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Frontiers in Microbiology 2022DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic...
DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the mutants in combination with mutants. The results show that knockout mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the and/or mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in HB27, thus, playing a key role in its genome stability.
PubMed: 36532486
DOI: 10.3389/fmicb.2022.1005862 -
The Journal of Biological Chemistry Jan 2023Twinkle is the ring-shaped replicative helicase within the human mitochondria with high homology to bacteriophage T7 gp4 helicase-primase. Unlike many orthologs of...
Twinkle is the ring-shaped replicative helicase within the human mitochondria with high homology to bacteriophage T7 gp4 helicase-primase. Unlike many orthologs of Twinkle, the N-terminal domain (NTD) of human Twinkle has lost its primase activity through evolutionarily acquired mutations. The NTD has no demonstrated activity thus far; its role has remained unclear. Here, we biochemically characterize the isolated NTD and C-terminal domain (CTD) with linker to decipher their contributions to full-length Twinkle activities. This novel CTD construct hydrolyzes ATP, has weak DNA unwinding activity, and assists DNA polymerase γ (Polγ)-catalyzed strand-displacement synthesis on short replication forks. However, CTD fails to promote multikilobase length product formation by Polγ in rolling-circle DNA synthesis. Thus, CTD retains all the motor functions but struggles to implement them for processive translocation. We show that NTD has DNA-binding activity, and its presence stabilizes Twinkle oligomerization. CTD oligomerizes on its own, but the loss of NTD results in heterogeneously sized oligomeric species. The CTD also exhibits weaker and salt-sensitive DNA binding compared with full-length Twinkle. Based on these results, we propose that NTD directly contributes to DNA binding and holds the DNA in place behind the central channel of the CTD like a "doorstop," preventing helicase slippages and sustaining processive unwinding. Consistent with this model, mitochondrial single-stranded DNA-binding protein (mtSSB) compensate for the NTD loss and partially restore kilobase length DNA synthesis by CTD and Polγ. The implications of our studies are foundational for understanding the mechanisms of disease-causing Twinkle mutants that lie in the NTD.
Topics: Humans; DNA; DNA Helicases; DNA Primase; DNA Replication; DNA, Mitochondrial; Mitochondria; Mitochondrial Proteins
PubMed: 36528058
DOI: 10.1016/j.jbc.2022.102797 -
Nucleic Acids Research Nov 2022DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during...
DNA polymerase α (Polα) is essential for DNA replication initiation and makes a notable contribution to genome mutagenesis. The activity and fidelity of Polα during the early steps of DNA replication have not been well studied. Here we show that at the beginning of DNA synthesis, when extending the RNA primer received from primase, Polα is more mutagenic than during the later DNA elongation steps. Kinetic and binding studies revealed substantially higher activity and affinity to the template:primer when Polα interacts with ribonucleotides of a chimeric RNA-DNA primer. Polα activity greatly varies during first six steps of DNA synthesis, and the bias in the rates of correct and incorrect dNTP incorporation leads to impaired fidelity, especially upon the second step of RNA primer extension. Furthermore, increased activity and stability of Polα/template:primer complexes containing RNA-DNA primers result in higher efficiency of mismatch extension.
Topics: Humans; DNA Polymerase I; Mutagens; DNA Replication; DNA Primase; Mutagenesis; DNA; DNA Primers; RNA
PubMed: 36454017
DOI: 10.1093/nar/gkac1101 -
Viruses Oct 2022Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa...
Poxviruses are large DNA viruses with a linear double-stranded DNA genome circularized at the extremities. The helicase-primase D5, composed of six identical 90 kDa subunits, is required for DNA replication. D5 consists of a primase fragment flexibly attached to the hexameric C-terminal polypeptide (res. 323-785) with confirmed nucleotide hydrolase and DNA-binding activity but an elusive helicase activity. We determined its structure by single-particle cryo-electron microscopy. It displays an AAA+ helicase core flanked by N- and C-terminal domains. Model building was greatly helped by the predicted structure of D5 using AlphaFold2. The 3.9 Å structure of the N-terminal domain forms a well-defined tight ring while the resolution decreases towards the C-terminus, still allowing the fit of the predicted structure. The N-terminal domain is partially present in papillomavirus E1 and polyomavirus LTA helicases, as well as in a bacteriophage NrS-1 helicase domain, which is also closely related to the AAA+ helicase domain of D5. Using the Pfam domain database, a D5_N domain followed by DUF5906 and Pox_D5 domains could be assigned to the cryo-EM structure, providing the first 3D structures for D5_N and Pox_D5 domains. The same domain organization has been identified in a family of putative helicases from large DNA viruses, bacteriophages, and selfish DNA elements.
