315 results match your criteria cerevisiae rpa

Generation of Fluorescent Versions of Saccharomyces cerevisiae RPA to Study the Conformational Dynamics of Its ssDNA-Binding Domains.

Methods Mol Biol 2021 ;2281:151-168

Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA.

Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that sequesters ssDNA and protects it from nucleolytic degradation. The RPA-ssDNA nucleoprotein acts as a hub to recruit over two dozen DNA metabolic enzymes onto ssDNA to coordinate DNA replication, repair, and recombination. RPA functions as a heterotrimer composed of RPA70, RPA32, and RPA14 subunits and has multiple DNA-binding and protein-interaction domains. Read More

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January 2021

Structural parameters of palindromic repeats determine the specificity of nuclease attack of secondary structures.

Nucleic Acids Res 2021 04;49(7):3932-3947

School of Biological Sciences and Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GE 30332, USA.

Palindromic sequences are a potent source of chromosomal instability in many organisms and are implicated in the pathogenesis of human diseases. In this study, we investigate which nucleases are responsible for cleavage of the hairpin and cruciform structures and generation of double-strand breaks at inverted repeats in Saccharomyces cerevisiae. We demonstrate that the involvement of structure-specific nucleases in palindrome fragility depends on the distance between inverted repeats and their transcriptional status. Read More

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Genome stability is guarded by yeast Rtt105 through multiple mechanisms.

Genetics 2021 Feb;217(2)

CNRS UMR7258, INSERM U1068, Aix-Marseille Université UM105, Institut Paoli-Calmettes, CRCM, Marseille, France.

Ty1 mobile DNA element is the most abundant and mutagenic retrotransposon present in the genome of the budding yeast Saccharomyces cerevisiae. Protein regulator of Ty1 transposition 105 (Rtt105) associates with large subunit of RPA and facilitates its loading onto a single-stranded DNA at replication forks. Here, we dissect the role of RTT105 in the maintenance of genome stability under normal conditions and upon various replication stresses through multiple genetic analyses. Read More

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February 2021

Single-molecule studies illuminate the function of RAD51 paralogs.

Petr Cejka

Mol Cell 2021 03;81(5):898-900

Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, Bellinzona, 6500, Switzerland; Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH) Zürich, 8093, Switzerland. Electronic address:

In this issue of Molecular Cell, Roy et al. (2021) and Belan et al. (2021) demonstrate that the yeast and nematode RAD51 paralog complexes function as chaperones to promote the assembly of the RAD51 nucleoprotein filament on RPA-coated ssDNA. Read More

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Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis.

J Biol Chem 2020 11 10;295(47):15883-15891. Epub 2020 Sep 10.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri USA. Electronic address:

Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Read More

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November 2020

Rad54 Drives ATP Hydrolysis-Dependent DNA Sequence Alignment during Homologous Recombination.

Cell 2020 06 4;181(6):1380-1394.e18. Epub 2020 Jun 4.

Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address:

Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Read More

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Relocation of Collapsed Forks to the Nuclear Pore Complex Depends on Sumoylation of DNA Repair Proteins and Permits Rad51 Association.

Cell Rep 2020 05;31(6):107635

Department of Biology, Tufts University, Medford, MA 02155, USA; Program in Genetics, Tufts University, Boston, MA 02111, USA. Electronic address:

Expanded CAG repeats form stem-loop secondary structures that lead to fork stalling and collapse. Previous work has shown that these collapsed forks relocalize to nuclear pore complexes (NPCs) in late S phase in a manner dependent on replication, the nucleoporin Nup84, and the Slx5 protein, which prevents repeat fragility and instability. Here, we show that binding of the Smc5/6 complex to the collapsed fork triggers Mms21-dependent sumoylation of fork-associated DNA repair proteins, and that RPA, Rad52, and Rad59 are the key sumoylation targets that mediate relocation. Read More

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RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand.

