Topics: DNA Primase; DNA Primers; DNA Replication; DNA, Archaeal; DNA-Directed DNA Polymerase; Fluorescence Polarization; Kinetics; Models, Biological; Nucleotides; Polymerization; RNA, Archaeal; Stochastic Processes
PubMed: 35064114
DOI: 10.1038/s41467-022-28093-2 -
Nature Cell Biology Jan 2022The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit...
The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit single-stranded DNA at double-strand breaks (DSBs) by blocking resection and/or through CST-Polα-primase-mediated fill-in. We show that primase (like 53BP1-shieldin and CST-Polα) promotes radial chromosome formation in PARPi-treated BRCA1-deficient cells and demonstrate shieldin-CST-Polα-primase-dependent incorporation of BrdU at DSBs. In the absence of 53BP1 or shieldin, radial formation in BRCA1-deficient cells was restored by the tethering of CST near DSBs, arguing that in this context, shieldin acts primarily by recruiting CST. Furthermore, a SHLD1 mutant defective in CST binding (SHLD1Δ) was non-functional in BRCA1-deficient cells and its function was restored after reconnecting SHLD1Δ to CST. Interestingly, at dysfunctional telomeres and at DNA breaks in class switch recombination where CST has been implicated, SHLD1Δ was fully functional, perhaps because these DNA ends carry CST recognition sites that afford SHLD1-independent binding of CST. These data establish that in BRCA1-deficient cells, CST-Polα-primase is the major effector of shieldin-dependent DSB processing.
Topics: Animals; BRCA1 Protein; Binding Sites; CRISPR-Cas Systems; Cell Line, Tumor; DNA; DNA Breaks, Double-Stranded; DNA Polymerase I; DNA Primase; DNA Repair; Gene Knockout Techniques; Humans; Mice; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rad51 Recombinase; Shelterin Complex; Telomere-Binding Proteins; Tumor Suppressor p53-Binding Protein 1
PubMed: 35027730
DOI: 10.1038/s41556-021-00812-9 -
Cell & Bioscience Jan 2022Ranaviruses (family Iridoviridae) are promiscuous pathogens that can infect across species barriers in poikilotherms and can replicate in amphibian and fish cells and...
BACKGROUND
Ranaviruses (family Iridoviridae) are promiscuous pathogens that can infect across species barriers in poikilotherms and can replicate in amphibian and fish cells and even in cultured mammalian cells. However, as nucleocytoplasmic large DNA viruses (NCLDVs), their replication and transcription mechanisms remain largely unknown. Here, we screened and uncovered the replication and transcription machinery of two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV), by a combination of methods, including the isolation of proteins on nascent DNA, recombinant virus-based affinity, and NanoLuc complementation assay.
RESULTS
The ranavirus replication and transcription machinery was deeply dissected and identified as a complicated apparatus containing at least 30 viral and 6 host proteins. The viral proteins ADRV-47L/RGV-63R (DNA polymerase, vDPOL), ADRV-23L/RGV-91R (proliferating cell nuclear antigen, vPCNA), ADRV-85L/RGV-27R (single-stranded DNA binding protein, vSSB), ADRV-88L/RGV-24R (vhelicase/primase), etc., constitute the core replisome. Specifically, the core of the transcription complex, the viral RNA polymerase, contain the host RNAPII subunits Rpb3, Rpb6, and Rpb11, which was a first report in NCLDVs. Furthermore, correlations and interactions among these factors in the machinery were described. Significantly, the replisome core protein vDPOL (ADRV-47L) can interact with numerous viral and host proteins and could act as a linker and regulation center in viral DNA replication and transcription. Thus, these results depicted an architecture for ranavirus replication and transcription.
CONCLUSIONS
Up to 36 components from ranavirus and their host were found to form viral replisomes and transcription complexes using a series of precise methods, which further constructed an architecture for ranavirus replication and transcription in which vDPOL was a key central factor and various components correlated and cooperated. Therefore, it provides a cornerstone for further understanding the mechanisms of the replication and transcription of ranaviruses which can ensure the efficient production of progeny virus and adaptation to cross-species infection.
PubMed: 34991685
DOI: 10.1186/s13578-021-00742-x -
Frontiers in Microbiology 2021PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative...
PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative polymerase from NrS-1 phage (NrSPol) is a representative of the PrimPols. In this study, we identified key residues for the catalytic activity of NrSPol and found that a loop in NrSPol functionally replaces the zinc finger motif that is commonly found in other AEP family proteins. A helix bundle domain (HBD), conserved in the AEP superfamily, was recently reported to bind to the primase recognition site and to be crucial for initiation of primer synthesis. We found that NrSPol can recognize different primase recognition sites, and that the initiation site for primer synthesis is not stringent, suggesting that the HBD conformation is flexible. More importantly, we found that although the HBD-inactivating mutation impairs the primase activity of NrSPol, it significantly enhances the DNA polymerase activity, indicating that the HBD hinders the DNA polymerase activity. The conflict between the primase activity and the DNA polymerase activity in a single protein with the same catalytic domain may be one reason for why DNA polymerases are generally unable to synthesize DNA .
PubMed: 34975792
DOI: 10.3389/fmicb.2021.766612 -
Frontiers in Genetics 2021Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the...
Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, NUBPL, the known ISC donor for respiratory complex I and a putative donor for mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.
PubMed: 34950192
DOI: 10.3389/fgene.2021.790521