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Nature Aug 2022Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of...
Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand and TPP1 (in complex with TIN2) recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The telomere DNA ends are replicated and maintained by telomerase, for the G-strand, and subsequently DNA polymerase α-primase (PolαPrim), for the C-strand. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN1 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.
Topics: Cryoelectron Microscopy; DNA; DNA Primase; Holoenzymes; Protein Binding; Shelterin Complex; Telomerase; Telomere; Tetrahymena
PubMed: 35831498
DOI: 10.1038/s41586-022-04931-7 -
Nucleic Acids Research Oct 2011The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the...
The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA-DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA-DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.
Topics: Amino Acid Sequence; DNA Polymerase I; DNA Primase; Models, Molecular; Molecular Sequence Data; Protein Subunits; RNA; Saccharomyces cerevisiae
PubMed: 21715379
DOI: 10.1093/nar/gkr534 -
Nucleic Acids Research May 2012Conserved Iron-Sulfur (Fe-S) clusters are found in a growing family of metalloproteins that are implicated in prokaryotic and eukaryotic DNA replication and repair.... (Review)
Review
Conserved Iron-Sulfur (Fe-S) clusters are found in a growing family of metalloproteins that are implicated in prokaryotic and eukaryotic DNA replication and repair. Among these are DNA helicase and helicase-nuclease enzymes that preserve chromosomal stability and are genetically linked to diseases characterized by DNA repair defects and/or a poor response to replication stress. Insight to the structural and functional importance of the conserved Fe-S domain in DNA helicases has been gleaned from structural studies of the purified proteins and characterization of Fe-S cluster site-directed mutants. In this review, we will provide a current perspective of what is known about the Fe-S cluster helicases, with an emphasis on how the conserved redox active domain may facilitate mechanistic aspects of helicase function. We will discuss testable models for how the conserved Fe-S cluster might operate in helicase and helicase-nuclease enzymes to conduct their specialized functions that help to preserve the integrity of the genome.
Topics: Amino Acid Sequence; DNA; DNA Glycosylases; DNA Helicases; DNA Primase; Deoxyribonucleases; Iron-Sulfur Proteins; Molecular Sequence Data; Protein Structure, Tertiary
PubMed: 22287629
DOI: 10.1093/nar/gks039 -
Scientific Reports Oct 2021PrimPol is a novel Primase-Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in...
Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast.
PrimPol is a novel Primase-Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3'-GTCG-5' DNA sequence, where the 3'-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.
Topics: Arabidopsis; Arabidopsis Proteins; Cell Nucleus; DNA; DNA Damage; DNA Primase; DNA Repair; DNA Replication; DNA, Single-Stranded; DNA-Directed DNA Polymerase; Mitochondria; Multifunctional Enzymes
PubMed: 34663822
DOI: 10.1038/s41598-021-00151-7 -
Acta Crystallographica. Section F,... Feb 2014Human primase synthesizes RNA primers and transfers them to the active site of Pol α with subsequent extension with dNTPs. Human primase is a heterodimer of two...
Human primase synthesizes RNA primers and transfers them to the active site of Pol α with subsequent extension with dNTPs. Human primase is a heterodimer of two subunits: a small catalytic subunit (p49) and a large subunit (p58). The structural details of the initiation and elongation steps of primer synthesis, as well as primer length counting, are not known. To address these questions, structural studies of human primase were initiated. Two types of crystals were obtained. The best diffracting crystals belonged to space group P1, with unit-cell parameters a = 86.2, b = 88.9, c = 94.68 Å, α = 93.82, β = 96.57, γ = 111.72°, and contained two heterodimers of full-length p49 and p59 subunits in the asymmetric unit.
Topics: Crystallization; Crystallography, X-Ray; DNA Primase; Electrophoresis, Polyacrylamide Gel; Humans; Protein Conformation
PubMed: 24637758
DOI: 10.1107/S2053230X13034432 -
Molecular Cell Jun 2020DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork...
DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.
Topics: Cell Line, Tumor; DNA; DNA Damage; DNA Primase; DNA Repair; DNA Replication; DNA-Binding Proteins; DNA-Directed DNA Polymerase; HEK293 Cells; Humans; K562 Cells; Multifunctional Enzymes; Nucleotidyltransferases; Transcription Factors
PubMed: 32442397
DOI: 10.1016/j.molcel.2020.04.031 -
Scientific Reports Oct 2017For DNA replication in vivo, DNA primase uses a complementary single-stranded DNA template to synthesize RNA primers ranging from 4 to 20 nucleotides in length, which...