Cell Stress 2020 Jan 17;4(3):48-63. Epub 2020 Jan 17.

Aix-Marseille Univ, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille, France. Equipe Labellisée par la Ligue Nationale contre le Cancer.

In , the absence of Pif1 helicase induces the instability of G4-containing CEB1 minisatellite during leading strand but not lagging strand replication. We report that RPA and Pif1 cooperate to maintain CEB1 stability when the G4 forming strand is either on the leading or lagging strand templates. At the leading strand, RPA acts in the same pathway as Pif1 to maintain CEB1 stability. Read More

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January 2020

Interaction of yeast Rad51 and Rad52 relieves Rad52-mediated inhibition of de novo telomere addition.

PLoS Genet 2020 02 3;16(2):e1008608. Epub 2020 Feb 3.

Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America.

DNA double-strand breaks (DSBs) are toxic forms of DNA damage that must be repaired to maintain genome integrity. Telomerase can act upon a DSB to create a de novo telomere, a process that interferes with normal repair and creates terminal deletions. We previously identified sequences in Saccharomyces cerevisiae (SiRTAs; Sites of Repair-associated Telomere Addition) that undergo unusually high frequencies of de novo telomere addition, even when the original chromosome break is several kilobases distal to the eventual site of telomerase action. Read More

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February 2020

Histone H3 Lysine 56 Acetylation Is Required for Formation of Normal Levels of Meiotic DNA Breaks in .

Front Cell Dev Biol 2019 10;7:364. Epub 2020 Jan 10.

MTA-DE Momentum Genome Architecture and Recombination Research Group, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.

Meiotic recombination is initiated by Spo11-catalyzed DNA double-strand breaks (DSBs) that are promoted by histone modifications and histone modifying enzymes. Herein we investigated the role of histone H3 lysine 56 acetylation (H3K56ac) located near the entry/exit points of the DNA in the globular H3 domain. We generated a series of mutant cells (Δ, Δ, Δ, and H3K56A) in which the endogenous level of H3K56ac was manipulated and tracked during meiotic growth. Read More

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January 2020

Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks.

Cell Mol Life Sci 2020 Aug 15;77(15):3041-3058. Epub 2019 Nov 15.

State Key Laboratory of Agro-Biotechnology and Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Biological Sciences China Agricultural University, Beijing, China.

The pre-initiation complex (pre-IC) has been proposed for two decades as an intermediate right before the maturation of the eukaryotic DNA replication fork. However, its existence and biochemical nature remain enigmatic. Here, through combining several enrichment strategies, we are able to isolate an endogenous dimeric CMG-containing complex (designated as d-CMG) distinct from traditional single CMG (s-CMG) and in vitro reconstituted dimeric CMG. Read More

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Activation of ATR-related protein kinase upon DNA damage recognition.

Curr Genet 2020 Apr 17;66(2):327-333. Epub 2019 Oct 17.

Department of Microbiology, Biochemistry and Molecular Genetics, International Center for Public Health, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, USA.

Chromosomes are constantly damaged by exogenous and endogenous factors. To cope with DNA damage, eukaryotic cells are equipped with three phosphatidylinositol 3-kinase-related kinases (PIKKs), such as ATM, ATR, and DNA-PK. PIKKs are structurally related to phosphatidylinositol 3-kinase (lipid kinase), however possess protein kinase activities. Read More

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The roles of fission yeast exonuclease 5 in nuclear and mitochondrial genome stability.

DNA Repair (Amst) 2019 11 21;83:102720. Epub 2019 Sep 21.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

The Exo5 family consists of bi-directional, single-stranded DNA-specific exonucleases that contain an iron-sulfur cluster as a structural motif and have multiple roles in DNA metabolism. S. cerevisiae Exo5 is essential for mitochondrial genome maintenance, while the human ortholog is important for nuclear genome stability and DNA repair. Read More

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November 2019

Actin and Nuclear Envelope Components Influence Ectopic Recombination in the Absence of Swr1.