Topics: DNA Primase; Cryoelectron Microscopy; Vaccinia virus; DNA Helicases; DNA; DNA Replication; Nucleotides
PubMed: 36298761
DOI: 10.3390/v14102206 -
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 -
Journal of Medicinal Chemistry Oct 2022When the nucleoside analogue acyclovir was introduced in the early 1980s, it presented a game-changing treatment modality for herpes simplex virus infections. Since...
When the nucleoside analogue acyclovir was introduced in the early 1980s, it presented a game-changing treatment modality for herpes simplex virus infections. Since then, work has been ongoing to improve the weaknesses that have now been identified: a narrow time window for therapeutic success, resistance in immunocompromised patients, little influence on frequency of recurrences, relatively fast elimination, and poor bioavailability. The present Drug Annotation focuses on the helicase-primase inhibitor pritelivir currently in development for the treatment of acyclovir-resistant HSV infections and describes how a change of the molecular target (from viral DNA polymerase to the HSV helicase-primase complex) afforded improvement of the shortcomings of nucleoside analogs. Details are presented for the discovery process leading to the final drug candidate, the pivotal preclinical studies on mechanism of action and efficacy, and on how ongoing clinical research has been able to translate preclinical promises into clinical use.
Topics: Humans; Acyclovir; Nucleosides; DNA Primase; Antiviral Agents; Pyridines; Herpes Simplex; Drug Resistance, Viral
PubMed: 36202389
DOI: 10.1021/acs.jmedchem.2c00668 -
Proceedings of the National Academy of... Aug 2022Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how...
Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5'-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5'-triphosphate (ATP) at the subunit interface stabilizes the subunit-subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5'-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein-protein and protein-DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein-ssDNA force field and elucidate the molecular basis of replicative helicase translocation.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Bacteriophage T7; DNA Helicases; DNA Primase; DNA, Single-Stranded; Protein Conformation
PubMed: 35914145
DOI: 10.1073/pnas.2202239119 -
Cancers Jul 2022p53 is a common tumor suppressor, and its mutation drives tumorigenesis. What is more, p53 mutations have also been reported to be indicative of poor prognosis in lung...
p53 is a common tumor suppressor, and its mutation drives tumorigenesis. What is more, p53 mutations have also been reported to be indicative of poor prognosis in lung cancer, but the detailed mechanism has not been elucidated. In this study, we found that DNA primase subunit 2 (PRIM2) had a high expression level and associated with poor prognosis in lung cancer. Furthermore, we found that PRIM2 expression was abnormally increased in lung cancer cells with p53 mutation or altered the p53/RB pathway based on database. We also verified that PRIM2 expression was elevated by mutation or deletion of p53 in lung cancer cell lines. Lastly, silence p53 increased the expression of RPIM2. Thus, these data suggest that PRIM2 is a cancer-promoting factor which is regulated by the p53/RB pathway. The p53 tumor-suppressor gene integrates numerous signals that control cell proliferation, cell cycle, and cell death; and the p53/RB pathway determines the cellular localization of transcription factor E2F, which regulates the expression of downstream targets. Next, we explored the role of PRIM2 in lung cancer and found that knockdown of PRIM2 induced cell cycle arrest, increased DNA damage, and increased cell senescence, leading to decreased lung cancer cell proliferation. Lastly, the positive correlation between PRIM2 and E2F/CDK also indicated that PRIM2 was involved in promoting cell cycle mediated by p53/RB pathway. These results confirmed that the expression of PRIM2 is regulated by the p53/RB pathway in lung cancer cells, promotes DNA replication and mismatch repair, and activates the cell cycle. Overall, we found that frequent p53 mutations increased PRIM2 expression, activated the cell cycle, and promoted lung cancer progression.
PubMed: 35884433
DOI: 10.3390/cancers14143370 -
Nature Aug 2022Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins. Replication of telomeres allows continued proliferation of human...
Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells. This replication requires telomerase extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)); the synthesis of the complementary C-strand ((CCCTAA)) is much less well characterized. The CST (CTC1-STN1-TEN1) protein complex, a DNA polymerase α-primase accessory factor, is known to be required for telomere replication in vivo, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3' overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension and recruits Polα-primase to telomeric ssDNA but also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.
Topics: DNA Primase; DNA Replication; DNA, Single-Stranded; G-Quadruplexes; Humans; Replicon; Shelterin Complex; Telomerase; Telomere
PubMed: 35831508
DOI: 10.1038/s41586-022-04930-8 -
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