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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 -
Genes Apr 2023In their influential reviews, Hanahan and Weinberg coined the term 'Hallmarks of Cancer' and described genome instability as a property of cells enabling cancer... (Review)
Review
In their influential reviews, Hanahan and Weinberg coined the term 'Hallmarks of Cancer' and described genome instability as a property of cells enabling cancer development. Accurate DNA replication of genomes is central to diminishing genome instability. Here, the understanding of the initiation of DNA synthesis in origins of DNA replication to start leading strand synthesis and the initiation of Okazaki fragment on the lagging strand are crucial to control genome instability. Recent findings have provided new insights into the mechanism of the remodelling of the prime initiation enzyme, DNA polymerase α-primase (Pol-prim), during primer synthesis, how the enzyme complex achieves lagging strand synthesis, and how it is linked to replication forks to achieve optimal initiation of Okazaki fragments. Moreover, the central roles of RNA primer synthesis by Pol-prim in multiple genome stability pathways such as replication fork restart and protection of DNA against degradation by exonucleases during double-strand break repair are discussed.
Topics: Eukaryota; DNA Replication; Multigene Family; Humans; DNA-Directed DNA Polymerase; DNA
PubMed: 37239371
DOI: 10.3390/genes14051012 -
Nature Communications Jun 2021CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins...
CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.
Topics: Bacteria; Bacterial Proteins; Bacteroidetes; CRISPR-Associated Proteins; CRISPR-Cas Systems; Computational Biology; DNA Primase; DNA Primers; DNA-Directed DNA Polymerase; Dimerization; Escherichia coli; Gene Expression; Mutation; Phylogeny; Prokaryotic Cells; Recombinant Proteins; Ribonucleotides
PubMed: 34140468
DOI: 10.1038/s41467-021-23535-9 -
Nucleic Acids Research May 2021To bypass a diverse range of fork stalling impediments encountered during genome replication, cells possess a variety of DNA damage tolerance (DDT) mechanisms including... (Review)
Review
To bypass a diverse range of fork stalling impediments encountered during genome replication, cells possess a variety of DNA damage tolerance (DDT) mechanisms including translesion synthesis, template switching, and fork reversal. These pathways function to bypass obstacles and allow efficient DNA synthesis to be maintained. In addition, lagging strand obstacles can also be circumvented by downstream priming during Okazaki fragment generation, leaving gaps to be filled post-replication. Whether repriming occurs on the leading strand has been intensely debated over the past half-century. Early studies indicated that both DNA strands were synthesised discontinuously. Although later studies suggested that leading strand synthesis was continuous, leading to the preferred semi-discontinuous replication model. However, more recently it has been established that replicative primases can perform leading strand repriming in prokaryotes. An analogous fork restart mechanism has also been identified in most eukaryotes, which possess a specialist primase called PrimPol that conducts repriming downstream of stalling lesions and structures. PrimPol also plays a more general role in maintaining efficient fork progression. Here, we review and discuss the historical evidence and recent discoveries that substantiate repriming as an intrinsic replication restart pathway for maintaining efficient genome duplication across all domains of life.
Topics: Animals; DNA; DNA Damage; DNA Primase; DNA Replication; DNA-Directed DNA Polymerase; Genome; History, 20th Century; Models, Genetic; Stress, Physiological
PubMed: 33744934
DOI: 10.1093/nar/gkab176 -
BioRxiv : the Preprint Server For... Aug 2023DNA replication in eukaryotes relies on the synthesis of a ~30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase...
DNA replication in eukaryotes relies on the synthesis of a ~30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ~20 nucleotides of DNA. Here we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
PubMed: 37577606
DOI: 10.1101/2023.08.01.551538 -
Nature Communications Mar 2024DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed...
DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed and respond to lesions that hinder its progression. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2), regulate Replication Termination Factor 2 (RTF2) levels at stalled replisomes, allowing fork stabilization and restart. Here, we show that during unperturbed replication, RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme that removes RNA from RNA-DNA heteroduplexes. RTF2, like RNase H2, is essential for mammalian development and maintains normal replication speed. However, persistent RTF2 and RNase H2 at stalled replication forks prevent efficient replication restart, which is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for RTF2-dependent regulation of replication-coupled ribonucleotide removal and reveal the existence of PRIM1-mediated direct replication restart in mammalian cells.
Topics: Animals; DNA Replication; DNA; DNA Damage; Cell Cycle Proteins; RNA; Ribonucleases; Mammals
PubMed: 38431617
DOI: 10.1038/s41467-024-45947-z -
Journal of Molecular Biology Jan 2022The ParABS system is supposed to be responsible for plasmid partitioning and chromosome segregation in bacteria. ParABS ensures a high degree of fidelity in inheritance...
The ParABS system is supposed to be responsible for plasmid partitioning and chromosome segregation in bacteria. ParABS ensures a high degree of fidelity in inheritance by dividing the genetic material equally between daughter cells during cell division. However, the molecular mechanisms underlying the assembly of the partition complex, representing the core of the ParABS system, are still far from being understood. Here we demonstrate that the partition complex is formed via liquid-liquid phase separation. Assembly of the partition complex is initiated by the formation of oligomeric ParB species, which in turn are regulated by CTP-binding. Phase diagrams and in vivo analysis show how the partition complex can further be spatially regulated by parS. By investigating the phylogenetic variation in phase separation and its regulation by CTP, we find a high degree of evolutionary conservation among distantly related prokaryotes. These results advance the understanding of partition complex formation and regulation in general, by confirming and extending recently proposed models.
