-
Current Opinion in Structural Biology Oct 2023Members of the primase-polymerase (Prim-Pol) superfamily are found in all domains of life and play diverse roles in genome stability, including primer synthesis during... (Review)
Review
Members of the primase-polymerase (Prim-Pol) superfamily are found in all domains of life and play diverse roles in genome stability, including primer synthesis during DNA replication, lesion repair and damage tolerance. This review focuses primarily on Prim-Pol members capable of de novo primer synthesis that have experimentally derived structural models available. We discuss the mechanism of DNA primer synthesis initiation by Prim-Pol catalytic domains, based on recent structural and functional studies. We also describe a general model for primer initiation that also includes the ancillary domains/subunits, which stimulate the initiation of primer synthesis.
Topics: DNA Primase; DNA Replication; Catalytic Domain
PubMed: 37459807
DOI: 10.1016/j.sbi.2023.102652 -
Proceedings of the National Academy of... Nov 2007Poxviruses are large enveloped viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. At least six virus-encoded proteins are required for...
Poxviruses are large enveloped viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. At least six virus-encoded proteins are required for synthesis and processing of the double-stranded DNA genome of vaccinia virus, the prototype member of the family. One of these proteins, D5, is an NTPase that contains an N-terminal archaeoeukaryotic primase domain and a C-terminal superfamily III helicase domain. Here we report that individual conserved aspartic acid residues in the predicted primase active site were required for in vivo complementation of infectious virus formation as well as genome and plasmid replication. Furthermore, purified recombinant D5 protein synthesized oligoribonucleotides in vitro. Incorporation of label from [alpha-(32)P]CTP or [alpha-(32)P]UTP into a RNase-sensitive and DNase-resistant product was demonstrated by using single-stranded circular bacteriophage DNA templates and depended on ATP or GTP and a divalent cation. Mutagenesis studies showed that the primase and NTPase activities of the recombinant D5 protein could be independently inactivated. Highly conserved orthologs of D5 are present in all poxviruses that have been sequenced, and more diverged orthologs are found in members of all other families of nucleocytoplasmic large DNA viruses. These viral primases may have roles in initiation of DNA replication or lagging-strand synthesis and represent potential therapeutic targets.
Topics: Acid Anhydride Hydrolases; Amino Acids; Binding Sites; Catalysis; DNA Primase; Genetic Vectors; Genome, Viral; HeLa Cells; Humans; Mutation; Oligonucleotides; Plasmids; Poxviridae
PubMed: 18000036
DOI: 10.1073/pnas.0709276104 -
Open Biology Jun 2021Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell.... (Review)
Review
Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell. Among these are the herpes virus DNA helicase and primase that are essential components of its replication machinery. In the HSV-1 replisome, the helicase-primase complex is composed of three components including UL5 (helicase), UL52 (primase) and UL8 (non-catalytic subunit). UL5 and UL52 subunits are functionally interdependent, and the UL8 component is required for the coordination of UL5 and UL52 activities proceeding in opposite directions with respect to the viral replication fork. Anti-viral compounds currently under development target the functions of UL5 and UL52. Here, we review the structural and functional properties of the UL5/UL8/UL52 complex and highlight the gaps in knowledge to be filled to facilitate molecular characterization of the structure and function of the helicase-primase complex for development of alternative anti-viral treatments.
Topics: Animals; Antiviral Agents; DNA Helicases; DNA Primase; Drug Development; Herpes Simplex; Herpesvirus 1, Human; Humans; Models, Molecular; Multienzyme Complexes; Protein Binding; Protein Interaction Domains and Motifs; Protein Subunits; Structure-Activity Relationship; Virus Replication
PubMed: 34102080
DOI: 10.1098/rsob.210011 -
International Journal of Molecular... Nov 2020Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and...
Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.
Topics: DNA; DNA Primase; DNA-Directed DNA Polymerase; Flap Endonucleases; Humans; Manganese; Multifunctional Enzymes; Nuclear Proteins; RNA; Replication Protein A; Substrate Specificity
PubMed: 33261049
DOI: 10.3390/ijms21239027 -
Science Advances Oct 2016PrimPol is a novel human enzyme that contains both DNA primase and DNA polymerase activities. We present the first structure of human PrimPol in ternary complex with a...
