We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol ...is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.
DNA replication forks that collapse during the process of genomic duplication lead to double-strand breaks and constitute a threat to genomic stability. The risk of fork collapse is higher in the ...presence of replication inhibitors or after UV irradiation, which introduces specific modifications in the structure of DNA. In these cases, fork progression may be facilitated by error-prone translesion synthesis (TLS) DNA polymerases. Alternatively, the replisome may skip the damaged DNA, leaving an unreplicated gap to be repaired after replication. This mechanism strictly requires a priming event downstream of the lesion. Here we show that PrimPol, a new human primase and TLS polymerase, uses its primase activity to mediate uninterrupted fork progression after UV irradiation and to reinitiate DNA synthesis after dNTP depletion. As an enzyme involved in tolerance to DNA damage, PrimPol might become a target for cancer therapy.
Human PrimPol activity is enhanced by RPA Martínez-Jiménez, María I; Lahera, Antonio; Blanco, Luis
Scientific reports,
04/2017, Letnik:
7, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Human PrimPol is a primase belonging to the AEP superfamily with the unique ability to synthesize DNA primers de novo, and a non-processive DNA polymerase able to bypass certain DNA lesions. PrimPol ...facilitates both mitochondrial and nuclear replication fork progression either acting as a conventional TLS polymerase, or repriming downstream of blocking lesions. In vivo assays have shown that PrimPol is rapidly recruited to sites of DNA damage by interaction with the human replication protein A (RPA). In agreement with previous findings, we show here that the higher affinity of RPA for ssDNA inhibits PrimPol activities in short ssDNA templates. In contrast, once the amount of ssDNA increases up to a length in which both proteins can simultaneously bind ssDNA, as expected during replicative stress conditions, PrimPol and RPA functionally interact, and their binding capacities are mutually enhanced. When using M13 ssDNA as template, RPA stimulated both the primase and polymerase activities of PrimPol, either alone or in synergy with Polε. These new findings supports the existence of a functional PrimPol/RPA association that allows repriming at the exposed ssDNA regions formed in the leading strand upon replicase stalling.
Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome. Here we introduce a novel multiple displacement amplification ...(MDA) method based on the unique DNA primase features of Thermus thermophilus (Tth) PrimPol. TthPrimPol displays a potent primase activity preferring dNTPs as substrates unlike conventional primases. A combination of TthPrimPol's unique ability to synthesize DNA primers with the highly processive Phi29 DNA polymerase (Φ29DNApol) enables near-complete whole genome amplification from single cells. This novel method demonstrates superior breadth and evenness of genome coverage, high reproducibility, excellent single-nucleotide variant (SNV) detection rates with low allelic dropout (ADO) and low chimera formation as exemplified by sequencing HEK293 cells. Moreover, copy number variant (CNV) calling yields superior results compared with random primer-based MDA methods. The advantages of this method, which we named TruePrime, promise to facilitate and improve single-cell genomic analysis.
Objective
To demonstrate that delayed cord clamping (DCC) is safe in mothers with confirmed SARS‐CoV‐2 infection.
Design, setting and participants
Prospective observational study involving ...epidemiological information from 403 pregnant women with SARS‐CoV‐2 between 1 March and 31 May 2020. Data were collected from 70 centres that participate in the Spanish Registry of COVID‐19.
Methods
Patients' information was collected from their medical chart.
Main outcomes and measures
The rate of perinatal transmission of SARS‐CoV‐2 and development of the infection in neonates within 14 days postpartum.
Results
The early cord clamping (ECC) group consisted of 231 infants (57.3%) and the DCC group consisted of 172 infants (42.7%). Five positive newborns (1.7% of total tests performed) were identified with the nasopharyngeal PCR tests performed in the first 12 hours postpartum, two from the ECC group (1.7%) and three from the DCC group (3.6%). No significant differences between groups were found regarding neonatal tests for SARS‐CoV‐2. No confirmed cases of vertical transmission were detected. The percentage of mothers who made skin‐to‐skin contact within the first 24 hours after delivery was significantly higher in the DCC group (84.3% versus 45.9%). Breastfeeding in the immediate postpartum period was also significantly higher in the DCC group (77.3% versus 50.2%).
Conclusions
The results of our study show no differences in perinatal outcomes when performing ECC or DCC, and skin‐to‐skin contact, or breastfeeding.
Tweetable
This study demonstrates that delayed cord clamping is safe in mothers with confirmed SARS‐CoV‐2 infection.
Tweetable
This study demonstrates that delayed cord clamping is safe in mothers with confirmed SARS‐CoV‐2 infection.
PrimPol is a recently described DNA polymerase that has the virtue of initiating DNA synthesis. In addition of being a sensu stricto DNA primase, PrimPol's polymerase activity has a large capacity to ...tolerate different kind of lesions. The different strategies used by PrimPol for DNA damage tolerance are based on its capacity to “read” certain lesions, to skip unreadable lesions, and as an ultimate solution, to restart DNA synthesis beyond the lesion thus acting as a TLS primase. This lesion bypass potential, revised in this article, is strengthened by the preferential use of moderate concentrations of manganese ions as the preferred metal activator. We show here that PrimPol is able to extend RNA primers with ribonucleotides, even when bypassing 8oxoG lesions, suggesting a potential new scenario for PrimPol as a TLS polymerase assisting transcription. We also show that PrimPol displays a high degree of versatility to accept or induce distortions of both primer and template strands, creating alternative alignments based on microhomology that would serve to skip unreadable lesions and to connect separate strands. In good agreement, PrimPol is highly prone to generate indels at short nucleotide repeats. Finally, an evolutionary view of the relationship between translesion synthesis and primase functions is briefly discussed.
