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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 -
Advances in Experimental Medicine and... 2017Eukaryotic replication proteins are highly conserved, and thus study of Saccharomyces cerevisiae replication can inform about this central process in higher eukaryotes... (Review)
Review
Eukaryotic replication proteins are highly conserved, and thus study of Saccharomyces cerevisiae replication can inform about this central process in higher eukaryotes including humans. The S. cerevisiae replisome is a large and dynamic assembly comprised of ~50 proteins. The core of the replisome is composed of 31 different proteins including the 11-subunit CMG helicase; RFC clamp loader pentamer; PCNA clamp; the heteroligomeric DNA polymerases ε, δ, and α-primase; and the RPA heterotrimeric single strand binding protein. Many additional protein factors either travel with or transiently associate with these replisome proteins at particular times during replication. In this chapter, we summarize several recent structural studies on the S. cerevisiae replisome and its subassemblies using single particle electron microscopy and X-ray crystallography. These recent structural studies have outlined the overall architecture of a core replisome subassembly and shed new light on the mechanism of eukaryotic replication.
Topics: Animals; DNA Helicases; DNA Polymerase II; DNA Replication; Humans; Protein Multimerization; Protein Structure, Quaternary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 29357060
DOI: 10.1007/978-981-10-6955-0_10 -
Current Biology : CB Mar 2016The machinery at the eukaryotic replication fork has seen many new structural advances using electron microscopy and crystallography. Recent structures of eukaryotic... (Review)
Review
The machinery at the eukaryotic replication fork has seen many new structural advances using electron microscopy and crystallography. Recent structures of eukaryotic replisome components include the Mcm2-7 complex, the CMG helicase, DNA polymerases, a Ctf4 trimer hub and the first look at a core replisome of 20 different proteins containing the helicase, primase, leading polymerase and a lagging strand polymerase. The eukaryotic core replisome shows an unanticipated architecture, with one polymerase sitting above the helicase and the other below. Additionally, structures of Mcm2 bound to an H3/H4 tetramer suggest a direct role of the replisome in handling nucleosomes, which are important to DNA organization and gene regulation. This review provides a summary of some of the many recent advances in the structure of the eukaryotic replisome.
Topics: Animals; Crystallography; DNA Helicases; DNA Replication; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Microscopy, Electron; Minichromosome Maintenance Proteins; Nucleosomes; Saccharomyces cerevisiae Proteins
PubMed: 27003891
DOI: 10.1016/j.cub.2016.02.034 -
Essays in Biochemistry Aug 2023Viruses have developed sophisticated biochemical and genetic mechanisms to manipulate and exploit their hosts. Enzymes derived from viruses have been essential research... (Review)
Review
Viruses have developed sophisticated biochemical and genetic mechanisms to manipulate and exploit their hosts. Enzymes derived from viruses have been essential research tools since the first days of molecular biology. However, most viral enzymes that have been commercialized are derived from a small number of cultivated viruses, which is remarkable considering the extraordinary diversity and abundance of viruses revealed by metagenomic analysis. Given the explosion of new enzymatic reagents derived from thermophilic prokaryotes over the past 40 years, those obtained from thermophilic viruses should be equally potent tools. This review discusses the still-limited state of the art regarding the functional biology and biotechnology of thermophilic viruses with a focus on DNA polymerases, ligases, endolysins, and coat proteins. Functional analysis of DNA polymerases and primase-polymerases from phages infecting Thermus, Aquificaceae, and Nitratiruptor has revealed new clades of enzymes with strong proofreading and reverse transcriptase capabilities. Thermophilic RNA ligase 1 homologs have been characterized from Rhodothermus and Thermus phages, with both commercialized for circularization of single-stranded templates. Endolysins from phages infecting Thermus, Meiothermus, and Geobacillus have shown high stability and unusually broad lytic activity against Gram-negative and Gram-positive bacteria, making them targets for commercialization as antimicrobials. Coat proteins from thermophilic viruses infecting Sulfolobales and Thermus strains have been characterized, with diverse potential applications as molecular shuttles. To gauge the scale of untapped resources for these proteins, we also document over 20,000 genes encoded by uncultivated viral genomes from high-temperature environments that encode DNA polymerase, ligase, endolysin, or coat protein domains.
Topics: Viruses; Bacteriophages; Bacteria; DNA-Directed DNA Polymerase; Biotechnology; Ligases; Biology
PubMed: 37222046
DOI: 10.1042/EBC20220209 -
Genes Jan 2017The complex molecular machines responsible for genome replication encounter many obstacles during their progression along DNA. Tolerance of these obstructions is... (Review)
Review
The complex molecular machines responsible for genome replication encounter many obstacles during their progression along DNA. Tolerance of these obstructions is critical for efficient and timely genome duplication. In recent years, primase-polymerase (PrimPol) has emerged as a new player involved in maintaining eukaryotic replication fork progression. This versatile replicative enzyme, a member of the archaeo-eukaryotic primase (AEP) superfamily, has the capacity to perform a range of template-dependent and independent synthesis activities. Here, we discuss the emerging roles of PrimPol as a leading strand repriming enzyme and describe the mechanisms responsible for recruiting and regulating the enzyme during this process. This review provides an overview and update of the current PrimPol literature, as well as highlighting unanswered questions and potential future avenues of investigation.
