34 results match your criteria rpa70 rpa32


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

View Article and Full-Text PDF
January 2021

Determinants of replication protein A subunit interactions revealed using a phosphomimetic peptide.

J Biol Chem 2020 12 30;295(52):18449-18458. Epub 2020 Oct 30.

Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea. Electronic address:

Replication protein A (RPA) is a eukaryotic ssDNA-binding protein and contains three subunits: RPA70, RPA32, and RPA14. Phosphorylation of the N-terminal region of the RPA32 subunit plays an essential role in DNA metabolism in processes such as replication and damage response. Phosphorylated RPA32 (pRPA32) binds to RPA70 and possibly regulates the transient RPA70-Bloom syndrome helicase (BLM) interaction to inhibit DNA resection. Read More

View Article and Full-Text PDF
December 2020

Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers.

Viruses 2020 10 2;12(10). Epub 2020 Oct 2.

BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA.

During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics of VRC formation over the course of infection. We used live cell fluorescence microscopy to analyze VRC formation during murine PyV (MuPyV) infection of a mouse fibroblast cell line that constitutively expresses a GFP-tagged replication protein A complex subunit (GFP-RPA32). Read More

View Article and Full-Text PDF
October 2020

LIM Protein Ajuba associates with the RPA complex through direct cell cycle-dependent interaction with the RPA70 subunit.

Sci Rep 2018 06 22;8(1):9536. Epub 2018 Jun 22.

Department of Biological Sciences, Hunter College, 695 Park Avenue, New York, NY, 10065, USA.

DNA damage response pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress. An early event in this activation is the recruitment and phosphorylation of RPA, a single stranded DNA binding complex composed of three subunits, RPA70, RPA32 and RPA14. Read More

View Article and Full-Text PDF

RPA inhibition increases replication stress and suppresses tumor growth.

Cancer Res 2014 Sep 28;74(18):5165-72. Epub 2014 Jul 28.

Department of Oral Biology, University of Nebraska Medical Center, Omaha, Nebraska. Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska.

The ATR/Chk1 pathway is a critical surveillance network that maintains genomic integrity during DNA replication by stabilizing the replication forks during normal replication to avoid replication stress. One of the many differences between normal cells and cancer cells is the amount of replication stress that occurs during replication. Cancer cells with activated oncogenes generate increased levels of replication stress. Read More

View Article and Full-Text PDF
September 2014

Nucleotide excision repair by mutant xeroderma pigmentosum group A (XPA) proteins with deficiency in interaction with RPA.

J Biol Chem 2011 Feb 9;286(7):5476-83. Epub 2010 Dec 9.

Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan.

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. Read More

View Article and Full-Text PDF
February 2011

Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase α.

Cell Res 2011 Feb 28;21(2):258-74. Epub 2010 Sep 28.

Howard Hughes Medical Institute, University of Michigan Medical School, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Read More

View Article and Full-Text PDF
February 2011

Arabidopsis replication protein A 70a is required for DNA damage response and telomere length homeostasis.

Plant Cell Physiol 2009 Nov 6;50(11):1965-76. Epub 2009 Oct 6.

Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

Replication protein A1 (RPA1/RPA70) forms a heterotrimeric complex together with RPA2/RPA32 and RPA3/RPA14 subunits which plays essential roles in various aspects of DNA metabolism including replication, repair, recombination and telomere maintenance. Compared with RPA70 in yeast and mammals, limited information is available about the factor in plants. In this study, we analyzed the functions of AtRPA70a, which is most similar to human RPA70 among four paralogs in Arabidopsis thaliana. Read More

View Article and Full-Text PDF
November 2009

A role for E1B-AP5 in ATR signaling pathways during adenovirus infection.

J Virol 2008 Aug 14;82(15):7640-52. Epub 2008 May 14.

CR-UK Institute for Cancer Studies, The Medical School, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.

E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Read More

View Article and Full-Text PDF

Epstein-Barr virus immediate-early protein Zta co-opts mitochondrial single-stranded DNA binding protein to promote viral and inhibit mitochondrial DNA replication.

