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1. Single molecule microscopy reveals key physical features of repair foci in living cells

Author

Thierry Mora, Chloé Guedj, Mathias L. Heltberg, Marie Villemeur, Aleksandra M. Walczak, Angela Taddei, Maxime Dahan, Judith Miné-Hattab, Laboratoire de physique de l'ENS - ENS Paris (LPENS (UMR_8023)), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Laboratoire de physique de l'ENS - ENS Paris (LPENS), Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Sorbonne Université (SU)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Dynamique du noyau [Institut Curie], Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Université Paris Diderot - Paris 7 (UPD7)

Subjects
Saccharomyces cerevisiae Proteins, Focus (geometry), DNA repair, QH301-705.5, [SDV]Life Sciences [q-bio], Science, RAD52, genetic processes, single particle tracking, S. cerevisiae, Saccharomyces cerevisiae, Physics of Living Systems, General Biochemistry, Genetics and Molecular Biology, Single-stranded binding protein, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Replication Protein A, Biology (General), 030304 developmental biology, Double strand, [PHYS]Physics [physics], 0303 health sciences, General Immunology and Microbiology, biology, General Neuroscience, fungi, General Medicine, liquid-liquid phase separation, Single Molecule Imaging, Chromatin, nuclear sub-compartments, Rad52 DNA Repair and Recombination Protein, enzymes and coenzymes (carbohydrates), Single Molecule Microscopy, single molecule microscopy, chemistry, biology.protein, Biophysics, Medicine, 030217 neurology & neurosurgery, DNA, Research Article, DNA Damage
Abstract

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus possibly reflecting the existence of a liquid droplet around damaged DNA.; In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus possibly reflecting the existence of a liquid droplet around damaged DNA.

Published
2021
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2. Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1

Author

Paul Victor Sauer, Elisabetta Boeri-Erba, Norbert Mücke, Françoise Ochsenbein, David Sitbon, Jennifer Timm, Danni Liu, Jörg Langowski, Geneviève Almouzni, Daniel Panne, Christophe Velours, European Molecular Biology Laboratory [Grenoble] (EMBL), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Dynamique du noyau, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Deutsches Krebsforschungszentrum, Heidelberg, Universität Heidelberg [Heidelberg] = Heidelberg University, ANR-16-CE11-0028,REPLICAF,Structure et mechanisme de l'assemblage de la chromatine associée à la réplication(2016), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Universität Heidelberg [Heidelberg], Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), European Molecular Biology Laboratory [Grenoble] ( EMBL ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Service de Bioénergétique, Biologie Stucturale, et Mécanismes ( SB2SM ), Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -INSTITUT CURIE-Centre National de la Recherche Scientifique ( CNRS ), Université Pierre et Marie Curie - Paris 6 ( UPMC ), Institut de biologie structurale ( IBS - UMR 5075 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), and Commissariat à l'Énergie Atomique et aux Énergies Alternatives (CEA) - Grenoble

Subjects
0301 basic medicine, Saccharomyces cerevisiae Proteins, QH301-705.5, [SDV]Life Sciences [q-bio], Science, S. cerevisiae, Saccharomyces cerevisiae, Biology, DNA replication, Biochemistry, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Ribonucleases, histones, Histone H2A, Histone methylation, Nucleosome, Histone code, Protein–DNA interaction, Histone octamer, Biology (General), DNA, Fungal, 2. Zero hunger, Genetics, [ SDV ] Life Sciences [q-bio], General Immunology and Microbiology, General Neuroscience, General Medicine, Biophysics and Structural Biology, Linker DNA, chromatin assembly factor 1, 3. Good health, Cell biology, 030104 developmental biology, Histone, histone chaperone, biology.protein, chromatin, Medicine, Protein Multimerization, Protein Binding, Research Article
Abstract

How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3–H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization. DOI: http://dx.doi.org/10.7554/eLife.23474.001, eLife digest Animal and plant cells contain very long DNA molecules that are tightly packaged by being wrapped around proteins called histones to form structures known as nucleosomes. While this is a useful way to store DNA, it also makes it inaccessible to many proteins and other molecules that activate genes, copy DNA or perform other important cell processes. To enable these processes to take place, the cell can selectively disassemble particular nucleosomes and remove the histone proteins. Afterwards, the nucleosomes must reassemble to repackage the DNA. A single nucleosome contains four pairs of histones, with two pairs consisting of a H3 and a H4 histone. Histone chaperones assemble nucleosomes in a two-step process. First, two of these histone H3-H4 pairs (collectively known as a tetramer) interact with DNA to form a group or “complex”. Then, two more pairs of different histones bind to complete the nucleosome. An enzyme called CAF1 is known to attach H3-H4 tetramers onto DNA as the DNA is being copied, which allows nucleosomes to form on the newly made DNA. However, it is not known how CAF1 deposits H3-H4 tetramers onto the DNA. Sauer et al. explored how yeast CAF1 works by carrying out a series of experiments in a cell-free system. The experiments showed that each CAF1 enzyme binds to a single H3-H4 pair. When attached to their histone cargo, two CAF1 enzymes bind to DNA and attach a H3-H4 tetramer onto it. The tetramer has to form in this way for histones to be correctly delivered to DNA after the DNA has been copied. Sauer et al. also identified a new region of the CAF1 enzyme that binds to DNA. Together with another region, this enables CAF1 to bind to an extended stretch of DNA that accommodates the H3-H4 tetramer. Together, the findings explain the sequence of events that take place when CAF1 attaches H3-H4 tetramers onto DNA in the first step of nucleosome formation. Future work will be required to understand the structure of CAF1 in different situations and to find out how the cell targets this enzyme to stretches of DNA that have just been copied. DOI: http://dx.doi.org/10.7554/eLife.23474.002

Published
2017
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