For DNA replication in vivo, DNA primase uses a complementary single-stranded DNA template to synthesize RNA primers ranging from 4 to 20 nucleotides in length, which are then elongated by DNA polymerase. Here, we report that, in the presence of double-stranded DNA, the thermophilic DNA primase TtDnaG2 synthesizes RNA primers of around 100 nucleotides with low initiation specificity at 70 °C. Analysing the structure of TtDnaG2, we identified that it adopts a compact conformation. The conserved sites in its zinc binding domain are sequestered away from its RNA polymerase domain, which might give rise to the low initiation specificity and synthesis of long RNA segments by TtDnaG2. Based on these unique features of TtDnaG2, a DNA amplification method has been developed. We utilized TtDnaG2 to synthesize RNA primers at 70 °C after 95 °C denaturation, followed by isothermal amplification with the DNA polymerase Bst3.0 or phi29. Using this method, we successfully amplified genomic DNA of a virus with 100% coverage and low copy number variation. Our data also demonstrate that this method can efficiently amplify circular DNA from a mixture of circular DNA and linear DNA, thus providing a tool to amplify low-copy-number circular DNA such as plasmids.
Topics: Amino Acid Sequence; Bacterial Proteins; DNA; DNA Primase; DNA, Circular; Genome, Viral; Nucleic Acid Amplification Techniques; Nucleic Acid Denaturation; RNA; RNA, Bacterial; Temperature; Templates, Genetic; Thermoanaerobacter
PubMed: 28993626
DOI: 10.1038/s41598-017-12241-6 -
Nature Communications Jan 2024Transcription-replication conflicts (TRCs), especially Head-On TRCs (HO-TRCs) can introduce R-loops and DNA damage, however, the underlying mechanisms are still largely...
Transcription-replication conflicts (TRCs), especially Head-On TRCs (HO-TRCs) can introduce R-loops and DNA damage, however, the underlying mechanisms are still largely unclear. We previously identified a chloroplast-localized RNase H1 protein AtRNH1C that can remove R-loops and relax HO-TRCs for genome integrity. Through the mutagenesis screen, we identify a mutation in chloroplast-localized primase ATH that weakens the binding affinity of DNA template and reduces the activities of RNA primer synthesis and delivery. This slows down DNA replication, and reduces competition of transcription-replication, thus rescuing the developmental defects of atrnh1c. Strand-specific DNA damage sequencing reveals that HO-TRCs cause DNA damage at the end of the transcription unit in the lagging strand and overexpression of ATH can boost HO-TRCs and exacerbates DNA damage. Furthermore, mutation of plastid DNA polymerase Pol1A can similarly rescue the defects in atrnh1c mutants. Taken together these results illustrate a potentially conserved mechanism among organisms, of which the primase activity can promote the occurrence of transcription-replication conflicts leading to HO-TRCs and genome instability.
Topics: DNA Primase; DNA Replication; DNA-Directed DNA Polymerase; DNA Damage; Mutation
PubMed: 38168108
DOI: 10.1038/s41467-023-44443-0 -
The Journal of Biological Chemistry Sep 1985A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at...
A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at least a primase and DNA polymerase gamma and require that rNTPs as well as dNTPs be present in the reaction mixture. Poly(dC) and poly(dT), as well as M13 phage DNA, are excellent templates for the primase activity. A single-stranded DNA containing the cloned origin of mitochondrial light-strand synthesis can be a more efficient template than M13 phage DNA alone. Primase and DNA polymerase activities were separated from each other by sedimentation in a glycerol density gradient. Using M13 phage DNA as template, these mitochondrial enzymes synthesize RNA primers that are 9 to 12 nucleotides in size and are covalently linked to nascent DNA. The formation of primers appears to be the rate-limiting step in the replication process. Replication of M13 DNA is sensitive to N-ethylmaleimide and dideoxynucleoside triphosphates, but insensitive to rifampicin, alpha-amanitin, and aphidicolin.
Topics: DNA Primase; DNA Replication; DNA-Directed DNA Polymerase; Humans; In Vitro Techniques; Mitochondria; Oligonucleotides; RNA Nucleotidyltransferases
PubMed: 4044569
DOI: No ID Found -
ELife Sep 2023The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA...
The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA and binds to more than 20 partner proteins. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimic ssDNA. NMR chemical-shift studies also provided valuable information on the interaction of RPA70N residues with target sequences. However, it is still unclear how RPA70N recognizes and distinguishes such a diverse group of target proteins. Here, we present high-resolution crystal structures of RPA70N in complex with peptides from eight DNA damage response proteins. The structures show that, in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners. Our results advance the mechanistic understanding of RPA70N-mediated recruitment of DNA damage response proteins.
Topics: Humans; Crystallography; DNA Damage; DNA Primase; DNA, Single-Stranded; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Eukaryota; Multifunctional Enzymes; Replication Protein A
PubMed: 37668474
DOI: 10.7554/eLife.81639