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Science Advances Apr 2023Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different...
Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different translesion DNA synthesis (TLS) polymerases by the use of tools for their depletion, we identified a unique role of Pol ι in regulating such a pathway choice. Pol ι deficiency unleashes PrimPol-dependent repriming, which accelerates DNA replication in a pathway that is epistatic with ZRANB3 knockdown. In Pol ι-depleted cells, the excess participation of PrimPol in nascent DNA elongation reduces replication stress signals, but thereby also checkpoint activation in S phase, triggering chromosome instability in M phase. This TLS-independent function of Pol ι requires its PCNA-interacting but not its polymerase domain. Our findings unravel an unanticipated role of Pol ι in protecting the genome stability of cells from detrimental changes in DNA replication dynamics caused by PrimPol.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Replication; DNA; DNA Repair; DNA Damage; Chromosomal Instability; DNA Primase; Multifunctional Enzymes
PubMed: 37058556
DOI: 10.1126/sciadv.ade7997 -
Frontiers in Plant Science 2023The family of consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their...
The family of consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their genome in the nucleus of a plant cell, taking advantage of the host's DNA replication machinery. For converting their DNA into double-stranded DNA, and subsequent replication, these viruses rely on host DNA polymerases. However, the priming of the very first step of this process, i.e. the conversion of incoming circular ssDNA into a dsDNA molecule, has remained elusive for almost 30 years. In this study, sequencing of melon () accession K18 carrying the Tomato leaf curl New Delhi virus (ToLCNDV) recessive resistance quantitative trait locus (QTL) in chromosome 11, and analyses of DNA sequence data from 100 melon genomes, showed a conservation of a shared mutation in the () of all accessions that exhibited resistance upon a challenge with ToLCNDV. Silencing of (native) and subsequent challenging with three different geminiviruses showed a severe reduction in titers of all three viruses, altogether emphasizing an important role of in geminiviral replication. A model is presented explaining the role of during initiation of geminiviral DNA replication, i.e. as a regulatory subunit of primase that generates an RNA primer at the onset of DNA replication in analogy to -mediated initiation of DNA replication in all living organisms.
PubMed: 37008458
DOI: 10.3389/fpls.2023.1130723 -
Frontiers in Molecular Biosciences 2023Mobile genetic elements can encode a wide variety of genes that support their own stability and mobility as well as genes that provide accessory functions to their...
Mobile genetic elements can encode a wide variety of genes that support their own stability and mobility as well as genes that provide accessory functions to their hosts. Such genes can be adopted from host chromosomes and can be exchanged with other mobile elements. Due to their accessory nature, the evolutionary trajectories of these genes can differ from those of essential host genes. The mobilome therefore provides a rich source of genetic innovation. We previously described a new type of primase encoded by SCC elements that is composed of an A-family polymerase catalytic domain in complex with a small second protein that confers single-stranded DNA binding. Here we use new structure prediction methods in conjunction with sequence database searches to show that related primases are widespread among putative mobile genetic elements in the Bacillota. Structure predictions show that the second protein adopts an OB fold (common among single-stranded DNA binding (SSB) proteins) and these predictions were far more powerful than simple sequence comparisons in identifying its homologs. The protein-protein interaction surface varies among these polymerase-SSB complexes appear to have arisen repeatedly by exploiting partial truncations of the polymerase's N-terminal accessory domains.
PubMed: 37006622
DOI: 10.3389/fmolb.2023.1113960 -
BioRxiv : the Preprint Server For... Mar 2023Genetic information is duplicated via the highly regulated process of DNA replication. The machinery coordinating this process, the replisome, encounters many...