J Virol 2008 May 27;82(9):4647-55. Epub 2008 Feb 27.

The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104, USA.

Disruption of cellular metabolic processes and usurpation of host proteins are hallmarks of herpesvirus lytic infection. Epstein-Barr virus (EBV) lytic replication is initiated by the immediate-early protein Zta. Zta is a multifunctional DNA binding protein that stimulates viral gene transcription, nucleates a replication complex at the viral origin of lytic replication, and inhibits cell cycle proliferation. Read More

View Article and Full-Text PDF

Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G2 checkpoint activation by the DNA strand-breaking nucleoside analogue 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine.

Mol Cancer Ther 2008 Jan;7(1):133-42

Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Box 71, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC), the prodrug (sapacitabine) of which is in clinical trials, has the novel mechanism of action of causing single-strand breaks after incorporating into DNA. Cells respond to this unique lesion by activating the G2 checkpoint, affected by the Chk1-Cdc25C-cyclin-dependent kinase 1/cyclin B pathway. This study aims at defining DNA damage checkpoint sensors that activate this response to CNDAC, particularly focusing on the major phosphatidylinositol 3-kinase-like protein kinase family proteins. Read More

View Article and Full-Text PDF
January 2008

Structure of the full-length human RPA14/32 complex gives insights into the mechanism of DNA binding and complex formation.

J Mol Biol 2007 Dec 2;374(4):865-76. Epub 2007 Oct 2.

The Eppley Institute for Research in Cancer and Allied Diseases, 987696 Nebraska Medical Center, Omaha, NE 68198-7696, USA.

Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1-42) and C-terminal (residues 175-270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Read More

View Article and Full-Text PDF
December 2007

Structural characterization of human RPA sequential binding to single-stranded DNA using ssDNA as a molecular ruler.

Biochemistry 2007 Jul 21;46(28):8226-33. Epub 2007 Jun 21.

Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.

Human replication protein A (RPA), a heterotrimer composed of RPA70, RPA32, and RPA14 subunits, contains four single-stranded DNA (ssDNA) binding domains (DBD): DBD-A, DBD-B, and DBD-C in RPA70 and DBD-D in RPA32. Although crystallographic or NMR structures of these DBDs and a trimerization core have been determined, the structure of the full length of RPA or the RPA-ssDNA complex remains unknown. In this article, we have examined the structural features of RPA interaction with ssDNA by fluorescence spectroscopy. Read More

View Article and Full-Text PDF

The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein.

Nucleic Acids Res 2007 20;35(3):755-70. Epub 2006 Dec 20.

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110 067, India.

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Read More

View Article and Full-Text PDF

Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases.

Arthritis Res Ther 2006 ;8(4):R111

Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Florida, PO Box 100221, Gainesville, Florida 32610, USA.

Replication protein A (RPA), a heterotrimer with subunits of molecular masses 70, 32, and 14 kDa, is a single-stranded-DNA-binding factor involved in DNA replication, repair, and recombination. There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS). This study sought to clarify the clinical significance of autoantibodies against RPA. Read More

View Article and Full-Text PDF
February 2007

A higher plant has three different types of RPA heterotrimeric complex.

J Biochem 2006 Jan;139(1):99-104

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510.

Replication protein A (RPA) is a protein complex composed of three subunits known as RPA70, RPA32, and RPA14. Generally, only one version of each of the three RPA genes is present in animals and yeast (with the exception of the human RPA32 ortholog). In rice (Oryza sativa L. Read More

View Article and Full-Text PDF
January 2006

Phosphorylation of replication protein A by S-phase checkpoint kinases.

DNA Repair (Amst) 2006 Mar 18;5(3):369-80. Epub 2006 Jan 18.

Department of Microbiology & Immunology, and the Witebsky Center for Microbial Pathogenesis & Immunology, University at Buffalo, School of Medicine & Biomedical Sciences, Buffalo, NY 14214, USA.