Genetics 2019 11 18;213(3):819-834. Epub 2019 Sep 18.

Department of Genome Biology, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), Consejo Superior de Investigaciones Científicas-University of Seville-University Pablo de Olavide, Spain

The accuracy of most DNA processes depends on chromatin integrity and dynamics. Our analyses in the yeast show that an absence of Swr1 (the catalytic and scaffold subunit of the chromatin-remodeling complex SWR) leads to the formation of long-duration Rad52, but not RPA, foci and to an increase in intramolecular recombination. These phenotypes are further increased by MMS, zeocin, and ionizing radiation, but not by double-strand breaks, HU, or transcription/replication collisions, suggesting that they are associated with specific DNA lesions. Read More

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November 2019

Regulation of DNA Double Strand Breaks Processing: Focus on Barriers.

Front Mol Biosci 2019 16;6:55. Epub 2019 Jul 16.

Dipartimento di Bioscienze, Università degli studi di Milano, Milan, Italy.

In all the eukaryotic cells, nucleolytic processing (resection) of a double strand DNA break (DSB) is a key step to channel the repair of the lesion toward the homologous recombination, at the expenses of the non-homologous end joining (NHEJ). The coordinated action of several nucleases and helicases generates 3' single strand (ss) DNA, which is covered by RPA and recombination factors. Molecular details of the process have been first dissected in the model organism . Read More

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Ddc2ATRIP promotes Mec1ATR activation at RPA-ssDNA tracts.

PLoS Genet 2019 08 1;15(8):e1008294. Epub 2019 Aug 1.

Department of Microbiology, Biochemistry and Molecular Genetics, International Center for Public Health, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, United States of America.

The DNA damage checkpoint response is controlled by the phosphatidylinositol 3-kinase-related kinases (PIKK), including ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR). ATR forms a complex with its partner ATRIP. In budding yeast, ATR and ATRIP correspond to Mec1 and Ddc2, respectively. Read More

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Complementary roles of Pif1 helicase and single stranded DNA binding proteins in stimulating DNA replication through G-quadruplexes.

Nucleic Acids Res 2019 09;47(16):8595-8605

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.

G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5' to 3' helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. Read More

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September 2019

Competing interaction partners modulate the activity of Sgs1 helicase during DNA end resection.

EMBO J 2019 07 7;38(13):e101516. Epub 2019 Jun 7.

Peter Debye Institute for Soft Matter Physics, Universität Leipzig, Leipzig, Germany.

DNA double-strand break repair by homologous recombination employs long-range resection of the 5' DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay between them has been shown, it remains unclear whether and how these proteins cooperate on the molecular level. Read More

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Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52.

Genes Dev 2019 07 6;33(13-14):814-827. Epub 2019 Jun 6.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, 75390, USA.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. Read More

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RPA Phosphorylation Inhibits DNA Resection.

Mol Cell 2019 07 29;75(1):145-153.e5. Epub 2019 May 29.

Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, USA. Electronic address:

Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded DNA (ssDNA) overhangs. Resection regulation in bacteria is programmed by a DNA sequence, but a general mechanism limiting resection in eukaryotes has remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify phosphorylated RPA (pRPA) as a negative resection regulator. Read More

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S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates.

Chromosoma 2019 09 9;128(3):249-265. Epub 2019 May 9.

Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, S10 2TN, UK.

We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Read More

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September 2019

The RecQ helicase Sgs1 drives ATP-dependent disruption of Rad51 filaments.

Nucleic Acids Res 2019 05;47(9):4694-4706

Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032, USA.

DNA helicases of the RecQ family are conserved among the three domains of life and play essential roles in genome maintenance. Mutations in several human RecQ helicases lead to diseases that are marked by cancer predisposition. The Saccharomyces cerevisiae RecQ helicase Sgs1 is orthologous to human BLM, defects in which cause the cancer-prone Bloom's Syndrome. Read More

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Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection.

