-
The FEBS Journal Apr 2024Eukaryotic 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...
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.
Topics: Humans; DNA Primase; Cryoelectron Microscopy; DNA Polymerase I; RNA; DNA Replication
PubMed: 38335062
DOI: 10.1111/febs.17082 -
Biochemistry. Biokhimiia Nov 2023Human 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...
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.
Topics: Humans; DNA-Directed DNA Polymerase; A549 Cells; Cisplatin; Hydrogen Peroxide; DNA Replication; DNA Damage; Adenocarcinoma of Lung; DNA Primase; Multifunctional Enzymes
PubMed: 38105210
DOI: 10.1134/S0006297923110214 -
Nucleic Acids Research Apr 2024DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate...
DNA replication stress, caused by various endogenous and exogenous agents, halt or stall DNA replication progression. Cells have developed diverse mechanisms to tolerate and overcome replication stress, enabling them to continue replication. One effective strategy to overcome stalled replication involves skipping the DNA lesion using a specialized polymerase known as PrimPol, which reinitiates DNA synthesis downstream of the damage. However, the mechanism regulating PrimPol repriming is largely unclear. In this study, we observe that knockdown of STN1 or CTC1, components of the CTC1/STN1/TEN1 complex, leads to enhanced replication progression following UV exposure. We find that such increased replication is dependent on PrimPol, and PrimPol recruitment to stalled forks increases upon CST depletion. Moreover, we find that p21 is upregulated in STN1-depleted cells in a p53-independent manner, and p21 depletion restores normal replication rates caused by STN1 deficiency. We identify that p21 interacts with PrimPol, and STN1 depletion stimulates p21-PrimPol interaction and facilitates PrimPol recruitment to stalled forks. Our findings reveal a previously undescribed interplay between CST, PrimPol and p21 in promoting repriming in response to stalled replication, and shed light on the regulation of PrimPol repriming at stalled forks.
Topics: Humans; DNA Replication; Cyclin-Dependent Kinase Inhibitor p21; Ultraviolet Rays; DNA Primase; DNA-Directed DNA Polymerase; Telomere-Binding Proteins; Multifunctional Enzymes; Tumor Suppressor Protein p53; DNA Damage
PubMed: 38348929
DOI: 10.1093/nar/gkae078 -
International Journal of Molecular... Dec 2023PrimPol is a DNA primase/polymerase from the Archaeo-Eukaryotic Primase (AEP) superfamily that enables the progression of stalled replication forks by synthesizing DNA...
PrimPol is a DNA primase/polymerase from the Archaeo-Eukaryotic Primase (AEP) superfamily that enables the progression of stalled replication forks by synthesizing DNA primers ahead of blocking lesions or abnormal structures in the ssDNA template. PrimPol's active site is formed by three AEP-conserved motifs: A, B and C. Motifs A and C of human PrimPol (PrimPol) harbor the catalytic residues (Asp, Glu, Asp) acting as metal ligands, whereas motif B includes highly conserved residues (Lys, Ser and His), which are postulated to stabilize 3' incoming deoxynucleotides (dNTPs). Additionally, other putative nucleotide ligands are situated close to motif C: Lys, almost invariant in the whole AEP superfamily, and Lys, specifically conserved in eukaryotic PrimPols. Here, we demonstrate that His is absolutely essential for 3'dNTP binding and, hence, for both primase and polymerase activities of PrimPol, whereas Ser and Lys are crucial for the dimer synthesis initiation step during priming, but dispensable for subsequent dNTP incorporation on growing primers. Conversely, the elimination of Lys does not affect the overall primase function; however, it is required for damage avoidance via primer-template realignments. Finally, Lys is identified as an extra anchor residue to stabilize the 3' incoming dNTP. Collectively, these results demonstrate that individual ligands modulate the stabilization of 3' incoming dNTPs to optimize DNA primer synthesis efficiency during initiation and primer maturation.
Topics: Humans; DNA Primase; Nucleotidyltransferases; Catalysis; Cognition; DNA Primers; Nucleotides; DNA-Directed DNA Polymerase; Multifunctional Enzymes
PubMed: 38203225
DOI: 10.3390/ijms25010051 -
ISME Communications Jan 2024Combining multiple displacement amplification (MDA) with metagenomics enables the analysis of samples with extremely low DNA concentrations, making them suitable for...
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.
PubMed: 38500705
DOI: 10.1093/ismeco/ycae024 -
Nature Structural & Molecular Biology Jan 2024The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a...
The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a vital role in viral replication and genome uncoating. Here we determined multiple cryo-EM structures of full-length hexameric D5 in diverse states. These states were captured during ATP hydrolysis while moving along the single-stranded DNA (ssDNA) track. Through comprehensive structural analysis combined with the helicase activity system, we revealed that when the primase domain is truncated or the interaction between the primase and helicase domains is disrupted, the double-stranded DNA (dsDNA) unwinds into ssDNA, suggesting a critical regulatory role of the primase domain. Two transition states bound with ssDNA substrate during unwinding reveals that two ATP molecules were consumed to drive DNA moving forward two nucleotides. Collectively, our findings shed light on the molecular mechanism that links ATP hydrolysis to the DNA unwinding in poxviruses.
Topics: DNA Primase; Monkeypox virus; DNA Helicases; DNA; DNA, Single-Stranded; Adenosine Triphosphate
PubMed: 38177671
DOI: 10.1038/s41594-023-01142-0 -
Nucleic Acids Research Dec 2023Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere...
Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.