The major eukaryotic single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), is a heterotrimer with subunits of 70, 32 and 14 kDa (RPA70, RPA32 and RPA14). RPA-coated ssDNA has been implicated as one of the triggers for intra-S-phase checkpoint activation. Phosphorylation of RPA occurs in cells with damaged DNA or stalled replication forks. Read More

View Article and Full-Text PDF

Modulation of replication protein A function by its hyperphosphorylation-induced conformational change involving DNA binding domain B.

J Biol Chem 2005 Sep 9;280(38):32775-83. Epub 2005 Jul 9.

Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.

Human replication protein A (RPA), composed of RPA70, RPA32, and RPA14 subunits, undergoes hyperphosphorylation in cells in response to DNA damage. Hyperphosphorylation that occurs predominately in the N-terminal region of RPA32 is believed to play a role in modulating the cellular activities of RPA essential for almost all DNA metabolic pathways. To understand how the hyperphosphorylation modulates the functions of RPA, we compared the structural characteristics of full-length native and hyperphosphorylated RPAs using mass spectrometric protein footprinting, fluorescence spectroscopy, and limited proteolysis. Read More

View Article and Full-Text PDF
September 2005

Interaction and colocalization of Rad9/Rad1/Hus1 checkpoint complex with replication protein A in human cells.

Oncogene 2005 Jul;24(29):4728-35

Department of Biochemistry and Molecular Biology, James H Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA.

Replication protein A (RPA) is a eukaryotic single-stranded DNA-binding protein consisting of three subunits of 70-, 32-, and 14-kDa (RPA70, RPA32, RPA14, respectively). It is a protein essential for most cellular DNA metabolic pathways. Checkpoint proteins Rad9, Rad1, and Hus1 form a clamp-like complex which plays a central role in the DNA damage-induced checkpoint response. Read More

View Article and Full-Text PDF

Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes.

Mol Cancer Res 2003 May;1(7):519-31

CEA, Commissariat à l'Energie Atomique, Laboratoire de Génétique de la Radiosensibilité, Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, BP 6, 92265 Fontenay-aux-Roses, France.

The Homo sapiens kin17 ((HSA)kin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that (HSA)kin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. Read More

View Article and Full-Text PDF

Human replication protein A. The C-terminal RPA70 and the central RPA32 domains are involved in the interactions with the 3'-end of a primer-template DNA.

J Biol Chem 2003 May 24;278(19):17515-24. Epub 2003 Feb 24.

Abteilung Biochemie, Institut für Molekulare Biotechnologie, D-07745 Jena, Germany.

Although the mechanical aspects of the single-stranded DNA (ssDNA) binding activity of human replication protein A (RPA) have been extensively studied, only limited information is available about its interaction with other physiologically relevant DNA structures. RPA interacts with partial DNA duplexes that resemble DNA intermediates found in the processes of DNA replication and DNA repair. Limited proteolysis of RPA showed that RPA associated with ssDNA is less protected against proteases than RPA bound to a partial duplex DNA containing a 5'-protruding tail that had the same length as the ssDNA. Read More

View Article and Full-Text PDF

Cloning of the large subunit of replication protein A (RPA) from yeast Saccharomyces cerevisiae and its DNA binding activity through redox potential.

J Biochem Mol Biol 2002 Mar;35(2):194-8

Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Pusan 609-735, Korea.

Eukaryotic replication protein A (RPA) is a single-stranded(ss) DNA binding protein with multiple functions in DNA replication, repair, and genetic recombination. The 70-kDa subunit of eukaryotic RPA contains a conserved four cysteine-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Recently, we described a novel function for the zinc-finger motif in the regulation of human RPA's ssDNA binding activity through reduction-oxidation (redox). Read More

View Article and Full-Text PDF

Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA.

J Mol Biol 2002 Aug;321(1):133-48

Department of Chemistry, University of Toledo, 2801 West Bancroft Street, OH 43606-3390, USA.

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential for DNA replication, and plays important roles in DNA repair and DNA recombination. Rad52 and RPA, along with other members of the Rad52 epistasis group of genes, repair double-stranded DNA breaks (DSBs). Two repair pathways involve RPA and Rad52, homologous recombination and single-strand annealing. Read More

View Article and Full-Text PDF

Molecular anatomy of the human excision nuclease assembled at sites of DNA damage.

