G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent ...genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments.
Display omitted
•G4s block replication in cells lacking PrimPol causing local epigenetic instability•PrimPol binds G4s but cannot directly replicate them•PrimPol reprimes DNA synthesis closely coupled to G4s•Repriming preserves epigenetic stability in proximity G4 sequences
Schiavone, Jozwiakowski et al. demonstrate that the recently described primase-polymerase in vertebrates, PrimPol, plays an important role in replicating G quadruplex (G4) structures by binding them and repriming DNA-leading strand synthesis close by. This maintains processive replication and prevents disruption of histone recycling, ensuring epigenetic stability around G4s.
DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but ...identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.
Display omitted
•PrimPol is a DNA primase-polymerase catalyzing bypass of UV and oxidative lesions•PrimPol operates in a UV lesion tolerance pathway that is non-epistatic with Pol η•PrimPol null cells are defective in fork progression, particularly after UV treatment•Loss of PrimPol leads to increased mitotic chromosomal breaks
Knife crime is a source of concern for the police in England and Wales, however little published research exists on this crime type. Who are the offenders who use knives to commit crime, when and ...why? Who are their victims, and is there a victim-offender overlap? What is the social network formation for people who are exposed to knife crime? Using a multidimensional approach, our aim is to answer these questions about one of England and Wales' largest jurisdictions: Thames Valley. We first provide a state-of-the-art narrative review of the knife crime literature, followed by an analysis of population-level data on central tendency and dispersion of knife crimes reported to the police (2015-2019), on offences, offenders, victims, victim-offender overlaps and gang-related assaults. Social network analysis was used to explore the formations of offender-victim networks. Our findings show that knife crime represents a small proportion of crime (1.86%) and is associated largely with violence offenses. 16-34 year-old white males are at greatest risk of being the victims, offenders or victim-offenders of knife crime, with similar relative risks between these three categories. Both knife offenders and victims are likely to have a criminal record. Knife crimes are usually not gang-related (less than 20%), and experienced mostly between strangers, with the altercation often a non-retaliatory 'one-off event'. Even gang-related knife crimes do not follow 'tit-for-tat' relationships-except when the individuals involved have extensive offending histories and then are likely to retaliate instantaneously. We conclude that while rare, an incident of knife crime remains predicable, as a substantial ratio of offenders and victims of future knife crime can be found in police records. Prevention strategies should not be focused on gang-related criminals, but on either prolific violent offenders or repeat victims who are known to the police-and therefore more susceptible to knife crime exposure.
PrimPol,
ase-
ymerase), the most recently identified eukaryotic polymerase, has roles in both nuclear and mitochondrial DNA maintenance. PrimPol is capable of acting as a DNA polymerase, with the ...ability to extend primers and also bypass a variety of oxidative and photolesions. In addition, PrimPol also functions as a primase, catalysing the preferential formation of DNA primers in a zinc finger-dependent manner. Although PrimPol's catalytic activities have been uncovered
, we still know little about how and why it is targeted to the mitochondrion and what its key roles are in the maintenance of this multicopy DNA molecule. Unlike nuclear DNA, the mammalian mitochondrial genome is circular and the organelle has many unique proteins essential for its maintenance, presenting a differing environment within which PrimPol must function. Here, we discuss what is currently known about the mechanisms of DNA replication in the mitochondrion, the proteins that carry out these processes and how PrimPol is likely to be involved in assisting this vital cellular process.
PrimPol is a primase–polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc ...finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol−/−
cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication.
Abstract
Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To ...elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation frequency, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication. Deletion of PrimPol in XPV cells, lacking functional polymerase Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells.
Liquid chromatography (LC)-mass spectrometry (MS)/MS lipidomic normalization is generally performed by equalizing pre-extraction sample materials or via DNA or protein pre-quantitation methods, which ...have known measurement inaccuracies. We propose the use of the sulfo-phospho-vanillin assay (SPVA), a total lipid colorimetric analysis, as a pre-quantitation method to normalize lipids in lipidomic LC-MS/MS applications. The assay has been applied to a 300 μL well volume in a 96-well plate and tested using Avanti total lipid standards of porcine brain and
. Assay parameters for lipid sample volume, sulfuric acid, vanillin/phosphoric acid, post-reaction incubation time, and wavelength are optimized for robust application to biologically sourced lipid samples. Standard test samples were prepared using three concentrations covering approximately 100 μg/mL range. The optimized assay yielded test sample errors less than 10%, indicating a precise and accurate assay performance. The test samples were then analyzed by LC-MS/MS and normalized using SPVA pre-quantitation and pseudo-mass normalization. The detected lipids showed smaller standard deviations and greater relative concentration differences compared to the pseudo-mass normalized lipids, showing promise as a normalization method.
PrimPol is a DNA damage tolerance enzyme possessing both translesion synthesis (TLS) and primase activities. To uncover its potential role in TLS-mediated IgVλ hypermutation and define its interplay ...with other TLS polymerases, PrimPol(-/-) and PrimPol(-/-)/Polη(-/-)/Polζ (-/-) gene knockouts were generated in avian cells. Loss of PrimPol had no significant impact on the rate of hypermutation or the mutation spectrum of IgVλ. However, PrimPol(-/-) cells were sensitive to methylmethane sulfonate, suggesting that it may bypass abasic sites at the IgVλ segment by repriming DNA synthesis downstream of these sites. PrimPol(-/-) cells were also sensitive to cisplatin and hydroxyurea, indicating that it assists in maintaining / restarting replication at a variety of lesions. To accurately measure the relative contribution of the TLS and primase activities, we examined DNA damage sensitivity in PrimPol(-/-) cells complemented with polymerase or primase-deficient PrimPol. Polymerase-defective, but not primase-deficient, PrimPol suppresses the hypersensitivity of PrimPol(-/-) cells. This indicates that its primase, rather than TLS activity, is pivotal for DNA damage tolerance. Loss of TLS polymerases, Polη and Polζ has an additive effect on the sensitivity of PrimPol(-/-) cells. Moreover, we found that PrimPol and Polη-Polζ redundantly prevented cell death and facilitated unperturbed cell cycle progression. PrimPol(-/-) cells also exhibited increased sensitivity to a wide variety of chain-terminating nucleoside analogs (CTNAs). PrimPol could perform close-coupled repriming downstream of CTNAs and oxidative damage in vitro. Together, these results indicate that PrimPol's repriming activity plays a central role in reinitiating replication downstream from CTNAs and other specific DNA lesions.
DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to ...translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.
PDF