Genetic information is duplicated via the highly regulated process of DNA replication. The machinery coordinating this process, the replisome, encounters many challenges, including replication fork-stalling lesions that threaten the accurate and timely transmission of genetic information. Cells have multiple mechanisms to repair or bypass lesions that would otherwise compromise DNA replication. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2) function to regulate Replication Termination Factor 2 (RTF2) at the stalled replisome, allowing for replication fork stabilization and restart. Here we show that RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme responsible for removing RNA in the context of RNA-DNA heteroduplexes. We show that during unperturbed DNA replication, RTF2, like RNase H2, is required to maintain normal replication fork speeds. However, persistent RTF2 and RNase H2 at stalled replication forks compromises the replication stress response, preventing efficient replication restart. Such restart is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for regulation of replication-coupled ribonucleotide incorporation during normal replication and the replication stress response that is achieved through RTF2. We also provide evidence for PRIM1 function in direct replication restart following replication stress in mammalian cells.
PubMed: 36993543
DOI: 10.1101/2023.03.13.532415 -
BioRxiv : the Preprint Server For... Sep 2023The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity...
The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5'-end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.
PubMed: 36993335
DOI: 10.1101/2023.03.16.533013 -
Molecular Cell Apr 2023Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish...
Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku barrier to nascent strand degradation. RNase H2 cooperates with the MRN-Ctp1 axis to sustain cell resistance to replication stress in a Ku-dependent manner. Mechanistically, the need of RNaseH2 in nascent strand degradation requires the primase activity that allows establishing the Ku barrier to Exo1, whereas impairing Okazaki fragment maturation reinforces the Ku barrier. Finally, replication stress induces Ku foci in a primase-dependent manner and favors Ku binding to RNA:DNA hybrids. We propose a function for the RNA:DNA hybrid originating from Okazaki fragments in controlling the Ku barrier specifying nuclease requirement to engage fork resection.
Topics: RNA; DNA Primase; DNA; DNA Replication; Schizosaccharomyces; Ribonucleases
PubMed: 36868227
DOI: 10.1016/j.molcel.2023.02.008 -
Nucleic Acids Research Feb 2023Bacterial genomes contain an abundance of transposable insertion sequence (IS) elements that are essential for genome evolution and fitness. Among them, IS629 is present...
Bacterial genomes contain an abundance of transposable insertion sequence (IS) elements that are essential for genome evolution and fitness. Among them, IS629 is present in most strains of enterohemorrhagic Escherichia coli O157 and accounts for many polymorphisms associated with gene inactivation and/or genomic deletions. The excision of IS629 from the genome is promoted by IS-excision enhancer (IEE) protein. Despite IEE has been identified in the most pathogenic serotypes of E. coli, its biochemical features that could explain its role in IS excision are not yet understood. We show that IEE is present in >30% of all available E. coli genome assemblies, and is highly conserved and very abundant within enterohemorrhagic, enteropathogenic and enterotoxigenic genomes. In vitro analysis of the recombinant protein from E. coli O157:H7 revealed the presence of a Mn2+-dependent error-prone DNA polymerase activity in its N-terminal archaeo-eukaryotic primase (AEP) domain able to promote dislocations of the primer and template strands. Importantly, IEE could efficiently perform in vitro an end-joining reaction of 3'-single-strand DNA overhangs with ≥4 bp of homology requiring both the N-terminal AEP and C-terminal helicase domains. The proposed role for IEE in the novel IS excision mechanism is discussed.
Topics: DNA Transposable Elements; Enterohemorrhagic Escherichia coli; Escherichia coli O157; Regulatory Sequences, Nucleic Acid; DNA-Directed DNA Polymerase; Escherichia coli Proteins
PubMed: 36715333
DOI: 10.1093/nar/gkad017 -
MicroPublication Biology 2023Geminiviruses are causal agents of devastating diseases in crops. Geminiviral genomes are single-stranded (ss) circular DNA molecules that replicate in the nucleus of...
Geminiviruses are causal agents of devastating diseases in crops. Geminiviral genomes are single-stranded (ss) circular DNA molecules that replicate in the nucleus of the infected cell through double-stranded (ds) intermediates by co-opting the plant DNA replication machinery. However, the identity of the plant DNA polymerases enabling geminiviral replication has remained largely elusive. Recently, we showed that DNA polymerase α mediates the ss-to-ds conversion of tomato yellow leaf curl virus (TYLCV), and is therefore essential for the infection. Here, we provide data indicating that the primase subunits of DNA polymerase α, PRIM1 and PRIM2, are also required for TYLCV replication.
