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Cell Cycle (Georgetown, Tex.) Feb 2023DNA double-strand breaks (DSBs) pose a major threat to the genome, so the efficient repair of such breaks is essential. DSB processing and repair is affected by 53BP1,...
DNA double-strand breaks (DSBs) pose a major threat to the genome, so the efficient repair of such breaks is essential. DSB processing and repair is affected by 53BP1, which has been proposed to determine repair pathway choice and/or promote repair fidelity. 53BP1 and its downstream effectors, RIF1 and shieldin, control 3' overhang length, and the mechanism has been a topic of intensive research. Here, we highlight recent evidence that 3' overhang control by 53BP1 occurs through fill-in synthesis of resected DSBs by CST/Polα/primase. We focus on the crucial role of fill-in synthesis in BRCA1-deficient cells treated with PARPi and discuss the notion of fill-in synthesis in other specialized settings and in the repair of random DSBs. We argue that - in addition to other determinants - repair pathway choice may be influenced by the DNA sequence at the break which can impact CST binding and therefore the deployment of Polα/primase fill-in.
Topics: DNA Breaks, Double-Stranded; DNA Primase; Tumor Suppressor p53-Binding Protein 1; DNA Repair; DNA End-Joining Repair
PubMed: 36205622
DOI: 10.1080/15384101.2022.2123886 -
Postepy Biochemii Mar 2019Primases are responsible for the synthesis of a short oligo RNA, which serves as primer for DNA polymerase. Primases play an essential role in the initiation of DNA... (Review)
Review
Primases are responsible for the synthesis of a short oligo RNA, which serves as primer for DNA polymerase. Primases play an essential role in the initiation of DNA replication at the origins, in the synthesis of Okazaki fragments and in the restart of stalled replication forks. Prokaryotic primases based on their structure and sequence alignments are classified as a family of DnaG proteins. Primases from this family contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either interacts with a helicase or is itself a DNA helicase. In this review, we are presenting the comparison of the representative primases from bacteria and bacteriophages, their mode of action and their involvement in DNA replication at the replication fork.
Topics: Bacteria; Bacteriophages; DNA Primase; DNA Replication; Structure-Activity Relationship
PubMed: 30901180
DOI: 10.18388/pb.2019_253 -
PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy.Biomolecules Feb 2022DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One... (Review)
Review
DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with conventional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a DNA primase, elongating primers that PrimPol itself sythesizes, or as translesion synthesis (TLS) DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only acts as a DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer.
Topics: DNA Primase; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Multifunctional Enzymes; Neoplasms
PubMed: 35204749
DOI: 10.3390/biom12020248 -
PloS One 2018The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C...
The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C construct revealed that its affinity for DNA is sensitive to the redox state of the [4Fe4S] cluster. Concerns about the validity of this conclusion have been raised, based in part on differences in X-ray crystal structures of the p58C272-464 construct used for that study and that of a N-terminally shifted p58C266-456 construct and consequently, an assumption that p58C272-464 has abnormal physical and functional properties. To address this controversy, a new p58C266-464 construct containing all residues was crystallized under the conditions previously used for crystallizing p58C272-464, and the solution structures of both constructs were assessed using circular dichroism and NMR spectroscopy. In the new crystal structure, p58C266-464 exhibits the same elements of secondary structure near the DNA binding site as observed in the crystal structure of p58C272-464. Moreover, in solution, circular dichroism and 15N,1H-heteronuclear single quantum coherence (HSQC) NMR spectra show there are no significant differences in the distribution of secondary structures or in the tertiary structure or the two constructs. To validate that the two constructs have the same functional properties, binding of a primed DNA template was measured using a fluorescence-based DNA binding assay, and the affinities for this substrate were the same (3.4 ± 0.5 μM and 2.7 ± 0.3 μM, respectively). The electrochemical properties of p58C266-464 were also measured and this p58C construct was able to engage in redox switching on DNA with the same efficiency as p58C272-464. Together, these results show that although p58C can be stabilized in different conformations in the crystalline state, in solution there is effectively no difference in the structure and functional properties of p58C constructs of different lengths.