Mol Cell Biol 2002 Aug;22(16):5938-45

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599,USA.

Human nucleotide excision repair is initiated by six repair factors (XPA, RPA, XPC-HR23B, TFIIH, XPF-ERCC1, and XPG) which sequentially assemble at sites of DNA damage and effect excision of damage-containing oligonucleotides. We here describe the molecular anatomy of the human excision nuclease assembled at the site of a psoralen-adducted thymine. Three polypeptides, primarily positioned 5' to the damage, are in close physical proximity to the psoralen lesion and thus are cross-linked to the damaged DNA: these proteins are RPA70, RPA32, and the XPD subunit of TFIIH. Read More

View Article and Full-Text PDF

Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA.

EMBO J 2002 Apr;21(7):1855-63

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

The human single-stranded DNA-binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8-10 nucleotides (nt)] and stable ( approximately 30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2. Read More

View Article and Full-Text PDF

Replication protein A in Pyrococcus furiosus is involved in homologous DNA recombination.

Authors:
K Komori Y Ishino

J Biol Chem 2001 Jul 7;276(28):25654-60. Epub 2001 May 7.

Department of Molecular Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan.

Single-stranded DNA-binding protein in Bacteria and replication protein A (RPA) in Eukarya play crucial roles in DNA replication, repair, and recombination processes. We identified an RPA complex from the hyperthermophilic archaeon, Pyrococcus furiosus. Unlike the single-peptide RPAs from the methanogenic archaea, Methanococcus jannaschii and Methanothermobacter thermoautotrophicus, P. Read More

View Article and Full-Text PDF

The majority of human replication protein A remains complexed throughout the cell cycle.

Authors:
Y M Loo T Melendy

Nucleic Acids Res 2000 Sep;28(17):3354-60

Department of Microbiology, School of Medicine and Biomedical Sciences, SUNY Buffalo, 138 Farber Hall, Buffalo, NY 14214-3000, USA.

Replication Protein A (RPA), the replicative single-strand DNA binding protein from eukaryotic cells, is a stable heterotrimeric complex consisting of three polypeptides. Cytological studies have investigated the subcellular distribution and association characteristics of the three RPA subunits during different stages of the cell cycle with varying results. In this study, various HeLa cell fractions were subjected to separation by either immunoprecipitation or velocity sedimentation. Read More

View Article and Full-Text PDF
September 2000

The role for zinc in replication protein A.

J Biol Chem 2000 Sep;275(35):27332-8

Department of Biochemistry and Molecular Biology, the University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.

Heterotrimeric human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), is a central player in DNA replication, recombination, and repair. The C terminus of the largest subunit, RPA70, contains a putative zinc-binding motif and is implicated in complex formation with two smaller subunits, RPA14 and RPA32. The C-terminal domain of RPA70 (RPA70-CTD) was characterized using proteolysis and x-ray fluorescence emission spectroscopy. Read More

View Article and Full-Text PDF
September 2000

Human replication protein A: global fold of the N-terminal RPA-70 domain reveals a basic cleft and flexible C-terminal linker.

J Biomol NMR 1999 Aug;14(4):321-31

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.

Human Replication Protein A (hsRPA) is required for multiple cellular processes in DNA metabolism including DNA repair, replication and recombination. It binds single-stranded DNA with high affinity and interacts specifically with multiple proteins. hsRPA forms a heterotrimeric complex composed of 70-, 32- and 14-kDa subunits (henceforth RPA70, RPA32, and RPA14). Read More

View Article and Full-Text PDF

The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding.

EMBO J 1999 Aug;18(16):4498-504

Department of Medical Genetics and Microbiology, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8.

Replication protein A (RPA), the eukaryote single-stranded DNA-binding protein (SSB), is a heterotrimer. The largest subunit, RPA70, which harbours the major DNA-binding activity, has two DNA-binding domains that each adopt an OB-fold. The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA-binding activity but the mechanism of DNA binding is unknown. Read More

View Article and Full-Text PDF