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Marc S Wold - One of the best experts on this subject based on the ideXlab platform.

  • Single-Molecule AnAlysis of ReplicAtion Protein A-DNA InterActions.
    Methods in enzymology, 2018
    Co-Authors: Fletcher E. Bain, Ran Chen, Laura A. Fischer, Marc S Wold
    Abstract:

    AbstrAct ReplicAtion Protein A (RPA) is A highly conserved, eukAryotic ssDNA-binding Protein essentiAl for genome stAbility. RPA interActs with ssDNA And with Protein pArtners to coordinAte DNA ReplicAtion, repAir, And recombinAtion. Single-molecule AnAlysis of RPA–DNA interActions is leAding to A better understAnding of the moleculAr interActions And dynAmics responsible for RPA function in cells. Here, we first describe how to express, purify, And lAbel RPA. We then describe how to prepAre mAteriAls And cArry out single-molecule experiments exAmining RPA–DNA interActions using totAl internAl reflection fluorescence microscopy (TIRFM). FinAlly, the lAst section describes how to AnAlyze TIRFM dAtA. This chApter will focus on humAn RPA. However, these methods cAn be Applied to RPA homologs from other species.

  • ReplicAtion Protein A single strAnded dnA s first responder dynAmic dnA interActions Allow ReplicAtion Protein A to direct single strAnd dnA intermediAtes into different pAthwAys for synthesis or repAir
    BioEssays, 2014
    Co-Authors: Ran Chen, Marc S Wold
    Abstract:

    ReplicAtion Protein A (RPA), the mAjor single-strAnded DNA-binding Protein in eukAryotic cells, is required for processing of single-strAnded DNA (ssDNA) intermediAtes found in ReplicAtion, repAir And recombinAtion. Recent studies hAve shown thAt RPA binding to ssDNA is highly dynAmic And thAt more thAn high-Affinity binding is needed for function. AnAlysis of DNA binding mutAnts identified forms of RPA with reduced Affinity for ssDNA thAt Are fully Active, And other mutAnts with higher Affinity thAt Are inActive. Single molecule studies showed thAt while RPA binds ssDNA with high Affinity, the RPA complex cAn rApidly diffuse Along ssDNA And be displAced by other Proteins thAt Act on ssDNA. FinAlly, dynAmic DNA binding Allows RPA to prevent error-prone repAir of double-strAnded breAks And promote error-free repAir. Together, these findings suggest A new pArAdigm where RPA Acts As A first responder At sites with ssDNA, thereby Actively coordinAting DNA repAir And DNA synthesis.

  • diffusion of humAn ReplicAtion Protein A Along single strAnded dnA
    Journal of Molecular Biology, 2014
    Co-Authors: Binh Nguyen, Marc S Wold, Joshua E Sokoloski, Roberto Galletto, Elliot L Elson, Timothy M Lohman
    Abstract:

    AbstrAct ReplicAtion Protein A (RPA) is A eukAryotic single-strAnded DNA (ssDNA) binding Protein thAt plAys criticAl roles in most Aspects of genome mAintenAnce, including ReplicAtion, recombinAtion And repAir. RPA binds ssDNA with high Affinity, destAbilizes DNA secondAry structure And fAcilitAtes binding of other Proteins to ssDNA. However, RPA must be removed from or redistributed Along ssDNA during these processes. To probe the dynAmics of RPA–DNA interActions, we combined ensemble And single-molecule fluorescence ApproAches to exAmine humAn RPA (hRPA) diffusion Along ssDNA And find thAt An hRPA heterotrimer cAn diffuse rApidly Along ssDNA. Diffusion of hRPA is functionAl in thAt it provides the mechAnism by which hRPA cAn trAnsiently disrupt DNA hAirpins by diffusing in from ssDNA regions AdjAcent to the DNA hAirpin. hRPA diffusion wAs Also monitored by the fluctuAtions in fluorescence intensity of A Cy3 fluorophore AttAched to the end of ssDNA. Using A novel method to cAlibrAte the Cy3 fluorescence intensity As A function of hRPA position on the ssDNA, we estimAte A one-dimensionAl diffusion coefficient of hRPA on ssDNA of D 1  ~ 5000 nt 2 s − 1 At 37 °C. Diffusion of hRPA while bound to ssDNA enAbles it to be reAdily repositioned to Allow other Proteins Access to ssDNA.

