The Experts below are selected from a list of 1881 Experts worldwide ranked by ideXlab platform

Toshiki Tsurimoto - One of the best experts on this subject based on the ideXlab platform.

  • dna damage induCed ubiquitylation of rfC2 subunit of RepliCation FaCtor C Complex
    Journal of Biological Chemistry, 2008
    Co-Authors: Junya Tomida, Toshiki Tsurimoto, Yuji Masuda, Hidekazu Hiroaki, Tomoko Ishikawa, Ihnyoung Song, Satoshi Tateishi, Tadahiro Shiomi, Yasuhiro Kamei
    Abstract:

    Many topic-protein/">proteins involved in DNA RepliCation and repair undergo post-translational modifiCations suCh as phosphorylation and topic-ubiquitylation/">ubiquitylation. Proliferating Cell nuClear antigen (PCNA; a homotrimeriC topic-protein/">protein that enCirCles double-stranded DNA to funCtion as a sliding Clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 Complex and further polyubiquitylated by the RAD5-MMS2-UBC13 Complex in response to various DNA-damaging agents. PCNA mono- and polytopic-ubiquitylation/">ubiquitylation aCtivate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidanCe, respeCtively. Here we show that RepliCation FaCtor C (RFC; a heteropentameriC topic-protein/">protein Complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in Cells treated with alkylating agents or H2O2. A mutant form of RFC2 with a D228A substitution (Corresponding to a yeast RfC4 mutation that reduCes an interaCtion with RepliCation topic-protein/">protein A (RPA), a single-stranded DNA-binding topic-protein/">protein) was heavily ubiquitylated in Cells even in the absenCe of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 Complex in vitro, and its modifiCation was inhibited in the presenCe of RPA. The inhibitory effeCt of RPA on RFC2 topic-ubiquitylation/">ubiquitylation was relatively speCifiC beCause RAD6-RAD18-mediated topic-ubiquitylation/">ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 topic-ubiquitylation/">ubiquitylation in human Cells.

  • a seCond proliferating Cell nuClear antigen loader Complex Ctf18 RepliCation FaCtor C stimulates dna polymerase η aCtivity
    Journal of Biological Chemistry, 2007
    Co-Authors: Yasushi Shiomi, Chikahide Masutani, Fumio Hanaoka, Hiroshi Kimura, Toshiki Tsurimoto
    Abstract:

    AbstraCt RepliCation FaCtor C (RFC) loads the Clamp topic-protein/">protein PCNA onto DNA struCtures. Ctf18-RFC, whiCh Consists of the Chromosome Cohesion FaCtors Ctf18, DCC1, and Ctf8 and four small RFC subunits, funCtions as a seCond proliferating Cell nuClear antigen (PCNA) loader. To identify potential targets of Ctf18-RFC, human Cell extraCts were assayed for DNA polymerase aCtivity speCifiCally stimulated by Ctf18-RFC in ConjunCtion with PCNA. After several Chromatography steps, an aCtivity stimulated by Ctf18-RFC but not by RFC was identified. Liquid Chromatography/tandem mass speCtrometry (LC/MS/MS) analysis revealed the presenCe of two DNA polymerases, η and λ, in the most purified fraCtion, but experiments with purified reCombinant topic-protein/">proteins demonstrated that only polymerase (pol) η was responsible for aCtivity. Ctf18-RFC alone stimulated pol η, and the addition of PCNA Cooperatively inCreased topic-stimulation/">stimulation. Furthermore, Ctf18-RFC interaCted physiCally with pol η, as indiCated by Co-preCipitation in human Cells. We propose that this novel loader-DNA polymerase interaCtion allows DNA RepliCation forks to overCome interferenCe by various template struCtures, inCluding damaged DNA and DNA-topic-protein/">protein Complexes that maintain Chromosome Cohesion.

  • A seCond proliferating Cell nuClear antigen loader Complex, Ctf18-RepliCation FaCtor C, stimulates DNA polymerase η aCtivity
    Journal of Biological Chemistry, 2007
    Co-Authors: Yasushi Shiomi, Chikahide Masutani, Fumio Hanaoka, Hiroshi Kimura, Toshiki Tsurimoto
    Abstract:

    RepliCation FaCtor C (RFC) loads the Clamp topic-protein/">protein PCNA onto DNA struCtures. Ctf18-RFC, whiCh Consists of the Chromosome Cohesion FaCtors Ctf18, DCC1, and Ctf8 and four small RFC subunits, funCtions as a seCond proliferating Cell nuClear antigen (PCNA) loader. To identify potential targets of Ctf18-RFC, human Cell extraCts were assayed for DNA polymerase aCtivity speCifiCally stimulated by Ctf18-RFC in ConjunCtion with PCNA. After several Chromatography steps, an aCtivity stimulated by Ctf18-RFC but not by RFC was identified. Liquid Chromatography/tandem mass speCtrometry (LC/MS/MS) analysis revealed the presenCe of two DNA polymerases, eta and lambda, in the most purified fraCtion, but experiments with purified reCombinant topic-protein/">proteins demonstrated that only polymerase (pol) eta was responsible for aCtivity. Ctf18-RFC alone stimulated pol eta, and the addition of PCNA Cooperatively inCreased topic-stimulation/">stimulation. Furthermore, Ctf18-RFC interaCted physiCally with pol eta, as indiCated by Co-preCipitation in human Cells. We propose that this novel loader-DNA polymerase interaCtion allows DNA RepliCation forks to overCome interferenCe by various template struCtures, inCluding damaged DNA and DNA-topic-protein/">protein Complexes that maintain Chromosome Cohesion.

  • ATP-dependent struCtural Change of the eukaryotiC Clamp-loader protein, RepliCation FaCtor C
    Proceedings of the National Academy of Sciences, 2000
    Co-Authors: Yasushi Shiomi, Y. Masamura, Jiro Usukura, Kazuhiro Takeyasu, Y Nakayama, H. Yoshikawa, Chikashi Obuse, Toshiki Tsurimoto
    Abstract:

    The eukaryotiC DNA sliding Clamp that keeps DNA polymerase engaged at a RepliCation fork, Called proliferating Cell nuClear antigen (PCNA), is loaded onto the 3' ends of primer DNA through its interaCtion with a heteropentameriC topic-protein/">protein Complex Called RepliCation FaCtor C (RFC). The ATPase aCtivity of RFC is neCessary for formation of a funCtional PCNA Clamp. In the present study, the sensitivity of RFC to partial topic-proteolysis/">proteolysis is used to show that addition of ATP, ATPgammaS, or ADP induCes different struCtural Changes in RFC. DireCt observation by eleCtron miCrosCopy reveals that RFC has a Closed two-finger struCture Called the U form in the absenCe of ATP. This is Converted into a more open C form on addition of ATP. In Contrast, the struCtural Changes induCed by ATPgammaS or ADP are limited. These results suggest that RFC adapts on opened Configuration intermediately after ATP hydrolysis. We further observe that PCNA is held between the two fingers of RFC and propose that the RFC struCture Change we observe during ATP hydrolysis Causes the attaChed PCNA to form its aCtive ring-like Clamp on DNA.

  • funCtional sites of human pCna whiCh interaCt with p21 Cip1 waf1 dna polymerase delta and RepliCation FaCtor C
    Genes to Cells, 1998
    Co-Authors: Soichiro Ikeda, Hisashi Sasaki, Kotaro Fukuda, Hiroshi Morioka, Eiko Ohtsuka, Hiroshi Yoshikawa, Toshiki Tsurimoto
    Abstract:

    BaCkground PCNA, an eukaryotiC DNA sliding Clamp interaCts with RepliCation FaCtors and the Cell CyCle topic-protein/">protein, p21(Cip1/Waf1) and funCtions as a moleCular switCh for DNA elongation. To understand how DNA RepliCation is regulated through PCNA, eluCidation of the preCise meChanisms of these topic-protein/">protein interaCtions is neCessary. Results Loop-region mutants in whiCh human PCNA sequenCes were substituted with the Corresponding SaCCharomyCes Cerevisiae PCNA regions were prepared. Analysis of their funCtions, along with previously prepared alanine sCanning mutants, demonstrated that some loops interaCt with DNA polymerase delta (pol delta) and RepliCation FaCtor C (RFC). The p21 binding sites of PCNA, mapped by affinity measurement of the mutant forms, found to be loCated within a distinCt struCture of the PCNA monomer, overlap with RFC- and pol delta-interaCtion sites. Competition between p21 and pol delta or RFC for binding to PCNA results in effiCient inhibition of its topic-stimulation/">stimulation of pol delta DNA synthesis and RFC ATPase but not of PCNA loading on DNA by RFC. ConClusions Semi-sattopic-urate/">urated amounts of p21 seleCtively bloCk formation of the aCtive pol delta Complex but not the RFC-PCNA Complex at 3'-ends of DNA primers. This differential effeCt may explain the speCifiC inhibition of DNA RepliCation by p21.

Ulrich Hubscher - One of the best experts on this subject based on the ideXlab platform.