Proc Natl Acad Sci U S A 2019 03 8;116(13):6091-6100. Epub 2019 Mar 8.

Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032;

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3' single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. Read More

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Chaperoning RPA during DNA metabolism.

Curr Genet 2019 Aug 22;65(4):857-864. Epub 2019 Feb 22.

State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China.

Single-stranded DNA (ssDNA) is widely generated during DNA metabolisms including DNA replication, repair and recombination and is susceptible to digestion by nucleases and secondary structure formation. It is vital for DNA metabolism and genome stability that ssDNA is protected and stabilized, which are performed by the major ssDNA-binding protein, and replication protein A (RPA) in these processes. In addition, RPA-coated ssDNA also serves as a protein-protein-binding platform for coordinating multiple events during DNA metabolisms. Read More

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Dynamics and selective remodeling of the DNA-binding domains of RPA.

Nat Struct Mol Biol 2019 02 4;26(2):129-136. Epub 2019 Feb 4.

Department of Biological Sciences, Marquette University, Milwaukee, WI, USA.

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-stranded DNA (ssDNA) intermediates, activating the DNA-damage response and handing off ssDNA to the appropriate downstream players. Six DNA-binding domains (DBDs) in RPA promote high-affinity binding to ssDNA yet also allow RPA displacement by lower affinity proteins. We generated fluorescent versions of Saccharomyces cerevisiae RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. Read More

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February 2019

Replication protein A (RPA) sumoylation positively influences the DNA damage checkpoint response in yeast.

J Biol Chem 2019 02 27;294(8):2690-2699. Epub 2018 Dec 27.

From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065

The DNA damage response relies on protein modifications to elicit physiological changes required for coping with genotoxic conditions. Besides canonical DNA damage checkpoint-mediated phosphorylation, DNA damage-induced sumoylation has recently been shown to promote genotoxin survival. Cross-talk between these two pathways exists in both yeast and human cells. Read More

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February 2019

A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA.

Nat Commun 2018 12 21;9(1):5447. Epub 2018 Dec 21.

Section of Structural Biology, Department of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7 2AZ, UK.

Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Read More

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December 2018

MutSγ-Induced DNA Conformational Changes Provide Insights into Its Role in Meiotic Recombination.

Biophys J 2018 12 6;115(11):2087-2101. Epub 2018 Nov 6.

Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University, Middletown, Connecticut. Electronic address:

In many organisms, MutSγ plays a role in meiotic recombination, facilitating crossover formation between homologous chromosomes. Failure to form crossovers leads to improper segregation of chromosomes and aneuploidy, which in humans result in infertility and birth defects. To improve current understanding of MutSγ function, this study investigates the binding affinities and structures of MutSγ in complex with DNA substrates that model homologous recombination intermediates. Read More

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December 2018

RPA resolves conflicting activities of accessory proteins during reconstitution of Dmc1-mediated meiotic recombination.

Nucleic Acids Res 2019 01;47(2):747-761

Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL, USA.

Dmc1 catalyzes homology search and strand exchange during meiotic recombination in budding yeast and many other organisms including humans. Here we reconstitute Dmc1 recombination in vitro using six purified proteins from budding yeast including Dmc1 and its accessory proteins RPA, Rad51, Rdh54/Tid1, Mei5-Sae3 and Hop2-Mnd1 to promote D-loop formation between ssDNA and dsDNA substrates. Each accessory protein contributed to Dmc1's activity, with the combination of all six proteins yielding optimal activity. Read More

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January 2019

Dynamic interactions of the homologous pairing 2 (Hop2)-meiotic nuclear divisions 1 (Mnd1) protein complex with meiotic presynaptic filaments in budding yeast.

J Biol Chem 2019 01 12;294(2):490-501. Epub 2018 Nov 12.

From the Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032,

Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. Read More

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January 2019