  • in vitro AnAlysis of the role of ReplicAtion Protein A rpA And rpA phosphorylAtion in Atr mediAted checkpoint signAling
    Journal of Biological Chemistry, 2012
    Co-Authors: Laura A Lindseyboltz, Marc S Wold, Joyce T Reardon, Aziz Sancar
    Abstract:

    AbstrAct ReplicAtion Protein A (RPA) plAys essentiAl roles in DNA metAbolism, including ReplicAtion, checkpoint, And repAir. Recently, we described An in vitro system in which the phosphorylAtion of humAn Chk1 kinAse by ATR (AtAxiA telAngiectAsiA mutAted- And RAd3-relAted) is dependent on RPA bound to single-strAnded DNA. Here, we report thAt phosphorylAtion of other ATR tArgets, p53 And RAd17, hAs the sAme requirements And thAt RPA is Also phosphorylAted in this system. At high p53 or RAd17-RFC concentrAtions, RPA phosphorylAtion is inhibited And, in this system, RPA with phosphomimetic mutAtions cAnnot support ATR kinAse function while A non-phosphorylAtAble RPA mutAnt exhibits full Activity. PhosphorylAtion of these ATR substrAtes depends on the recruitment of ATR And the substrAtes by RPA to the RPA-ssDNA complex. FinAlly, mutAnt RPAs lAcking checkpoint function exhibit essentiAlly normAl Activity in nucleotide excision repAir, reveAling RPA sepArAtion of function for checkpoint And excision repAir.

  • Detection of PosttrAnslAtionAl ModificAtions of ReplicAtion Protein A
    Methods in molecular biology (Clifton N.J.), 2012
    Co-Authors: Cathy S. Hass, Ran Chen, Marc S Wold
    Abstract:

    ReplicAtion Protein A (RPA) is A single-strAnd DNA-binding Protein thAt is found in All eukAryotes. RPA is subjected to multiple posttrAnslAtionAl modificAtions including serine- And threonine-phosphorylAtion, poly-ADP ribosylAtion, And SUMOylAtion. These modificAtions Are believed to regulAte RPA Activity through modulAting interActions with DNA And pArtner Proteins. This Article describes two methods used to detect posttrAnslAtionAl modified RPA: immunofluorescence And immmuoblotting.

Walter J. Chazin - One of the best experts on this subject based on the ideXlab platform.

  • DiphenylpyrAzoles As ReplicAtion Protein A inhibitors.
    ACS medicinal chemistry letters, 2014
    Co-Authors: Alex G. Waterson, Michael D. Feldkamp, J. Phillip Kennedy, J.d. Patrone, N.f. Pelz, Andreas O. Frank, B. Vangamudi, Elaine M. Souza-fagundes, Olivia W. Rossanese, Walter J. Chazin
    Abstract:

    ReplicAtion Protein A is the primAry eukAryotic ssDNA binding Protein thAt hAs A centrAl role in initiAting the cellulAr response to DNA dAmAge. RPA recruits multiple Proteins to sites of DNA dAmAge viA the N-terminAl domAin of the 70 kDA subunit (RPA70N). Here we describe the optimizAtion of A diphenylpyrAzole cArboxylic Acid series of inhibitors of these RPA–Protein interActions. We evAluAted substituents on the AromAtic rings As well As the type And geometry of the linkers used to combine frAgments, ultimAtely leAding to submicromolAr inhibitors of RPA70N Protein–Protein interActions.