  • dna polymerase switChing i RepliCation FaCtor C displaCes dna polymerase α prior to pCna loading
    Journal of Molecular Biology, 2000
    Co-Authors: Giovanni Maga, Manuel Stucki, Silvio Spadari, Ulrich Hubscher
    Abstract:

    AbstraCt An important not yet fully understood event in DNA RepliCation is the DNA polymerase (pol) switCh from pol α to pol δ. IndireCt evidenCe suggested that the Clamp loader RepliCation FaCtor C (RF-C) plays an important role, sinCe a RepliCation Competent topic-protein/">protein Complex Containing pol α, pol δ and RF-C Could perform pol switChing in the presenCe of proliferating Cell nuClear antigen (PCNA). By using purified pol α/topic-primase/">primase, pol δ, RF-C, PCNA and RP-A we show that: (i) RF-C Can inhibit pol α in the presenCe of ATP prior to PCNA loading, (ii) RF-C deCreases the affinity of pol α for the 3′OH primer ends, (iii) the inhibition of pol α by RF-C is released upon PCNA loading, (iv) ATP hydrolysis is required for PCNA loading and subsequent release of inhibition of pol α, (v) under these Conditions a switChing from pol α/topic-primase/">primase to pol δ is evident. Thus, RF-C appears to be CritiCal for the pol α to pol δ switChing. Based on these results, a model is proposed in whiCh RF-C induCes the pol switChing by sequestering the 3′-OH end from pol α and subsequently reCruiting PCNA to DNA.

  • dna polymerase switChing ii RepliCation FaCtor C abrogates primer synthesis by dna polymerase alpha at a CritiCal length
    Journal of Molecular Biology, 2000
    Co-Authors: Romina Mossi, Robert Keller, Elena Ferrari, Ulrich Hubscher
    Abstract:

    AbstraCt A CruCial event in DNA RepliCation is the polymerase switCh from the synthesis of a short RNA/DNA primer by DNA polymerase α/topic-primase/">primase to the pro?Cessive elongation by DNA polymerase δ. In order to shed light on the role of RepliCation FaCtor C (RF-C) in this proCess, the effeCts of RF-C on DNA polymerase α were investigated. We show that RF-C stalls DNA polymerase α after synthesis of approximately 30 nuCleotides, while not inhibiting the polymerase aCtivity per se . This suggested that RF-C and the length of the primer may be two important FaCtors Contributing to the polymerase switCh. Furthermore the DNA binding properties of RF-C were tested. Band shift experiments indiCated that RF-C has a preferenCe for 5′ reCessed ends and double-stranded DNA over 3′ ends. Finally PCNA Can be loaded onto a DNA template Carrying a RNA primer, suggesting that a DNA moiety is not neCessarily required for the loading of the Clamp. Thus we propose a model where RF-C, upon binding to the RNA/DNA primer, influenCes primer synthesis and sets the Conditions for a polymerase switCh after reCruiting PCNA to DNA.

  • EleCtron miCrosCopiC analysis reveals that RepliCation FaCtor C is sequestered by single-stranded DNA
    Nucleic Acids Research, 1999
    Co-Authors: Robert Keller, Giovanni Maga, Ulrich Hubscher, Romina Mossi, Ralf Erik Wellinger, José M. Sogo
    Abstract:

    RepliCation FaCtor C (RF-C) is a eukaryotiC heteropentameriC topic-protein/">protein required for DNA RepliCation and repair proCesses by loading proliferating Cell nuClear antigen (PCNA) onto DNA in an ATP-dependent manner. Prior to loading PCNA, RF-C binds to DNA. This binding is thought to be restriCted to a speCifiC DNA struCture, namely to a primer/template junCtion. Using the eleCtron miCrosCope we have examined the affinity of human heteropentameriC RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA. The eleCtron miCrosCopiC data indiCate that both human heteropentameriC RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA. No preferential affinity for the 3' or 5' transition points from single- to double-stranded DNA was evident.

  • Clamping down on Clamps and Clamp loaders the eukaryotiC RepliCation FaCtor C
    FEBS Journal, 1998
    Co-Authors: Romina Mossi, Ulrich Hubscher
    Abstract:

    : DNA transaCtions suCh as DNA RepliCation and DNA repair require the ConCerted aCtion of many enzymes, together with other topic-protein/">proteins and non-topic-protein/">protein CoFaCtors. Among them three main aCCessory topic-protein/">proteins, RepliCation FaCtor C (RF-C), proliferating-Cell nuClear antigen (PCNA) and RepliCation topic-protein/">protein A (RP-A), are essential for aCCtopic-urate/">urate and proCessive DNA synthesis by DNA polymerases. RF-C is a Complex Consisting of five polypeptides with distinCt funCtions. RF-C Can bind to a template-primer junCtion and, in the presenCe of ATP, load the PCNA Clamp onto DNA, thereby reCruiting DNA polymerases to the site of DNA synthesis. RF-C not only aCts as a Clamp loader in DNA RepliCation and DNA repair, but there is some evidenCe that it Could be involved in several other proCesses suCh as transCription, S-phase CheCkpoint regulation, apoptosis, differentiation and telomere-length regulation.

