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DNA Repair Sep 2019What happens to DNA replication when it encounters a damaged or nicked DNA template has been under investigation for five decades. Initially it was thought that DNA... (Review)
Review
What happens to DNA replication when it encounters a damaged or nicked DNA template has been under investigation for five decades. Initially it was thought that DNA polymerase, and thus the replication-fork progression, would stall at road blocks. After the discovery of replication-fork helicase and replication re-initiation factors by the 1990s, it became clear that the replisome can "skip" impasses and finish replication with single-stranded gaps and double-strand breaks in the product DNA. But the mechanism for continuous fork progression after encountering roadblocks is entangled with translesion synthesis, replication fork reversal and recombination repair. The recently determined structure of the bacteriophage T7 replisome offers the first glimpse of how helicase, primase, leading-and lagging-strand DNA polymerases are organized around a DNA replication fork. The tightly coupled leading-strand polymerase and lagging-strand helicase provides a scaffold to consolidate data accumulated over the past five decades and offers a fresh perspective on how the replisome may skip lesions and complete discontinuous DNA synthesis. Comparison of the independently evolved bacterial and eukaryotic replisomes suggests that repair of discontinuous DNA synthesis occurs post replication in both.
Topics: Bacteriophage T7; DNA Damage; DNA Helicases; DNA Repair; DNA Replication; DNA, Viral; DNA-Directed DNA Polymerase; Escherichia coli; Genomic Instability; Recombinational DNA Repair; Viral Proteins
PubMed: 31303546
DOI: 10.1016/j.dnarep.2019.102658 -
Proceedings of the National Academy of... Apr 2022Human DNA polymerase α (Polα) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Polα extends the RNA primers...
Human DNA polymerase α (Polα) does not possess proofreading ability and plays an important role in genome replication and mutagenesis. Polα extends the RNA primers generated by primase and provides a springboard for loading other replication factors. Here we provide the structural and functional analysis of the human Polα interaction with a mismatched template:primer. The structure of the human Polα catalytic domain in the complex with an incoming deoxycytidine triphosphate (dCTP) and the template:primer containing a T-C mismatch at the growing primer terminus was solved at a 2.9 Å resolution. It revealed the absence of significant distortions in the active site and in the conformation of the substrates, except the primer 3′-end. The T-C mismatch acquired a planar geometry where both nucleotides moved toward each other by 0.4 Å and 0.7 Å, respectively, and made one hydrogen bond. The binding studies conducted at a physiological salt concentration revealed that Polα has a low affinity to DNA and is not able to discriminate against a mispaired template:primer in the absence of deoxynucleotide triphosphate (dNTP). Strikingly, in the presence of cognate dNTP, Polα showed a more than 10-fold higher selectivity for a correct duplex versus a mismatched one. According to pre-steady-state kinetic studies, human Polα extends the T-C mismatch with a 249-fold lower efficiency due to reduction of the polymerization rate constant by 38-fold and reduced affinity to the incoming nucleotide by 6.6-fold. Thus, a mismatch at the postinsertion site affects all factors important for primer extension: affinity to both substrates and the rate of DNA polymerization.
Topics: Catalytic Domain; DNA Polymerase I; DNA Primers; DNA Replication; Humans; Kinetics
PubMed: 35467978
DOI: 10.1073/pnas.2111744119 -
Molecular Cell Jun 2020DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork...
DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.
Topics: Cell Line, Tumor; DNA; DNA Damage; DNA Primase; DNA Repair; DNA Replication; DNA-Binding Proteins; DNA-Directed DNA Polymerase; HEK293 Cells; Humans; K562 Cells; Multifunctional Enzymes; Nucleotidyltransferases; Transcription Factors
PubMed: 32442397
DOI: 10.1016/j.molcel.2020.04.031 -
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 -
ELife Sep 2023The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA...
The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA and binds to more than 20 partner proteins. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimic ssDNA. NMR chemical-shift studies also provided valuable information on the interaction of RPA70N residues with target sequences. However, it is still unclear how RPA70N recognizes and distinguishes such a diverse group of target proteins. Here, we present high-resolution crystal structures of RPA70N in complex with peptides from eight DNA damage response proteins. The structures show that, in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners. Our results advance the mechanistic understanding of RPA70N-mediated recruitment of DNA damage response proteins.
Topics: Humans; Crystallography; DNA Damage; DNA Primase; DNA, Single-Stranded; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Eukaryota; Multifunctional Enzymes; Replication Protein A
PubMed: 37668474
DOI: 10.7554/eLife.81639 -
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 -
Nature Aug 2022The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication...
The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.
Topics: DNA; DNA Primase; DNA Primers; DNA Replication; Humans; Protein Domains; RNA; Shelterin Complex; Substrate Specificity; Telomere; Templates, Genetic
PubMed: 35830881
DOI: 10.1038/s41586-022-05040-1 -
Genes Mar 2024The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation... (Review)
Review
The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.
Topics: Humans; DNA; DNA Replication; DNA Polymerase III; Minichromosome Maintenance Proteins; Genomic Instability
PubMed: 38540419
DOI: 10.3390/genes15030360 -
Nucleic Acids Research Nov 2021DNA-protein interactions play essential roles in all living cells. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins...
DNA-protein interactions play essential roles in all living cells. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is both challenging and important, since it may contribute to finding the means to regulate metabolic pathways involving DNA-protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA-protein interactions. Effective binding of T7 primase to its specific DNA recognition sequences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication.
Topics: Binding Sites; DNA; DNA Primase; Machine Learning; Nucleotide Motifs; Protein Binding
PubMed: 34718733
DOI: 10.1093/nar/gkab956 -
International Journal of Molecular... Apr 2023Replicative DNA polymerases, such as DNA polymerase α-primase, δ and ε, are multi-subunit complexes that are responsible for the bulk of nuclear DNA replication... (Review)
Review
Replicative DNA polymerases, such as DNA polymerase α-primase, δ and ε, are multi-subunit complexes that are responsible for the bulk of nuclear DNA replication during the S phase. Over the last decade, extensive genome-wide association studies and expression profiling studies of the replicative DNA polymerase genes in human patients have revealed a link between the replicative DNA polymerase genes and various human diseases and disorders including cancer, intellectual disability, microcephalic primordial dwarfism and immunodeficiency. These studies suggest the importance of dissecting the mechanisms involved in the functioning of replicative DNA polymerases in understanding and treating a range of human diseases. Previous studies in have established this organism as a useful model to understand a variety of human diseases. Here, we review the studies on that explored the link between DNA polymerases and human disease. First, we summarize the recent studies linking replicative DNA polymerases to various human diseases and disorders. We then review studies on replicative DNA polymerases in . Finally, we suggest the possible use of models to study human diseases and disorders associated with replicative DNA polymerases.
Topics: Animals; Humans; Drosophila; Genome-Wide Association Study; DNA-Directed DNA Polymerase; DNA Replication; Mutation
PubMed: 37175782
DOI: 10.3390/ijms24098078