  • StructurAl AnAlysis of ReplicAtion Protein A recruitment of the DNA dAmAge response Protein SMARCAL1.
    Biochemistry, 2014
    Co-Authors: Michael D. Feldkamp, Aaron C. Mason, Brandt F. Eichman, Walter J. Chazin
    Abstract:

    SWI/SNF-relAted, mAtrix-AssociAted, Actin-dependent regulAtor of chromAtin, subfAmily A-like1 (SMARCAL1) is A recently identified DNA dAmAge response Protein involved in remodeling stAlled ReplicAtion forks. The eukAryotic single-strAnd DNA binding Protein ReplicAtion Protein A (RPA) recruits SMARCAL1 to stAlled forks in vivo And fAcilitAtes regression of forks contAining leAding strAnd gAps. Both Activities Are mediAted by A direct interAction between An RPA binding motif (RBM) At the N-terminus of SMARCAL1 And the C-terminAl winged-helix domAin of the RPA 32 kDA subunit (RPA32C). Here we report A biophysicAl And structurAl chArActerizAtion of the SMARCAL1–RPA interAction. IsothermAl titrAtion cAlorimetry And circulAr dichroism spectroscopy reveAled thAt RPA32C binds SMARCAL1-RBM with A Kd of 2.5 μM And induces A disorder-to-helix trAnsition. The crystAl structure of RPA32C wAs refined to 1.4 A resolution, And the SMARCAL1-RBM binding site wAs mApped on the structure on the bAsis of nucleAr mAgnetic resonAnce chemicAl shift perturbAtions. ConservAtion of the interAction surfAce to other RBM-contAining Proteins Allowed construction of A model for the RPA32C/SMARCAL1-RBM complex. The implicAtions of our results Are discussed with respect to the recruitment of SMARCAL1 And other DNA dAmAge response And repAir Proteins to stAlled ReplicAtion forks.

  • surfAce reengineering of rpA70n enAbles cocrystAllizAtion with An inhibitor of the ReplicAtion Protein A interAction motif of Atr interActing Protein
    Biochemistry, 2013
    Co-Authors: Andreas O. Frank, Alex G. Waterson, J.d. Patrone, B. Vangamudi, J.p. Kennedy, Stephen W. Fesik, Walter J. Chazin
    Abstract:

    ReplicAtion Protein A (RPA) is the primAry single-strAnded DNA (ssDNA) binding Protein in eukAryotes. The N-terminAl domAin of the RPA70 subunit (RPA70N) interActs viA A bAsic cleft with A wide rAnge of DNA processing Proteins, including severAl thAt regulAte DNA dAmAge response And repAir. SmAll molecule inhibitors thAt disrupt these Protein–Protein interActions Are therefore of interest As chemicAl probes of these criticAl DNA processing pAthwAys And As inhibitors to counter the upregulAtion of DNA dAmAge response And repAir AssociAted with treAtment of cAncer pAtients with rAdiAtion or DNA-dAmAging Agents. DeterminAtion of three-dimensionAl structures of Protein–ligAnd complexes is An importAnt step for elAborAtion of smAll molecule inhibitors. However, Although crystAl structures of free RPA70N And An RPA70N–peptide fusion construct hAve been reported, RPA70N–inhibitor complexes hAve been recAlcitrAnt to crystAllizAtion. AnAlysis of the P61 lAttice of RPA70N crystAls led us to hypothesize thAt the lig...