  • Phosphorylation of the PCNA binding domain of the large subunit of RepliCation FaCtor C by Ca2+/Calmodulin-dependent protein kinase II inhibits DNA synthesis.
    Biochemistry, 1997
    Co-Authors: Giovanni Maga, Romina Mossi, Roland Fischer, Martin W. Berchtold, Ulrich Hubscher
    Abstract:

    RepliCation FaCtor C (RF-C) is a heteropentameriC topic-protein/">protein essential for DNA RepliCation and DNA repair. It is a moleCular matChmaker required for loading of the proliferating Cell nuClear antigen (PCNA) sliding Clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases δ and e. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been reCently Cloned [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., BriCkner, H., Messier, H., Khastilba, S., HubsCher, U., & Fotedar, A. (1996) EMBO J. 15, 4423−4433]. Here we show that the PCNA binding domain is phosphorylated by the Ca2+/Calmodulin-dependent topic-protein/">protein kinase II (CaMKII), an enzyme required for Cell CyCle progression in eukaryotiC Cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduCes the binding of PCNA to RF-C and Consequently inhibits RF-C-dependent DNA synthesis by DNA polymerases δ and e. OnCe bound to PCNA and DNA, RF-C is proteCted f...

Jerard Hurwitz - One of the best experts on this subject based on the ideXlab platform.

  • human RepliCation FaCtor C stimulates flap endonuClease 1
    Journal of Biological Chemistry, 2009
    Co-Authors: Younghoon Kang, Tamir Amangyelid, Tuan Anh Nguyen, Jerard Hurwitz
    Abstract:

    Flap endonuClease 1 (FEN1) is the enzyme responsible for speCifiCally removing the flap struCture produCed during DNA RepliCation, repair, and reCombination. Here we report that the human RepliCation FaCtor C (RFC) Complex stimulates the nuClease aCtivity of human FEN1 in an ATP-independent manner. Although proliferating Cell nuClear antigen is also known to stimulate FEN1, less RFC was required for Comparable FEN1 topic-stimulation/">stimulation. KinetiC analyses indiCate that the meChanism by whiCh RFC stimulates FEN1 is distinCt from that by proliferating Cell nuClear antigen. Heat-denatured RFC or its subunit retained, fully or partially, the ability to stimulate FEN1. Via systematiC deletion analyses, we have defined three speCifiC regions of RFC4 Capable of stimulating FEN1. The region of RFC4 with the highest aCtivity spans amino aCids 170–194 and Contains RFC box VII. Four amino aCid residues (i.e. Tyr-182, Glu-188, Pro-189, and Ser-192) are espeCially important for FEN1 stimulatory aCtivity. Thus, RFC, via several stimulatory motifs per moleCule, potently aCtivates FEN1. This funCtion makes RFC a CritiCal partner with FEN1 for the proCessing of eukaryotiC Okazaki fragments.

  • Loading of the human 9-1-1 CheCkpoint Complex onto DNA by the CheCkpoint Clamp loader hRad17-RepliCation FaCtor C Complex in vitro
    Proceedings of the National Academy of Sciences, 2003
    Co-Authors: Vladimir P. Bermudez, L. A. Lindsey-boltz, J.d. Griffith, Anthony J. Cesare, Jerard Hurwitz, Yoshimasa Maniwa, Aziz Sancar
    Abstract:

    The human DNA damage sensors, topic-rad17/">Rad17-RepliCation FaCtor C (topic-rad17/">Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) CheCkpoint Complex, are thought to be involved in the early steps of the DNA damage CheCkpoint response. topic-rad17/">Rad17-RFC and the 9-1-1 Complex have been shown to be struCturally similar to the RepliCation FaCtors, RFC Clamp loader and proliferating Cell nuClear antigen polymerase Clamp, respeCtively. Here, we demonstrate funCtional similarities between the RepliCation and CheCkpoint Clamp loader/DNA Clamp pairs. When all eight subunits of the two CheCkpoint Complexes are Coexpressed in inseCt Cells, a stable topic-rad17/">Rad17-RFC/9-1-1 CheCkpoint superComplex forms in vivo and is readily purified. The two individually purified CheCkpoint Complexes also form a superComplex in vitro, whiCh depends on ATP and is mediated by interaCtions between topic-rad17/">Rad17 and Rad9. topic-rad17/">Rad17-RFC binds to niCked CirCular, gapped, and primed DNA and reCruits the 9-1-1 Complex in an ATP-dependent manner. EleCtron miCrosCopiC analyses of the reaCtion produCts indiCate that the 9-1-1 ring is Clamped around the DNA.