The objective of this work was on the one hand to assess the antibacterial activity of amines anchored to the external surface of mesoporous silica particles against Listeria monocytogenes in ...comparison with the same dose of free amines as well. It was also our aim to elucidate the mechanism of action of the new antimicrobial device. The suitability of silica nanoparticles to anchor, concentrate and improve the antimicrobial power of polyamines against L. monocytogenes has been demonstrated in a saline solution and in a food matrix. Moreover, through microscope observations it has been possible to determine that the attractive binding forces between the positive amine corona on the surface of nanoparticles and the negatively charged bacteria membrane provoke a disruption of the cell membrane. The surface concentration of amines on the surface of the nanoparticles is so effective that immobilized‐amines were 100 times more effective in killing L. monocytogenes bacteria than the same amount of free polyamines. This novel approach for the creation of antimicrobial nanodevices opens the possibility to put in value the antimicrobial power of natural molecules that have been discarded because of its low antimicrobial power.
Practical Application
Consumers demand for high‐quality products, free from chemical preservatives, with an extended shelf‐life. In this study, a really powerful antimicrobial agent based on a nanomaterial functionalized with a non‐antimicrobial organic molecule was developed as a proof of concept. Following this approach it could be possible to develop a new generation of natural and removable antimicrobials based on their anchoring to functional surfaces for food, agricultural or medical purposes.
Justification
In Mexico, the number of unidentified bodies has been steadily rising for years. By now, more than 50,000 bodies are considered unidentified. Forensic laboratories that could perform ...comparative molecular genetic investigation are often overburdened and examinations can take months. Therefore, pragmatic approaches that can help to identify more unknown bodies must be sought. The increased use of distinctive physical features might be one, and the high rate of tattooed people in Mexico points towards a great potential of tattoos as a tool for identification. The prerequisite for a comparison of antemortem (missing persons) and postmortem (unknown bodies) data is an objective description of the particularities, e.g., of the tattoos. The aim of this study was to establish an objective classification for tattoo motives, taking into consideration local preferences.
Methods
In the database of the medicolegal services of the Instituto Jaliscience de Ciencias Forenses (IJCF) in Guadalajara, postmortem data of 1000 tattooed bodies from 2019 were evaluated. According to sex and age, the tattooed body localization and the tattoo motives were categorized.
Results
The 1000 tattooed deceased showed tattoos on 2342 body localizations. The motives were grouped and linked to the following 11 keywords (with decreasing frequency): letters/numbers, human, symbol (other), plant, symbol (religious), animal, object, fantasy/demon/comic, tribal/ornament/geometry, other, unrecognizable.
Conclusion
Using the proposed classification, tattoo motives can be described objectively and classified in a practical way. If used for antemortem (missing persons) and postmortem (unknown bodies) documentation, motives can be searched and compared efficiently—helping to identify unknown bodies.
Abstract
Human PrimPol is a monomeric enzyme whose DNA primase activity is required to rescue stalled replication forks during nuclear and mitochondrial DNA replication. PrimPol contains an ...Archeal-Eukaryotic Primases (AEP) core followed by a C-terminal Zn finger-containing domain (ZnFD), that is exclusively required for primer formation and for PrimPol function in vivo. The present study describes the sequential substrate interactions of human PrimPol during primer synthesis, and the relevance of the ZnFD at each individual step. Both the formation of a PrimPol:ssDNA binary complex and the upcoming interaction with the 3′-nucleotide (pre-ternary complex) remained intact when lacking the ZnFD. Conversely, the ZnFD was required for the subsequent binding and selection of the 5′-nucleotide that will become the first nucleotide of the new primer strand. Providing different 5′-site nucleotides, we can conclude that the ZnFD of PrimPol most likely interacts with the γ-phosphate moiety of the 5′-site nucleotide, optimizing formation of the initial dimer. Moreover, the ZnFD also contributes to recognize the cryptic G at the preferred priming sequence 3′GTC5′. Dimer elongation to obtain long DNA primers occurs processively and is facilitated by the 5′-terminal triphosphate, indicating that the ZnFD is also essential in the subsequent translocation/elongation events during DNA primer synthesis.
A key component of antiretroviral therapy (ART) for HIV patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir. Recent reports of tenofovir toxicity in patients taking ART for ...HIV cannot be explained solely on the basis of off-target inhibition of mitochondrial DNA polymerase gamma (Polγ). PrimPol was discovered as a primase-polymerase localized to the mitochondria with repriming and translesion synthesis capabilities and, therefore, a potential contributor to mitochondrial toxicity. We established a possible role of PrimPol in tenofovir-induced toxicity in vitro and show that tenofovir-diphosphate incorporation by PrimPol is dependent on the n-1 nucleotide. We identified and characterized a PrimPol mutation, D114N, in an HIV+ patient on tenofovir-based ART with mitochondrial toxicity. This mutant form of PrimPol, targeting a catalytic metal ligand, was unable to synthesize primers, likely due to protein instability and weakened DNA binding. We performed cellular respiration and toxicity assays using PrimPol overexpression and shRNA knockdown strains in renal proximal tubular epithelial cells. The PrimPol-knockdown strain was hypersensitive to tenofovir treatment, indicating that PrimPol protects against tenofovir-induced mitochondrial toxicity. We show that a major cellular role of PrimPol is protecting against toxicity caused by ART and individuals with inactivating mutations may be predisposed to these effects.
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