PubMed: 36685730
DOI: 10.17912/micropub.biology.000735 -
Acta Biochimica Et Biophysica Sinica Nov 2022PRIMPOL (primase-polymerase) is a recently discovered DNA primase-polymerase involved in DNA damage tolerance and replication stress response in eukaryotic cells....
PRIMPOL (primase-polymerase) is a recently discovered DNA primase-polymerase involved in DNA damage tolerance and replication stress response in eukaryotic cells. However, the detailed mechanism of the PRIMPOL response to replication stress remains elusive. Here, we demonstrate that replication-related factors, including replication protein A (RPA), regulate the accumulation of PRIMPOL in subnuclear foci in response to replication stress induced by replication inhibitors. Moreover, PRIMPOL works at G-quadruplexes (G4s) in human cells to resolve the replication stress induced by G4s. The formation of PRIMPOL foci persists throughout the cell cycle. We further demonstrate that PRIMPOL competes with RAD51 to resolve G4-induced replication stress. In conclusion, our results provide novel insight into the mechanism of PRIMPOL in G4s to resolve replication stress and competition between PRIMPOL (repriming)- and RAD51 (fork reversal)-mediated pathways, which indicates a new strategy to improve the tumor response to DNA-damaging chemotherapy by targeting the PRIMPOL pathway.
Topics: Humans; DNA Replication; Replication Protein A; G-Quadruplexes; DNA-Directed DNA Polymerase; DNA; DNA Primase; Rad51 Recombinase; Multifunctional Enzymes
PubMed: 36647718
DOI: 10.3724/abbs.2022165 -
PLoS Genetics Dec 2022Genetic association studies of many heritable traits resulting from physiological testing often have modest sample sizes due to the cost and burden of the required...
Genetic association studies of many heritable traits resulting from physiological testing often have modest sample sizes due to the cost and burden of the required phenotyping. This reduces statistical power and limits discovery of multiple genetic associations. We present a strategy to leverage pleiotropy between traits to both discover new loci and to provide mechanistic hypotheses of the underlying pathophysiology. Specifically, we combine a colocalization test with a locus-level test of pleiotropy. In simulations, we show that this approach is highly selective for identifying true pleiotropy driven by the same causative variant, thereby improves the chance to replicate the associations in underpowered validation cohorts and leads to higher interpretability. Here, as an exemplar, we use Obstructive Sleep Apnea (OSA), a common disorder diagnosed using overnight multi-channel physiological testing. We leverage pleiotropy with relevant cellular and cardio-metabolic phenotypes and gene expression traits to map new risk loci in an underpowered OSA GWAS. We identify several pleiotropic loci harboring suggestive associations to OSA and genome-wide significant associations to other traits, and show that their OSA association replicates in independent cohorts of diverse ancestries. By investigating pleiotropic loci, our strategy allows proposing new hypotheses about OSA pathobiology across many physiological layers. For example, we identify and replicate the pleiotropy across the plateletcrit, OSA and an eQTL of DNA primase subunit 1 (PRIM1) in immune cells. We find suggestive links between OSA, a measure of lung function (FEV1/FVC), and an eQTL of matrix metallopeptidase 15 (MMP15) in lung tissue. We also link a previously known genome-wide significant peak for OSA in the hexokinase 1 (HK1) locus to hematocrit and other red blood cell related traits. Thus, the analysis of pleiotropic associations has the potential to assemble diverse phenotypes into a chain of mechanistic hypotheses that provide insight into the pathogenesis of complex human diseases.
Topics: Humans; Genome-Wide Association Study; Phenotype; Genetic Association Studies; Sleep Apnea, Obstructive; Sleep; Genetic Pleiotropy; Polymorphism, Single Nucleotide; DNA Primase
PubMed: 36574455
DOI: 10.1371/journal.pgen.1010557