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EMBO Reports Nov 2023DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier-Gorlin syndrome. The other 12 encode components of the CDC45-MCM-GINS...
DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier-Gorlin syndrome. The other 12 encode components of the CDC45-MCM-GINS helicase, around which the eukaryotic replisome forms, or are factors required for helicase assembly during DNA replication initiation. A role for DONSON in CDC45-MCM-GINS assembly was unanticipated, since DNA replication initiation can be reconstituted in vitro with purified proteins from budding yeast, which lacks DONSON. Using mouse embryonic stem cells as a model for the mammalian helicase, we show that DONSON binds directly but transiently to CDC45-MCM-GINS during S-phase and is essential for chromosome duplication. Rapid depletion of DONSON leads to the disappearance of the CDC45-MCM-GINS helicase from S-phase cells and our data indicate that DONSON is dispensable for loading of the MCM2-7 helicase core onto chromatin during G1-phase, but instead is essential for CDC45-MCM-GINS assembly during S-phase. These data identify DONSON as a missing link in our understanding of mammalian chromosome duplication and provide a molecular explanation for why mutations in human DONSON are associated with Meier-Gorlin syndrome.
Topics: Mice; Animals; Humans; Cell Cycle Proteins; Chromosome Duplication; Cell Cycle; DNA Replication; Minichromosome Maintenance Proteins; Mammals
PubMed: 37781960
DOI: 10.15252/embr.202357677 -
Molecular Cell Aug 2023DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing...
DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing and activation in which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on replication origins. Here, we report the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome and demonstrate that SUV420H1 directly interacts with H4 N-terminal tail, the DNA, and the acidic patch in the nucleosome. The H4 (1-24) forms a lasso-shaped structure that stabilizes the SUV420H1-nucleosome complex and precisely projects the H4K20 residue into the SUV420H1 catalytic center. In vitro and in vivo analyses reveal a crucial role of the SUV420H1 KR loop (residues 214-223), which lies close to the H2A.Z-specific residues D97/S98, in H2A.Z-nucleosome preferential recognition. Together, our findings elucidate how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification and provide insights into how SUV420H1 preferentially recognizes H2A.Z nucleosome.
Topics: Histones; Nucleosomes; Methylation; DNA; DNA Replication
PubMed: 37536340
DOI: 10.1016/j.molcel.2023.07.001 -
Nucleic Acids Research Oct 2023Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair....
Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair. During DNA replication, RPA is required for extended origin DNA unwinding and DNA synthesis. To determine the requirements for RPA during these processes, we tested ssDNA-binding proteins (SSBs) from different domains of life in reconstituted Saccharomyces cerevisiae origin unwinding and DNA replication reactions. Interestingly, Escherichia coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we demonstrated that specific ssDNA-binding properties of RPA are required for origin unwinding but that its protein-interaction domains are dispensable. In contrast, we found that each of these auxiliary RPA domains have distinct functions at the eukaryotic replication fork. The Rfa1 OB-F domain negatively regulates lagging-strand synthesis, while the Rfa2 winged-helix domain stimulates nascent strand initiation. Together, our findings reveal a requirement for specific modes of ssDNA binding in the transition to extensive origin DNA unwinding and identify RPA domains that differentially impact replication fork function.
Topics: DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins; Protein Binding; Replication Protein A; Saccharomyces cerevisiae; Bacteriophage T4
PubMed: 37739410
DOI: 10.1093/nar/gkad765 -
Current Opinion in Structural Biology Aug 2023Mammalian chromosomes are organized at different length scales within the cell nucleus. Topologically Associating Domains (TADs) are structural units of 3D genome... (Review)
Review
Mammalian chromosomes are organized at different length scales within the cell nucleus. Topologically Associating Domains (TADs) are structural units of 3D genome organization with functions in gene regulation, DNA replication, recombination and repair. Whereas TADs were initially interpreted as insulated domains, recent studies are revealing that these domains should be interpreted as dynamic collections of actively extruding loops. This process of loop extrusion is subsequently blocked at dedicated TAD boundaries, thereby promoting intra-domain interactions over their surroundings. In this review, we discuss how mammalian TAD structure can emerge from this dynamic process and we discuss recent evidence that TAD boundaries can have regulatory functions.
Topics: Animals; Chromatin; Cell Nucleus; Gene Expression Regulation; Chromosomes, Mammalian; Genome; Mammals
PubMed: 37302180
DOI: 10.1016/j.sbi.2023.102622 -
Molecular Cell Nov 2023The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45...
The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients' mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.