PubMed: 28067825
DOI: 10.3390/genes8010020 -
Advances in Experimental Medicine and... 2021The discovery of the nucleoside analogue, acyclovir, represented a milestone in the management of infections caused by herpes simplex virus and varicella-zoster virus....
The discovery of the nucleoside analogue, acyclovir, represented a milestone in the management of infections caused by herpes simplex virus and varicella-zoster virus. Ganciclovir, another nucleoside analogue, was then used for the management of systemic and organ-specific human cytomegalovirus diseases. The pyrophosphate analogue, foscarnet, and the nucleotide analogue, cidofovir, have been approved subsequently and constitute the second-line antiviral drugs. However, the viral DNA polymerase is the ultimate target of all these antiviral agents with a possible emergence of cross-resistance between these drugs. Recently, letermovir that targets the viral terminase complex was approved for the prophylaxis of human cytomegalovirus infections in hematopoietic stem cell transplant recipients. Other viral targets such as the protein kinase and the helicase-primase complex are also evaluated for the development of novel potent inhibitors against herpesviruses.
Topics: Antiviral Agents; Cidofovir; Cytomegalovirus; Drug Resistance, Viral; Humans; Simplexvirus
PubMed: 34258735
DOI: 10.1007/978-981-16-0267-2_1 -
Nature Aug 2022Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins. Replication of telomeres allows continued proliferation of human...
Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells. This replication requires telomerase extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)); the synthesis of the complementary C-strand ((CCCTAA)) is much less well characterized. The CST (CTC1-STN1-TEN1) protein complex, a DNA polymerase α-primase accessory factor, is known to be required for telomere replication in vivo, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3' overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension and recruits Polα-primase to telomeric ssDNA but also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.
Topics: DNA Primase; DNA Replication; DNA, Single-Stranded; G-Quadruplexes; Humans; Replicon; Shelterin Complex; Telomerase; Telomere
PubMed: 35831508
DOI: 10.1038/s41586-022-04930-8 -
The Enzymes 2016The initiation of chromosomal DNA replication starts at a replication origin, which in bacteria is a discrete locus that contains DNA sequence motifs recognized by an... (Review)
Review
The initiation of chromosomal DNA replication starts at a replication origin, which in bacteria is a discrete locus that contains DNA sequence motifs recognized by an initiator protein whose role is to assemble the replication fork machinery at this site. In bacteria with a single chromosome, DnaA is the initiator and is highly conserved in all bacteria. As an adenine nucleotide binding protein, DnaA bound to ATP is active in the assembly of a DnaA oligomer onto these sites. Other proteins modulate DnaA oligomerization via their interaction with the N-terminal region of DnaA. Following the DnaA-dependent unwinding of an AT-rich region within the replication origin, DnaA then mediates the binding of DnaB, the replicative DNA helicase, in a complex with DnaC to form an intermediate named the prepriming complex. In the formation of this intermediate, the helicase is loaded onto the unwound region within the replication origin. As DnaC bound to DnaB inhibits its activity as a DNA helicase, DnaC must dissociate to activate DnaB. Apparently, the interaction of DnaB with primase (DnaG) and primer formation leads to the release of DnaC from DnaB, which is coordinated with or followed by translocation of DnaB to the junction of the replication fork. There, DnaB is able to coordinate its activity as a DNA helicase with the cellular replicase, DNA polymerase III holoenzyme, which uses the primers made by primase for leading strand DNA synthesis.
Topics: Bacteria; Bacterial Proteins; DNA Helicases; DNA Replication; Replication Origin
PubMed: 27241926
DOI: 10.1016/bs.enz.2016.03.001 -
Protein Science : a Publication of the... Jun 2019DNA replication mechanisms are conserved across all organisms. The proteins required to initiate, coordinate, and complete the replication process are best characterized... (Review)
Review
DNA replication mechanisms are conserved across all organisms. The proteins required to initiate, coordinate, and complete the replication process are best characterized in model organisms such as Escherichia coli. These include nucleotide triphosphate-driven nanomachines such as the DNA-unwinding helicase DnaB and the clamp loader complex that loads DNA-clamps onto primer-template junctions. DNA-clamps are required for the processivity of the DNA polymerase III core, a heterotrimer of α, ε, and θ, required for leading- and lagging-strand synthesis. DnaB binds the DnaG primase that synthesizes RNA primers on both strands. Representative structures are available for most classes of DNA replication proteins, although there are gaps in our understanding of their interactions and the structural transitions that occur in nanomachines such as the helicase, clamp loader, and replicase core as they function. Reviewed here is the structural biology of these bacterial DNA replication proteins and prospects for future research.
Topics: DNA Replication; DNA, Bacterial; Escherichia coli; Models, Molecular
PubMed: 30945375
DOI: 10.1002/pro.3615