  • Discovery of Protein-Protein InterAction Inhibitors of ReplicAtion Protein A.
    ACS medicinal chemistry letters, 2013
    Co-Authors: J.d. Patrone, Walter J. Chazin, Alex G. Waterson, N.f. Pelz, Andreas O. Frank, B. Vangamudi, Olivia W. Rossanese, J.p. Kennedy, Feldkamp, Stephen W. Fesik
    Abstract:

    ReplicAtion Protein A (RPA) is A ssDNA binding Protein thAt is essentiAl for DNA ReplicAtion And repAir. The initiAtion of the DNA dAmAge response by RPA is mediAted by Protein–Protein interActions involving the N-terminAl domAin of the 70 kDA subunit with pArtner Proteins. Inhibition of these interActions increAses sensitivity towArd DNA dAmAge And ReplicAtion stress And mAy therefore be A potentiAl strAtegy for cAncer drug discovery. TowArd this end, we hAve discovered two leAd series of compounds, derived from hits obtAined from A frAgment-bAsed screen, thAt bind to RPA70N with low micromolAr Affinity And inhibit the binding of An ATRIP-derived peptide to RPA. These compounds mAy offer A promising stArting point for the discovery of clinicAlly useful RPA inhibitors.

  • A new structurAl frAmework for integrAting ReplicAtion Protein A into DNA processing mAchinery
    Nucleic Acids Research, 2013
    Co-Authors: Chris Brosey, Chunli Yan, Susan E Tsutakawa, William T Heller, Robert P Rambo, John A. Tainer, Ivaylo Ivanov, Walter J. Chazin
    Abstract:

    By coupling the protection And orgAnizAtion of single-strAnded DNA (ssDNA) with recruitment And Alignment of DNA processing fActors, ReplicAtion Protein A (RPA) lies At the heArt of dynAmic multi-Protein DNA processing mAchinery. Nevertheless, how RPA coordinAtes biochemicAl functions of its eight domAins remAins unknown. We exAmined the structurAl biochemistry of RPA’s DNA-binding Activity, combining smAll-Angle X-rAy And neutron scAttering with All-Atom moleculAr dynAmics simulAtions to investigAte the Architecture of RPA’s DNA-binding core. The scAttering dAtA reveAl compAction promoted by DNA binding; DNA-free RPA exists in An ensemble of stAtes with inter-domAin mobility And becomes progressively more condensed And less dynAmic on binding ssDNA. Our results contrAst with previous models proposing RPA initiAlly binds ssDNA in A condensed stAte And becomes more extended As it fully engAges the substrAte. Moreover, the consensus view thAt RPA engAges ssDNA in initiAl, intermediAte And finAl stAges conflicts with our dAtA reveAling thAt RPA undergoes two (not three) trAnsitions As it binds ssDNA with no evidence for A discrete intermediAte stAte. These results form A frAmework for understAnding how RPA integrAtes the ssDNA substrAte into DNA processing mAchinery, provides substrAte Access to its binding pArtners And promotes the progression And selection of DNA processing pAthwAys.

Ran Chen - One of the best experts on this subject based on the ideXlab platform.

  • Single-Molecule AnAlysis of ReplicAtion Protein A-DNA InterActions.
    Methods in enzymology, 2018
    Co-Authors: Fletcher E. Bain, Ran Chen, Laura A. Fischer, Marc S Wold
    Abstract:

    AbstrAct ReplicAtion Protein A (RPA) is A highly conserved, eukAryotic ssDNA-binding Protein essentiAl for genome stAbility. RPA interActs with ssDNA And with Protein pArtners to coordinAte DNA ReplicAtion, repAir, And recombinAtion. Single-molecule AnAlysis of RPA–DNA interActions is leAding to A better understAnding of the moleculAr interActions And dynAmics responsible for RPA function in cells. Here, we first describe how to express, purify, And lAbel RPA. We then describe how to prepAre mAteriAls And cArry out single-molecule experiments exAmining RPA–DNA interActions using totAl internAl reflection fluorescence microscopy (TIRFM). FinAlly, the lAst section describes how to AnAlyze TIRFM dAtA. This chApter will focus on humAn RPA. However, these methods cAn be Applied to RPA homologs from other species.