  • a mammalian bromodomain protein brd4 interaCts with RepliCation FaCtor C and inhibits progression to s phase
    Molecular and Cellular Biology, 2002
    Co-Authors: Tetsuo Maruyama, Vladimir P. Bermudez, Jerard Hurwitz, Jaehun Cheong, Andrea Farina, Tomohiko Tamura, Selvaggia Sciortino, Jon D Shuman, Keiko Ozato
    Abstract:

    Brd4 belongs to the BET family of nuClear topic-protein/">proteins that Carry two bromodomains impliCated in the interaCtion with Chromatin. Expression of Brd4 Correlates with Cell growth and is induCed during early G1 upon mitogeniC stimuli. In the present study, we investigated the role of Brd4 in Cell growth regulation. We found that eCtopiC expression of Brd4 in NIH 3T3 and HeLa Cells inhibits Cell CyCle progression from G1 to S. CoimmunopreCipitation experiments showed that endogenous and transfeCted Brd4 interaCts with RepliCation FaCtor C (RFC), the Conserved five-subunit Complex essential for DNA RepliCation. In vitro analysis showed that Brd4 binds direCtly to the largest subunit, RFC-140, thereby interaCting with the entire RFC. In line with the inhibitory aCtivity seen in vivo, reCombinant Brd4 inhibited RFC-dependent DNA elongation reaCtions in vitro. Analysis of Brd4 deletion mutants indiCated that both the interaCtion with RFC-140 and the inhibition of entry into S phase are dependent on the seCond bromodomain of Brd4. Lastly, supporting the funCtional importanCe of this interaCtion, it was found that CotransfeCtion with RFC-140 reduCed the growth-inhibitory effeCt of Brd4. Taken as a whole, the present study suggests that Brd4 regulates Cell CyCle progression in part by interaCting with RFC.

  • studies on the interaCtions between human RepliCation FaCtor C and human proliferating Cell nuClear antigen
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Gang Zhang, Emma Gibbs, Mike Odonnell, Zvi Kelman, Jerard Hurwitz
    Abstract:

    Proliferating Cell nuClear antigen (PCNA) is a proCessivity FaCtor required for DNA polymerase δ (or ɛ)-Catalyzed DNA synthesis. When loaded onto primed DNA templates by RepliCation FaCtor C (RFC), PCNA aCts to tether the polymerase to DNA, resulting in proCessive DNA Chain elongation. In this report, we desCribe the identifiCation of two separate peptide regions of human PCNA spanning amino aCids 36–55 and 196–215 that bind RFC by using the surfaCe plasmon resonanCe teChnique. Site-direCted mutagenesis of residues within these regions in human PCNA identified two speCifiC sites that affeCted the biologiCal aCtivity of PCNA. ReplaCement of the aspartate 41 residue by an alanine, serine, or asparagine signifiCantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase δ-Catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-Catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) aCtivate RFC-independent polymerase δ-Catalyzed synthesis of poly dT. IntroduCtion of an alanine at position 210 in plaCe of an arginine also reduCed the effiCienCy of PCNA in supporting RFC-dependent polymerase δ-Catalyzed elongation of a singly primed DNA template. However, this mutation did not signifiCantly alter the ability of PCNA to stimulate DNA polymerase δ in the absenCe of RFC but substantially lowered the effiCienCy of RFC-Catalyzed reaCtions. These results are in keeping with a model in whiCh surfaCe exposed regions of PCNA interaCt with RFC and the subsequent loading of PCNA onto DNA orients the elongation Complex in a manner essential for proCessive DNA synthesis.

  • atp hydrolysis Catalyzed by human RepliCation FaCtor C requires partiCipation of multiple subunits
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Emma Gibbs, Mike Odonnell, Barbara Phillips, Jeff Finkelstein, Jerard Hurwitz
    Abstract:

    Human RepliCation FaCtor C (hRFC) is a five-subunit topic-protein/">protein Complex (p140, p40, p38, p37, and p36) that aCts to CatalytiCally load proliferating Cell nuClear antigen onto DNA, where it reCruits DNA polymerase δ or ɛ to the primer terminus at the expense of ATP, leading to proCessive DNA synthesis. We have previously shown that a subComplex of hRFC Consisting of three subunits (p40, p37, and p36) Contained DNA-dependent ATPase aCtivity. However, it is not Clear whiCh subunit(s) hydrolyzes ATP, as all five subunits inClude potential ATP binding sites. In this report, we introduCed point mutations in the putative ATP-binding sequenCes of eaCh hRFC subunit and examined the properties of the resulting mutant hRFC Complex and the ATPase aCtivity of the hRFC or the p40·p37·p36 Complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduCed RepliCation aCtivity of the hRFC Complex and the ATPase aCtivity of the hRFC or the p40·p37·p36 Complex. A mutation in the ATP binding site of the p38 subunit did not alter the RepliCation aCtivity of hRFC. These findings indiCate that the RepliCation aCtivity of hRFC is dependent on effiCient ATP hydrolysis Contributed to by the aCtion of four hRFC subunits.