Topics: Bacteria; Bacterial Physiological Phenomena; Bacterial Proteins; Cell Division; Chromosome Segregation; Chromosomes, Bacterial; Corynebacterium glutamicum; Cytidine Triphosphate; DNA Primase; DNA, Bacterial; Phase Transition; Phylogeny
PubMed: 34902429
DOI: 10.1016/j.jmb.2021.167401 -
Journal of Biotechnology Jan 2024DNA damage tolerance (DDT) pathways mitigate the effects of DNA damage during replication by rescuing the replication fork stalled at a DNA lesion or other barriers and... (Review)
Review
DNA damage tolerance (DDT) pathways mitigate the effects of DNA damage during replication by rescuing the replication fork stalled at a DNA lesion or other barriers and also repair discontinuities left in the newly replicated DNA. From yeast to mammalian cells, RAD18-regulated translesion synthesis (TLS) and template switching (TS) represent the dominant pathways of DDT. Monoubiquitylation of the polymerase sliding clamp PCNA by HRAD6A-B/RAD18, an E2/E3 protein pair, enables the recruitment of specialized TLS polymerases that can insert nucleotides opposite damaged template bases. Alternatively, the subsequent polyubiquitylation of monoubiquitin-PCNA by Ubc13-Mms2 (E2) and HLTF or SHPRH (E3) can lead to the switching of the synthesis from the damaged template to the undamaged newly synthesized sister strand to facilitate synthesis past the lesion. When immediate TLS or TS cannot occur, gaps may remain in the newly synthesized strand, partly due to the repriming activity of the PRIMPOL primase, which can be filled during the later phases of the cell cycle. The first part of this review will summarize the current knowledge about RAD18-dependent DDT pathways, while the second part will offer a molecular toolkit for the identification and characterization of the cellular functions of a DDT protein. In particular, we will focus on advanced techniques that can reveal single-stranded and double-stranded DNA gaps and their repair at the single-cell level as well as monitor the progression of single replication forks, such as the specific versions of the DNA fiber and comet assays. This collection of methods may serve as a powerful molecular toolkit to monitor the metabolism of gaps, detect the contribution of relevant pathways and molecular players, as well as characterize the effectiveness of potential inhibitors.
Topics: Animals; DNA Replication; Proliferating Cell Nuclear Antigen; DNA Damage; DNA; Saccharomyces cerevisiae; DNA Repair; Mammals; DNA-Binding Proteins; Saccharomyces cerevisiae Proteins
PubMed: 38072328
DOI: 10.1016/j.jbiotec.2023.12.001 -
Cell & Bioscience Feb 2021DNA primase subunit 1 (PRIM1) has been reported as a novel oncogene in several cancer types. However, its roles in hepatocellular carcinoma (HCC) remain unclear. This...
BACKGROUND
DNA primase subunit 1 (PRIM1) has been reported as a novel oncogene in several cancer types. However, its roles in hepatocellular carcinoma (HCC) remain unclear. This study aimed to investigate underlying mechanisms of PRIM1 and identify it as a potential molecular target for HCC.
METHODS
Hub genes were screened between HCC tissues and normal liver tissues in 3 gene expression omnibus (GEO) datasets and the cancer genome atlas (TCGA). The expression features and prognostic value of one of the hub genes PRIM1 were analyzed by bioinformatic analyses and immunohistochemistry. Loss-of-function and gain-of-function studies were used to investigate the regulatory role of PRIM1 in HCC cells. Real-time (RT)-qPCR, western blotting, and ubiquitin immunoprecipitation assays were performed to explore the underlying mechanisms. The xenograft model was employed to detect the roles of PRIM1 in tumor growth in vivo. Finally, the 3D spheroid model was conducted to validate the role of PRIM1 in tumor growth and sorafenib resistance.
RESULTS
The hub genes of HCC were screened in multiple bioinformatic datasets. PRIM1, as one of the hub genes, was significantly overexpressed in HCC tissues in mRNA and protein levels. In addition, high expression of PRIM1 indicated poor prognosis of HCC patients in TCGA, ICGC, and Nantong cohorts. Overexpression of PRIM1 promoted the proliferation, migration/invasion, and sorafenib resistance of HCC cells, with the decrease in apoptosis and cell cycle arrest. Mechanically, PRIM1 facilitated epithelial-mesenchymal transition (EMT) process and the activity of PI3K/AKT/mTOR signaling of HCC cells. Additionally, PRIM1 could cause the ubiquitination and degradation of P53 by upregulating Ubiquitin Conjugating Enzyme E2 C (UBE2C). Furthermore, knockdown of PRIM1 significantly inhibited the growth of xenograft tumors and HCC cells-derived spheroids with enhanced sorafenib resistance.
CONCLUSION
This study implies that PRIM1 may play a key role in the progression of HCC and may serve as a potential target for HCC treatment.
PubMed: 33622397
DOI: 10.1186/s13578-021-00555-y -
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