Topics: Binding Sites; Circular Dichroism; Crystallography, X-Ray; DNA; DNA Primase; Molecular Docking Simulation; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Protein Binding; Protein Domains; Protein Structure, Secondary; RNA
PubMed: 30562384
DOI: 10.1371/journal.pone.0209345 -
The Enzymes 2016The replication system of bacteriophage T7 is remarkable in that the 40,000 nucleotide genome is replicated over 100-fold in a matter of minutes. In order to accomplish... (Review)
Review
The replication system of bacteriophage T7 is remarkable in that the 40,000 nucleotide genome is replicated over 100-fold in a matter of minutes. In order to accomplish this feat T7 has evolved an efficient and economical process for the replication of its DNA. The T7 replisome provides a model system to study DNA replication. Four proteins are sufficient for reconstitution of the functional replication complex, yet the assembled replisome recapitulates all the key features of more complex prokaryotic and eukaryotic systems. In this review, we describe chemical mechanisms employed by individual proteins at the replication fork. Integration of structural, biochemical, and single-molecule data reveals a compelling view on how a nearly 1-MDa molecular machine acts as a unit to synthetize the two antiparallel DNA strands in a coordinated fashion.
Topics: Bacteriophage T7; DNA Replication; DNA, Viral; DNA-Directed DNA Polymerase; Multienzyme Complexes; Viral Proteins
PubMed: 27241928
DOI: 10.1016/bs.enz.2016.02.001 -
The Journal of Biological Chemistry Nov 2017The T4 replisome has provided a unique opportunity to investigate the intricacies of DNA replication. We present a comprehensive review of this system focusing on the... (Comparative Study)
Comparative Study Review
The T4 replisome has provided a unique opportunity to investigate the intricacies of DNA replication. We present a comprehensive review of this system focusing on the following: its 8-protein composition, their individual and synergistic activities, and assembly and into a replisome capable of coordinated leading/lagging strand DNA synthesis. We conclude with a brief comparison with other replisomes with emphasis on how coordinated DNA replication is achieved.
Topics: Bacteriophage T4; Bacteriophage T7; Carbon-Nitrogen Ligases; DNA Replication; DNA-Directed DNA Polymerase; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Multienzyme Complexes; Protein Multimerization; Species Specificity; Viral Proteins
PubMed: 28972188
DOI: 10.1074/jbc.R117.811208 -
Scientific Reports Sep 2021Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA...
Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA structures. PrimPol possesses both DNA/RNA primase and DNA polymerase activities, and also bypasses a number of DNA lesions in vitro. In this work, we have analyzed translesion synthesis activity of PrimPol in vitro on DNA with an 1,2-intrastrand cisplatin cross-link (1,2-GG CisPt CL) or a model DNA-protein cross-link (DpCL). PrimPol was capable of the 1,2-GG CisPt CL bypass in the presence of Mn ions and preferentially incorporated two complementary dCMPs opposite the lesion. Nucleotide incorporation was stimulated by PolDIP2, and yeast Pol ζ efficiently extended from the nucleotides inserted opposite the 1,2-GG CisPt CL in vitro. DpCLs significantly blocked the DNA polymerase activity and strand displacement synthesis of PrimPol. However, PrimPol was able to reach the DpCL site in single strand template DNA in the presence of both Mg and Mn ions despite the presence of the bulky protein obstacle.