PrimPol is a novel human enzyme that contains both DNA primase and DNA polymerase activities. We present the first structure of human PrimPol in ternary complex with a DNA template-primer and an incoming deoxynucleoside triphosphate (dNTP). The ability of PrimPol to function as a DNA primase stems from a simple but remarkable feature-almost complete lack of contacts to the DNA primer strand. This, in turn, allows two dNTPs to bind initiation and elongation sites on the enzyme for the formation of the first dinucleotide. PrimPol shows the ability to synthesize DNA opposite ultraviolet (UV) lesions; however, unexpectedly, the active-site cleft of the enzyme is constrained, which precludes the bypass of UV-induced DNA lesions by conventional translesion synthesis. Together, the structure addresses long-standing questions about how DNA primases actually initiate synthesis and how primase and polymerase activities combine in a single enzyme to carry out DNA synthesis.
Topics: DNA; DNA Primase; DNA Repair; DNA-Directed DNA Polymerase; Humans; Multifunctional Enzymes; Protein Domains; Structure-Activity Relationship; Ultraviolet Rays
PubMed: 27819052
DOI: 10.1126/sciadv.1601317 -
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 -
Scientific Reports Nov 2016The structural differences between bacterial and human primases render the former an excellent target for drug design. Here we describe a technique for selecting small...
The structural differences between bacterial and human primases render the former an excellent target for drug design. Here we describe a technique for selecting small molecule inhibitors of the activity of T7 DNA primase, an ideal model for bacterial primases due to their common structural and functional features. Using NMR screening, fragment molecules that bind T7 primase were identified and then exploited in virtual filtration to select larger molecules from the ZINC database. The molecules were docked to the primase active site using the available primase crystal structure and ranked based on their predicted binding energies to identify the best candidates for functional and structural investigations. Biochemical assays revealed that some of the molecules inhibit T7 primase-dependent DNA replication. The binding mechanism was delineated via NMR spectroscopy. Our approach, which combines fragment based and virtual screening, is rapid and cost effective and can be applied to other targets.
Topics: Bacteriophage T7; Catalytic Domain; Crystallography, X-Ray; DNA Primase; DNA Replication; Enzyme Inhibitors; Humans; Models, Molecular; Molecular Docking Simulation; Nuclear Magnetic Resonance, Biomolecular; Small Molecule Libraries; Viral Proteins
PubMed: 27805033
DOI: 10.1038/srep36322 -
TheScientificWorldJournal Mar 2003DNA polymerase alpha (pol alpha) holds a special position among the growing family of eukaryotic DNA polymerases. In fact, pol alpha is associated with DNA primase to... (Review)
Review
DNA polymerase alpha (pol alpha) holds a special position among the growing family of eukaryotic DNA polymerases. In fact, pol alpha is associated with DNA primase to form a four subunit complex and, as a consequence, is the only enzyme able to start DNA synthesis de novo. Because of this peculiarity the major role of the DNA polymerase alpha-primase complex (pol-prim) is in the initiation of DNA replication at chromosomal origins and in the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. However, pol-prim seems to play additional roles in other complex cellular processes, such as the response to DNA damage, telomere maintenance, and the epigenetic control of higher order chromatin assembly.
Topics: Animals; Cell Cycle; DNA; DNA Damage; DNA Polymerase I; DNA Primase; DNA Replication; Eukaryotic Cells; Gene Expression Regulation; Macromolecular Substances; Models, Genetic
PubMed: 12806117
DOI: 10.1100/tsw.2003.05 -
Science Advances Apr 2023Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different...
Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different translesion DNA synthesis (TLS) polymerases by the use of tools for their depletion, we identified a unique role of Pol ι in regulating such a pathway choice. Pol ι deficiency unleashes PrimPol-dependent repriming, which accelerates DNA replication in a pathway that is epistatic with ZRANB3 knockdown. In Pol ι-depleted cells, the excess participation of PrimPol in nascent DNA elongation reduces replication stress signals, but thereby also checkpoint activation in S phase, triggering chromosome instability in M phase. This TLS-independent function of Pol ι requires its PCNA-interacting but not its polymerase domain. Our findings unravel an unanticipated role of Pol ι in protecting the genome stability of cells from detrimental changes in DNA replication dynamics caused by PrimPol.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Replication; DNA; DNA Repair; DNA Damage; Chromosomal Instability; DNA Primase; Multifunctional Enzymes
PubMed: 37058556
DOI: 10.1126/sciadv.ade7997 -
Nucleic Acids Research Aug 2023Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria....
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Primase; DNA Replication; DNA; DNA Primers; Catalysis; Multifunctional Enzymes
PubMed: 37326028
DOI: 10.1093/nar/gkad507