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Annual Review of Biochemistry Jun 2021The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a... (Review)
Review
The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein-nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.
Topics: Animals; DNA; DNA Polymerase III; DNA Replication; Eukaryota; Humans; Origin Recognition Complex; Proliferating Cell Nuclear Antigen
PubMed: 33784179
DOI: 10.1146/annurev-biochem-090120-125407 -
Critical Reviews in Biochemistry and... 2016The machines that decode and regulate genetic information require the translation, transcription and replication pathways essential to all living cells. Thus, it might... (Review)
Review
The machines that decode and regulate genetic information require the translation, transcription and replication pathways essential to all living cells. Thus, it might be expected that all cells share the same basic machinery for these pathways that were inherited from the primordial ancestor cell from which they evolved. A clear example of this is found in the translation machinery that converts RNA sequence to protein. The translation process requires numerous structural and catalytic RNAs and proteins, the central factors of which are homologous in all three domains of life, bacteria, archaea and eukarya. Likewise, the central actor in transcription, RNA polymerase, shows homology among the catalytic subunits in bacteria, archaea and eukarya. In contrast, while some "gears" of the genome replication machinery are homologous in all domains of life, most components of the replication machine appear to be unrelated between bacteria and those of archaea and eukarya. This review will compare and contrast the central proteins of the "replisome" machines that duplicate DNA in bacteria, archaea and eukarya, with an eye to understanding the issues surrounding the evolution of the DNA replication apparatus.
Topics: Animals; DNA; DNA Replication; Evolution, Molecular; Genetic Variation; Humans; Models, Molecular; Protein Biosynthesis; Proteins; RNA; Transcription, Genetic
PubMed: 27160337
DOI: 10.3109/10409238.2015.1125845 -
Nucleic Acids Research Aug 2023Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria....
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Primase; DNA Replication; DNA; DNA Primers; Catalysis; Multifunctional Enzymes
PubMed: 37326028
DOI: 10.1093/nar/gkad507 -
Molecular & Cellular Oncology 2014The DNA-directed primase-polymerase PrimPol of the archaeo-eukaryotic primase superfamily represents an ancient solution to the many problems faced during genome... (Review)
Review
The DNA-directed primase-polymerase PrimPol of the archaeo-eukaryotic primase superfamily represents an ancient solution to the many problems faced during genome duplication. This versatile enzyme is capable of initiating de novo DNA/RNA synthesis, DNA chain elongation, and has the capacity to bypass modifications that stall the replisome by trans-lesion synthesis or origin-independent re-priming, thus allowing discontinuous synthesis of the leading strand. Recent studies have shown that PrimPol is an important new player in replication fork progression in eukaryotic cells; this review summarizes our current understanding of PrimPol and highlights important questions that remain to be addressed.
PubMed: 27308331
DOI: 10.4161/23723548.2014.960754 -
Journal of Visualized Experiments : JoVE Oct 2019DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of...
DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.
Topics: Binding Sites; DNA; DNA Primase; DNA Replication; High-Throughput Nucleotide Sequencing; Protein Array Analysis; Protein Binding; RNA; Sequence Analysis, DNA
PubMed: 31657797
DOI: 10.3791/59737 -
Mitochondrion Nov 2014Higher plant mitochondrial genomes exhibit much greater structural complexity compared to most other organisms. Unlike well-characterized metazoan mitochondrial DNA... (Review)
Review
Higher plant mitochondrial genomes exhibit much greater structural complexity compared to most other organisms. Unlike well-characterized metazoan mitochondrial DNA (mtDNA) replication, an understanding of the mechanism(s) and proteins involved in plant mtDNA replication remains unclear. Several plant mtDNA replication proteins, including DNA polymerases, DNA primase/helicase, and accessory proteins have been identified. Mitochondrial dynamics, genome structure, and the complexity of dual-targeted and dual-function proteins that provide at least partial redundancy suggest that plants have a unique model for maintaining and replicating mtDNA when compared to the replication mechanism utilized by most metazoan organisms.
Topics: DNA Replication; Mitochondria; Mitochondrial Proteins; Plant Proteins; Plants
PubMed: 24681310
DOI: 10.1016/j.mito.2014.03.008 -
BioRxiv : the Preprint Server For... Jan 2024Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand...
Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand DNA synthesis ("the end-replication problem"). We report a second end-replication problem that originates from the incomplete duplication of the C-rich telomeric repeat strand by lagging-strand synthesis. This problem is solved by CST-Polymeraseα(Polα)-primase fill-in synthesis. priming for lagging-strand DNA replication does not occur on the 3' overhang and lagging-strand synthesis stops in an ~150-nt zone more than 26 nt from the end of the template. Consistent with the data, lagging-end telomeres of cells lacking CST-Polα-primase lost ~50-60 nt of CCCTAA repeats per population doubling (PD). The C-strands of leading-end telomeres shortened by ~100 nt/PD, reflecting the generation of 3' overhangs through resection. The measured overall C-strand shortening in absence of CST-Polα-primase fill-in is consistent with the combined effects of incomplete lagging-strand synthesis and 5' resection at the leading-ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-strand and CST-Polα-primase to maintain the C-strand.
PubMed: 37961611
DOI: 10.1101/2023.10.26.564248 -
Nature Structural & Molecular Biology May 2023The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural...
The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural rearrangements of multiple domains. Using cryogenic electron microscopy, we solved a 3.6 Å human primosome structure caught at an early stage of RNA primer elongation with deoxynucleotides. The structure confirms a long-standing role of primase large subunit and reveals new insights into how primosome is limited to synthesizing short RNA-DNA primers.
Topics: Humans; DNA Primase; DNA; DNA Replication; DNA Primers; RNA
PubMed: 37069376
DOI: 10.1038/s41594-023-00971-3 -
Frontiers in Bioscience (Landmark... Jan 2017Since 1970, the DNA polymerase gamma (PolG) has been known to be the DNA polymerase responsible for replication and repair of mitochondrial DNA, and until recently it... (Review)
Review
Since 1970, the DNA polymerase gamma (PolG) has been known to be the DNA polymerase responsible for replication and repair of mitochondrial DNA, and until recently it was generally accepted that this was the only polymerase present in mitochondria. However, recent data has challenged that opinion, as several polymerases are now proposed to have activity in mitochondria. To date, their exact role of these other DNA polymerases is unclear and the amount of evidence supporting their role in mitochondria varies greatly. Further complicating matters, no universally accepted standards have been set for definitive proof of the mitochondrial localization of a protein. To gain an appreciation of these newly proposed DNA polymerases in the mitochondria, we review the evidence and standards needed to establish the role of a polymerase in the mitochondria. Employing PolG as an example, we established a list of criteria necessary to verify the existence and function of new mitochondrial proteins. We then apply this criteria towards several other putative mitochondrial polymerases. While there is still a lot left to be done in this exciting new direction, it is clear that PolG is not acting alone in mitochondria, opening new doors for potential replication and repair mechanisms.
Topics: Animals; DNA Polymerase beta; DNA Polymerase gamma; DNA Primase; DNA, Mitochondrial; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Humans; Mitochondria; Mitochondrial Diseases; Mitochondrial Proteins; Multifunctional Enzymes; Mutation; DNA Polymerase theta
PubMed: 27814640
DOI: 10.2741/4510 -
Biochemical Society Transactions Feb 2019It has been known for decades that the principal replicative DNA polymerases that effect genome replication are incapable of starting DNA synthesis Rather, they require... (Review)
Review
It has been known for decades that the principal replicative DNA polymerases that effect genome replication are incapable of starting DNA synthesis Rather, they require a 3'-OH group from which to extend a DNA chain. Cellular DNA replication systems exploit a dedicated, limited processivity RNA polymerase, termed primase, that synthesizes a short oligoribonucleotide primer which is then extended by a DNA polymerase. Thus, primases can initiate synthesis, proceed with primer elongation for a short distance then transfer the primer to a DNA polymerase. Despite these well-established properties, the mechanistic basis of these dynamic behaviours has only recently been established. In the following, the author will describe recent insights from studies of the related eukaryotic and archaeal DNA primases. Significantly, the general conclusions from these studies likely extend to a broad class of extrachromosomal element-associated primases as well as the human primase-related DNA repair enzyme, PrimPol.
Topics: Archaeal Proteins; DNA Primase; DNA Replication; DNA-Directed DNA Polymerase; Humans; Multifunctional Enzymes
PubMed: 30647143
DOI: 10.1042/BST20180627