Topics: Cisplatin; Cross-Linking Reagents; DNA; DNA Damage; DNA Primase; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Multifunctional Enzymes
PubMed: 34475447
DOI: 10.1038/s41598-021-96692-y -
Trends in Biochemical Sciences Oct 2016The DNA replication machinery, or replisome, is a macromolecular complex that combines DNA unwinding, priming and synthesis activities. In eukaryotic cells, the helicase... (Review)
Review
The DNA replication machinery, or replisome, is a macromolecular complex that combines DNA unwinding, priming and synthesis activities. In eukaryotic cells, the helicase and polymerases are multi-subunit, highly-dynamic assemblies whose structural characterization requires an integrated approach. Recent studies have combined single-particle electron cryo-microscopy and protein crystallography to gain insights into the mechanism of DNA duplication by the eukaryotic replisome. We review current understanding of how replication fork unwinding by the CMG helicase is coupled to leading-strand synthesis by polymerase (Pol) ɛ and lagging-strand priming by Pol α/primase, and discuss emerging principles of replisome organization.
Topics: Cell Cycle; Cell Cycle Proteins; DNA; DNA Polymerase I; DNA Polymerase II; DNA Primase; DNA Replication; Gene Expression; Humans; Minichromosome Maintenance Proteins; Molecular Docking Simulation; Protein Binding; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Secondary; Replication Origin; Saccharomyces cerevisiae
PubMed: 27555051
DOI: 10.1016/j.tibs.2016.07.011 -
Nature Communications Jun 2023The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and...
The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA polymerase and RNA primase activities, respectively. It has been unclear how Pol α hands over an RNA primer from Pri1 to Pol1 for DNA primer extension, and how the primer length is defined. Here we report the cryo-EM analysis of yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states, revealing a series of very large movements. We reveal a critical point at which Pol1-core moves to take over the 3'-end of the RNA from Pri1. DNA extension is limited by a spiral motion of Pol1-core. Since both Pri1 and Pol1-core are flexibly attached to a stable platform, primer growth produces stress that limits the primer length.
Topics: DNA Primase; DNA-Directed DNA Polymerase; DNA Replication; DNA; RNA; Saccharomyces cerevisiae; DNA Primers
PubMed: 37344454
DOI: 10.1038/s41467-023-39441-1 -
Current Genetics Dec 2019This review elaborates on the findings of a new report which possibly resolves the biochemical nature of a novel type of DNA imprint as ribonucleotide and the mechanism... (Review)
Review
This review elaborates on the findings of a new report which possibly resolves the biochemical nature of a novel type of DNA imprint as ribonucleotide and the mechanism of its formation during cell differentiation in fission yeast. The process of mating-type switching in fission yeast, Schizosaccharomyces pombe, displays characteristics of a typical mammalian stem cell lineage, wherein a cell divides to produce an identical cell and a differentiated cell after every two cell divisions. This developmental asymmetry has been ascribed to play a role in generation of a DNA strand-specific imprint at the mat1 locus during lagging strand synthesis and its segregation to one of the two daughter cells by the process of asymmetric, semi-conservative DNA replication. The nature of this imprint and mechanisms of its generation have been a subject of research and debate. A recent report by Singh et al. (Nucleic Acids Res 47:3422-3433. https://doi.org/10.1093/nar/gkz092 , 2019) provides compelling evidence in support of a ribonucleotide as the imprint moiety within the mat1 DNA and demonstrates the role of Mcm10/Cdc23, an important, evolutionarily conserved component of DNA replication machinery in eukaryotes, in installing the imprint through a non-canonical primase activity and interaction with DNA Polα and Swi1. The high degree of conservation of DNA replication machinery, especially the presence of the T7 gene 4 helicase/primase domain in the mammalian orthologs of Mcm10 suggests that similar mechanisms of DNA imprinting may play a role during cell differentiation in metazoans.
Topics: Cell Cycle Proteins; DNA Primase; DNA Replication; DNA-Binding Proteins; Genes, Mating Type, Fungal; Genomic Imprinting; Minichromosome Maintenance Proteins; RNA; RNA, Fungal; Ribonucleotides; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 31076844
DOI: 10.1007/s00294-019-00991-x