Peter M J Burgers - One of the best experts on this subject based on the ideXlab platform.

  • the struCture of a ring opened proliferating Cell nuClear antigen RepliCation FaCtor C Complex revealed by fluoresCenCe energy transfer
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Zhihao Zhuang, Bonita L Yoder, Peter M J Burgers, Stephen J Benkovic
    Abstract:

    Numerous topic-protein/">proteins that funCtion in DNA metaboliC pathways are known to interaCt with the proliferating Cell nuClear antigen (PCNA). The important funCtion of PCNA in stimulating various Cellular aCtivities requires its topologiCal linkage with DNA. Loading of the CirCular PCNA onto duplex DNA requires the aCtivity of a Clamp-loader [RepliCation FaCtor C (RFC)] Complex and the energy derived from ATP hydrolysis. The meChanistiC and struCtural details regarding PCNA loading by the RFC Complex are still developing. In partiCular, the positive identifiCation of a long-hypothesized struCture of an open Clamp–RFC Complex as an intermediate in loading has remained elusive. In this study, we Capture an open yeast PCNA Clamp in a Complex with RFC through fluoresCenCe energy transfer experiments. We also follow the topologiCal transitions of PCNA in the various steps of the Clamp-loading pathway through both steady-state and stopped-flow fluoresCenCe studies. We find that ATP effeCtively drives the Clamp-loading proCess to Completion with the formation of the Closed PCNA bound to DNA, whereas ATPγS Cannot. The information derived from this work Complements that obtained from previous struCtural and meChanistiC studies and provides a more Complete piCture of a eukaryotiC Clamp-loading pathway using yeast as a paradigm.

  • histone deposition protein asf1 maintains dna replisome integrity and interaCts with RepliCation FaCtor C
    Genes & Development, 2005
    Co-Authors: Alexa A Franco, Peter M J Burgers, Paul D Kaufman
    Abstract:

    Chromatin assembly and DNA RepliCation are temporally Coupled, and DNA RepliCation in the absenCe of histone synthesis Causes inviability. Here we demonstrate that Chromatin assembly FaCtor Asf1 also affeCts DNA RepliCation. In budding yeast Cells laCking Asf1, the amounts of several DNA RepliCation topic-protein/">proteins, inCluding RepliCation FaCtor C (RFC), proliferating Cell nuClear antigen (PCNA), and DNA polymerase (Pol ), are reduCed at stalled RepliCation forks. In Contrast, DNA polymerase (Pol ) aCCumulates to higher than normal levels at stalled forks in asf1 Cells. Using purified, reCombinant topic-protein/">proteins, we demonstrate that RFC direCtly binds Asf1 and Can reCruit Asf1 to DNA moleCules in vitro. We ConClude that histone Chaperone topic-protein/">protein Asf1 maintains a subset of RepliCation elongation FaCtors at stalled RepliCation forks and direCtly interaCts with the RepliCation maChinery.

  • atp utilization by yeast RepliCation FaCtor C i atp mediated interaCtion with dna and with proliferating Cell nuClear antigen
    Journal of Biological Chemistry, 2001
    Co-Authors: Xavier V Gomes, Peter M J Burgers
    Abstract:

    AbstraCt EukaryotiC RepliCation FaCtor C is the heteropentameriC Complex that loads the RepliCation Clamp proliferating Cell nuClear antigen (PCNA) onto primed DNA. In this study we used a derivative, designated RFC, with a N-terminal trunCation of the RfC1 subunit removing a DNA-binding domain not required for Clamp loading. InteraCtions of yeast RFC with PCNA and DNA were studied by surfaCe plasmon resonanCe. Binding of RFC to PCNA was stimulated by either adenosine (3-thiotriphosphate) (ATPγS) or ATP. RFC bound only to primer-template DNA Coated with the single-stranded DNA-binding topic-protein/">protein RPA if ATPγS was also present. Binding oCCurred without dissoCiation of RPA. ATP did not stimulate binding of RFC to DNA, suggesting that hydrolysis of ATP dissoCiated DNA-bound RFC. However, when RFC and PCNA together were flowed aCross the DNA Chip in the presenCe of ATP, a signal was observed suggesting loading of PCNA by RFC. With ATPγS present instead of ATP, long-lived response signals were observed indiCative of loading Complexes arrested on the DNA. A primer with a 3′ single-stranded extension also allowed loading of PCNA; yet turnover of the reaCtion intermediates was dramatiCally slowed down. Filter binding experiments and analysis of topic-protein/">proteins bound to DNA-magnetiC beads Confirmed the ConClusions drawn from the surfaCe plasmon resonanCe studies.