Topics: DNA Replication; DNA-Binding Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Shelterin Complex; Telomere; Telomere-Binding Proteins; Chromosomes, Fungal
PubMed: 37953281
DOI: 10.1093/nar/gkad1036 -
BioRxiv : the Preprint Server For... Mar 2024The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), initiates DNA synthesis on both chromosome strands by generating chimeric...
The human primosome, a four-subunit complex of primase and DNA polymerase alpha (Polα), initiates DNA synthesis on both chromosome strands by generating chimeric RNA-DNA primers for loading DNA polymerases delta and epsilon (Polε). Replication protein A (RPA) tightly binds to single-stranded DNA strands, protecting them from nucleolytic digestion and unauthorized transactions. We report here that RPA plays a critical role for the human primosome during DNA synthesis across inverted repeats prone to hairpin formation. On other alternatively structured DNA forming a G-quadruplex, RPA provides no assistance for primosome. A stimulatory effect of RPA on DNA synthesis across hairpins was also observed for the catalytic domain of Polα but not of Polε. The important factors for an efficient hairpin bypass by primosome are the high affinity of RPA to DNA based on four DNA-binding domains and the interaction of the winged-helix-turn-helix domain of RPA with Polα. Binding studies indicate that this interaction stabilizes the RPA/Polα complex on the primed template. This work provides insight into a cooperative action of RPA and primosome on DNA, which is critical for DNA synthesis across inverted repeats.
PubMed: 38559116
DOI: 10.1101/2024.03.11.584335 -
BMC Plant Biology Feb 2024Grafting is widely used as an important agronomic approach to deal with environmental stresses. However, the molecular mechanism of grafted tomato scions in response to...
Integrated transcriptome and DNA methylome analysis reveal the biological base of increased resistance to gray leaf spot and growth inhibition in interspecific grafted tomato scions.
BACKGROUND
Grafting is widely used as an important agronomic approach to deal with environmental stresses. However, the molecular mechanism of grafted tomato scions in response to biotic stress and growth regulation has yet to be fully understood.
RESULTS
This study investigated the resistance and growth performance of tomato scions grafted onto various rootstocks. A scion from a gray leaf spot-susceptible tomato cultivar was grafted onto tomato, eggplant, and pepper rootstocks, creating three grafting combinations: one self-grafting of tomato/tomato (TT), and two interspecific graftings, namely tomato/eggplant (TE) and tomato/pepper (TP). The study utilized transcriptome and DNA methylome analyses to explore the regulatory mechanisms behind the resistance and growth traits in the interspecific graftings. Results indicated that interspecific grafting significantly enhanced resistance to gray leaf spot and improved fruit quality, though fruit yield was decreased compared to self-grafting. Transcriptome analysis demonstrated that, compared to self-grafting, interspecific graftings triggered stronger wounding response and endogenous immune pathways, while restricting genes related to cell cycle pathways, especially in the TP grafting. Methylome data revealed that the TP grafting had more hypermethylated regions at CHG (H = A, C, or T) and CHH sites than the TT grafting. Furthermore, the TP grafting exhibited increased methylation levels in cell cycle related genes, such as DNA primase and ligase, while several genes related to defense kinases showed decreased methylation levels. Notably, several kinase transcripts were also confirmed among the rootstock-specific mobile transcripts.
CONCLUSIONS
The study concludes that interspecific grafting alters gene methylation patterns, thereby activating defense responses and inhibiting the cell cycle in tomato scions. This mechanism is crucial in enhancing resistance to gray leaf spot and reducing growth in grafted tomato scions. These findings offer new insights into the genetic and epigenetic contributions to agronomic trait improvements through interspecific grafting.
Topics: Transcriptome; Solanum lycopersicum; Epigenome; Gene Expression Profiling; Fruit
PubMed: 38383283
DOI: 10.1186/s12870-024-04764-8 -
Molecular Microbiology Nov 2023Evolutionary studies often identify genes that have been exchanged between different organisms and the phrase Lateral or Horizontal Gene Transfer is often used in this... (Review)
Review
Evolutionary studies often identify genes that have been exchanged between different organisms and the phrase Lateral or Horizontal Gene Transfer is often used in this context. However, they rarely provide any mechanistic information concerning how these gene transfers might have occurred. With the astonishing increase in the number of sequences in public databases over the past two or three decades, identical antibiotic resistance genes have been identified in many different sequence contexts. One explanation for this would be that genes are initially transmitted by transposons which have subsequently decayed and can no longer be detected. Here, we provide an overview of a protein, IEE (Insertion Sequence Excision Enhancer) observed to facilitate high-frequency excision of IS629 from clinically important Escherichia coli O157:H7 and subsequently shown to affect a large class of bacterial insertion sequences which all transpose using the copy-out-paste-in transposition mechanism. Excision depends on both IEE and transposase indicating association with the transposition process itself. We review genetic and biochemical data and propose that IEE immobilizes genes carried by compound transposons by removing the flanking insertion sequence (IS) copies. The biochemical activities of IEE as a primase with the capacity to recognize DNA microhomologies and the observation that its effect appears restricted to IS families which use copy-out-paste-in transposition, suggests IS deletion occurs by abortive transposition involving strand switching (primer invasion) during the copy-out step. This reinforces the proposal made for understanding the widespread phenomenon loss of ISApl1 flanking mcr-1 in the compound transposon Tn6330 which we illustrate with a detailed model. This model also provides a convincing way to explain the high levels of IEE-induced precise IS excision.
Topics: Humans; DNA Transposable Elements; Anti-Bacterial Agents; Regulatory Sequences, Nucleic Acid; Bacteria; Drug Resistance, Microbial; DNA-Directed DNA Polymerase; DNA Primase; Multifunctional Enzymes
PubMed: 37574851
DOI: 10.1111/mmi.15140