Topics: Animals; Humans; Cell Cycle Proteins; Cryoelectron Microscopy; DNA; DNA Helicases; DNA Replication; Minichromosome Maintenance Proteins; Nuclear Proteins; Saccharomyces cerevisiae; Enzyme Activation
PubMed: 37820732
DOI: 10.1016/j.molcel.2023.09.029 -
Annual Review of Biochemistry Apr 2024Mammalian mitochondrial DNA (mtDNA) is replicated and transcribed by phage-like DNA and RNA polymerases, and our understanding of these processes has progressed... (Review)
Review
Mammalian mitochondrial DNA (mtDNA) is replicated and transcribed by phage-like DNA and RNA polymerases, and our understanding of these processes has progressed substantially over the last several decades. Molecular mechanisms have been elucidated by biochemistry and structural biology and essential in vivo roles established by cell biology and mouse genetics. Single molecules of mtDNA are packaged by mitochondrial transcription factor A into mitochondrial nucleoids, and their level of compaction influences the initiation of both replication and transcription. Mutations affecting the molecular machineries replicating and transcribing mtDNA are important causes of human mitochondrial disease, reflecting the critical role of the genome in oxidative phosphorylation system biogenesis. Mechanisms controlling mtDNA replication and transcription still need to be clarified, and future research in this area is likely to open novel therapeutic possibilities for treating mitochondrial dysfunction.
PubMed: 38594940
DOI: 10.1146/annurev-biochem-052621-092014 -
Biochemical Society Transactions Jun 2024Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the... (Review)
Review
Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals.
Topics: DNA, Mitochondrial; DNA Replication; Animals; Mitochondria; Humans; DNA-Directed RNA Polymerases; Transcription, Genetic; Drosophila; Exoribonucleases; Drosophila melanogaster
PubMed: 38884788
DOI: 10.1042/BST20230952 -
Nature Communications Aug 2023The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of...
The chromatin-based rule governing the selection and activation of replication origins in metazoans remains to be investigated. Here we report that NFIB, a member of Nuclear Factor I (NFI) family that was initially purified in host cells to promote adenoviral DNA replication but has since mainly been investigated in transcription regulation, is physically associated with the pre-replication complex (pre-RC) in mammalian cells. Genomic analyses reveal that NFIB facilitates the assembly of the pre-RC by increasing chromatin accessibility. Nucleosome binding and single-molecule magnetic tweezers shows that NFIB binds to and opens up nucleosomes. Transmission electron microscopy indicates that NFIB promotes nucleosome eviction on parental chromatin. NFIB deficiency leads to alterations of chromosome contacts/compartments in both G and S phase and affects the firing of a subset of origins at early-replication domains. Significantly, cancer-associated NFIB overexpression provokes gene duplication and genomic alterations recapitulating the genetic aberrance in clinical breast cancer and empowering cancer cells to dynamically evolve growth advantage and drug resistance. Together, these results point a role for NFIB in facilitating replication licensing by acting as a genome organizer, shedding new lights on the biological function of NFIB and on the replication origin selection in eukaryotes.
Topics: Animals; Adenoviridae; Cell Nucleus; Chromatin; Genomics; Mammals; NFI Transcription Factors; Nucleosomes; Humans
PubMed: 37604829
DOI: 10.1038/s41467-023-40846-1 -
Biochemistry. Biokhimiia Nov 2023The evolution of mitochondria has proceeded independently in different eukaryotic lines, which is reflected in the diversity of mitochondrial genomes and mechanisms of... (Review)
Review
The evolution of mitochondria has proceeded independently in different eukaryotic lines, which is reflected in the diversity of mitochondrial genomes and mechanisms of their expression in eukaryotic species. Mitochondria have lost most of bacterial ancestor genes by transferring them to the nucleus or eliminating them. However, mitochondria of almost all eukaryotic cells still retain relatively small genomes, as well as their replication, transcription, and translation apparatuses. The dependence on the nuclear genome, specific features of mitochondrial transcripts, and synthesis of highly hydrophobic membrane proteins in the mitochondria have led to significant changes in the translation apparatus inherited from the bacterial ancestor, which retained the basic structure necessary for protein synthesis but became more specialized and labile. In this review, we discuss specific properties of translation initiation in the mitochondria and how the evolution of mitochondria affected the functions of main factors initiating protein biosynthesis in these organelles.
Topics: Mitochondria; Protein Biosynthesis; Genome, Mitochondrial; Mitochondrial Proteins
PubMed: 38105202
DOI: 10.1134/S0006297923110135 -
Molecular Cell Aug 2023Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA...
Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.
Topics: Humans; BRCA2 Protein; Rad51 Recombinase; DNA-Binding Proteins; DNA, Single-Stranded; DNA; DNA Repair; Protein Binding
PubMed: 37499663
DOI: 10.1016/j.molcel.2023.06.031