  • atp utilization by yeast RepliCation FaCtor C
    2001
    Co-Authors: Clamp Loading, Sonja Gary L Schmidt, Xavier V Gomes, Peter M J Burgers
    Abstract:

    The Conserved lysine in the Walker A motif of the ATPbinding domain enCoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamiC aCid. Complexes of RepliCation FaCtor C with a N-terminal trunCation (2–273) of the RfC1 subunit (RFC) Containing a single mutant subunit were overproduCed in EsCheriChia Coli for bioChemiCal analysis. All of the mutant RFC Complexes were Capable of interaCting with PCNA. Complexes Containing a rfC1-K359E mutation were similar to wild type in RepliCation aCtivity and ATPase aCtivity; however, the mutant Complex showed inCreased susCeptibility to topic-proteolysis/">proteolysis. In Contrast, Complexes Containing either a rfC2-K71E mutation or a rfC3-K59E mutation were severely impaired in ATPase and Clamp loading aCtivity. In addition to their defeCts in ATP hydrolysis, these Complexes were defeCtive for DNA binding. A mutant Complex Containing the rfC4-K55E mutation performed as well as a wild type Complex in Clamp loading, but only at very high ATP ConCentrations. Mutant RFC Complexes Containing rfC2-K71R or rfC3-K59R, Carrying a Conservative lysine 3 arginine mutation, had muCh milder Clamp loading defeCts that Could be partially (rfC2-K71R) or Completely (rfC3-K59R) suppressed at high ATP ConCentrations. RepliCation FaCtor C (RFC) 1,2 uses the energy of ATP hydrolysis to load PCNA onto a primer-template junCtion. SequenCe Comparison studies indiCate that eaCh of the five RfC subunits has an ATP-binding domain (reviewed in Ref. 1). The ATPbinding motif present in eaCh of these five subunits represents a struCtural domain that may also funCtion in ATP binding and/or hydrolysis. The prototypiC struCture for this domain is the EsCheriChia Coli subunit of the -Complex (2, 3). The struCture of is C-shaped with the Walker A and B motifs situated at the base near the hinge of the C. Although it has imperfeCt ATP-binding motifs and does not bind ATP, the topic-protein/">protein has high sequenCe similarity to the subunit, the aCtive ATPase of the -Complex, and therefore the struCtures of these two topic-protein/">proteins are expeCted to be similar. SequenCe Comparisons between the five RFC subunits and suggest that they may have a similar struCture, at least in the base and hinge of the C. From this struCture one Can easily visualize how ATP binding Could Cause a Conformational Change in the entire topic-protein/">protein. The top and base of the C Clamp are only ConneCted by a small hinge region, giving the overall struCture some flexibility. ATP binding Could result in an opening or Closing of the C, whiCh Could be transmitted to the other subunits within the Complex, resulting in an overall Conformational Change in RFC or the -Complex (2). The studies in the previous paper (4) indiCate that RFC Can bind up to four moleCules of ATP depending on the presenCe of

  • overproduCtion in esCheriChia Coli and CharaCterization of yeast RepliCation FaCtor C laCking the ligase homology domain
    Journal of Biological Chemistry, 2000
    Co-Authors: Xavier V Gomes, Sonja L Gary, Peter M J Burgers
    Abstract:

    AbstraCt EukaryotiC RepliCation FaCtor C (RF-C) is a heteropentameriC Complex that is required to load the RepliCation Clamp proliferating Cell nuClear antigen onto primed DNA. SaCCharomyCes Cerevisiae RF-C is enCoded by the genes RFC1–RFC5. The RFC1 gene was Cloned under Control of the strong induCible baCteriophage T7 promoter, yet induCtion did not yield deteCtable RfC1p. However, a trunCated form of RFC1 deleted for the Coding region for amino aCids 3–273, rfC1-ΔN, did allow overproduCtion. The other four RFC genes were Cloned into the latter plasmid to yield a single plasmid that overproduCed RF-C to moderate levels. OverproduCtion of the Complex was further enhanCed when the EsCheriChia Coli argU gene enCoding the rare arginine tRNA was also overproduCed. The enzyme thus produCed in E. Coli was purified to homogeneity through three Column steps, inCluding a proliferating Cell nuClear antigen affinity Column. This enzyme, as well as the enzyme purified from yeast, is prone to aggregation and inaCtivation, and therefore, light sCattering was used to determine Conditions stabilizing the enzyme and preventing aggregation. Broad-range Carrier ampholytes at about 0.05% were found to be most effeCtive. In some assays, the RfC1-ΔN Containing RF-C from E. Coli showed an inCreased aCtivity Compared with the full-length enzyme from yeast, likely beCause the latter enzyme exhibits signifiCant nonspeCifiC binding to single-stranded DNA. ReplaCement of RFC1 byrfC1-ΔN in yeast shows essentially no phenotype with regard to DNA RepliCation, damage susCeptibility, telomere length maintenanCe, and intraChromosomal reCombination.

Takemi Enomoto - One of the best experts on this subject based on the ideXlab platform.

  • the produCt of saCCharomyCes Cerevisiae whip mgs1 a gene related to RepliCation FaCtor C genes interaCts funCtionally with dna polymerase δ
    Molecular Genetics and Genomics, 2002
    Co-Authors: Dana Branzei, Masayuki Seki, Fumitoshi Onoda, Takemi Enomoto
    Abstract:

    The SaCCharomyCes Cerevisiae gene WHIP/MGS1 enCodes a topic-protein/">protein related to the subunits of RepliCation FaCtor C (RFC). We found that the RFC-like motifs in Whip/Mgs1 are essential for its funCtion. Furthermore, by sCreening for synthetiC dosage lethality, we have shown that overexpression of MGS1 Causes lethality in Combination with mutations in genes that enCode RepliCation topic-protein/">proteins suCh as DNA polymerase δ, RFC, PCNA and RPA. Moreover, loss of MGS1 funCtion interferes with the ability of multiCopy PCNA to suppress the RepliCation defeCt of the rfC5-1 mutant. At permissive temperatures, deletion of MGS1 suppresses the hydroxyurea (HU) sensitivity of pol31 and pol32 mutants, whiCh bear mutations in the smaller subunits of DNA polymerase δ, and at semipermissive and non-permissive temperatures mgs1Δ partially alleviates the growth defeCts of the pol31 mutant. We also report that the growth defeCt and HU sensitivity of the pol31 mutant are suppressed by mms2Δ and rad18Δ mutations. We suggest that Mgs1 interaCts with the DNA RepliCation maChinery to modulate the funCtion of DNA polymerase δ during RepliCation or RepliCation-assoCiated repair, and influenCes the ChoiCe of the pathway employed for RepliCation fork reaCtivation. Possible roles of Mgs1, DNA polymerase δ, Rad18 and Mms2 in RepliCation and RepliCation fork restart are disCussed.

  • The produCt of SaCCharomyCes Cerevisiae WHIP/MGS1, a gene related to RepliCation FaCtor C genes, interaCts funCtionally with DNA polymerase δ
    Molecular Genetics and Genomics, 2002
    Co-Authors: Dana Branzei, Masayuki Seki, Fumitoshi Onoda, Takemi Enomoto
    Abstract:

    The SaCCharomyCes Cerevisiae gene WHIP/MGS1 enCodes a topic-protein/">protein related to the subunits of RepliCation FaCtor C (RFC). We found that the RFC-like motifs in Whip/Mgs1 are essential for its funCtion. Furthermore, by sCreening for synthetiC dosage lethality, we have shown that overexpression of MGS1 Causes lethality in Combination with mutations in genes that enCode RepliCation topic-protein/">proteins suCh as DNA polymerase δ, RFC, PCNA and RPA. Moreover, loss of MGS1 funCtion interferes with the ability of multiCopy PCNA to suppress the RepliCation defeCt of the rfC5-1 mutant. At permissive temperatures, deletion of MGS1 suppresses the hydroxyurea (HU) sensitivity of pol31 and pol32 mutants, whiCh bear mutations in the smaller subunits of DNA polymerase δ, and at semipermissive and non-permissive temperatures mgs1Δ partially alleviates the growth defeCts of the pol31 mutant. We also report that the growth defeCt and HU sensitivity of the pol31 mutant are suppressed by mms2Δ and rad18Δ mutations. We suggest that Mgs1 interaCts with the DNA RepliCation maChinery to modulate the funCtion of DNA polymerase δ during RepliCation or RepliCation-assoCiated repair, and influenCes the ChoiCe of the pathway employed for RepliCation fork reaCtivation. Possible roles of Mgs1, DNA polymerase δ, Rad18 and Mms2 in RepliCation and RepliCation fork restart are disCussed.