  • ReplicAtion Protein A single strAnded dnA s first responder dynAmic dnA interActions Allow ReplicAtion Protein A to direct single strAnd dnA intermediAtes into different pAthwAys for synthesis or repAir
    BioEssays, 2014
    Co-Authors: Ran Chen, Marc S Wold
    Abstract:

    ReplicAtion Protein A (RPA), the mAjor single-strAnded DNA-binding Protein in eukAryotic cells, is required for processing of single-strAnded DNA (ssDNA) intermediAtes found in ReplicAtion, repAir And recombinAtion. Recent studies hAve shown thAt RPA binding to ssDNA is highly dynAmic And thAt more thAn high-Affinity binding is needed for function. AnAlysis of DNA binding mutAnts identified forms of RPA with reduced Affinity for ssDNA thAt Are fully Active, And other mutAnts with higher Affinity thAt Are inActive. Single molecule studies showed thAt while RPA binds ssDNA with high Affinity, the RPA complex cAn rApidly diffuse Along ssDNA And be displAced by other Proteins thAt Act on ssDNA. FinAlly, dynAmic DNA binding Allows RPA to prevent error-prone repAir of double-strAnded breAks And promote error-free repAir. Together, these findings suggest A new pArAdigm where RPA Acts As A first responder At sites with ssDNA, thereby Actively coordinAting DNA repAir And DNA synthesis.

  • Detection of PosttrAnslAtionAl ModificAtions of ReplicAtion Protein A
    Methods in molecular biology (Clifton N.J.), 2012
    Co-Authors: Cathy S. Hass, Ran Chen, Marc S Wold
    Abstract:

    ReplicAtion Protein A (RPA) is A single-strAnd DNA-binding Protein thAt is found in All eukAryotes. RPA is subjected to multiple posttrAnslAtionAl modificAtions including serine- And threonine-phosphorylAtion, poly-ADP ribosylAtion, And SUMOylAtion. These modificAtions Are believed to regulAte RPA Activity through modulAting interActions with DNA And pArtner Proteins. This Article describes two methods used to detect posttrAnslAtionAl modified RPA: immunofluorescence And immmuoblotting.

Robert A. Bambara - One of the best experts on this subject based on the ideXlab platform.

  • CAtAlysis of strAnd AnneAling by ReplicAtion Protein A derives from its strAnd melting properties.
    The Journal of biological chemistry, 2008
    Co-Authors: Jeremy D. Bartos, Marc S Wold, Lyndsay J. Willmott, Sara K. Binz, Robert A. Bambara
    Abstract:

    EukAryotic DNA-binding Protein ReplicAtion Protein A (RPA) hAs A strAnd melting property thAt Assists polymerAses And helicAses in resolving DNA secondAry structures. Curiously, previous results suggested thAt humAn RPA (hRPA) promotes undesirAble recombinAtion by fAcilitAting AnneAling of flAps produced trAnsiently during DNA ReplicAtion; however, the mechAnism wAs not understood. We designed A series of substrAtes, representing displAced DNA flAps generAted during mAturAtion of OkAzAki frAgments, to investigAte the strAnd AnneAling properties of RPA. Until cleAved by FEN1 (flAp endonucleAse 1), such flAps cAn initiAte homologous recombinAtion. hRPA inhibited AnneAling of strAnds lAcking secondAry structure but promoted AnneAling of structured strAnds. AppArently, both processes primArily derive from the strAnd melting properties of hRPA. These properties slowed the spontAneous AnneAling of unstructured single strAnds, which occurred efficiently without hRPA. However, structured strAnds without hRPA displAyed very slow spontAneous AnneAling becAuse of stAble intrAmoleculAr hydrogen bonding. hRPA AppeAred to trAnsiently melt the single strAnds so thAt they could bind to form double strAnds. In this wAy, melting ironicAlly promoted AnneAling. Time course meAsurements in the presence of hRPA suggest thAt structured single strAnds Achieve An equilibrium with double strAnds, A consequence of RPA driving both AnneAling And melting. Promotion of AnneAling reAched A mAximum At A specific hRPA concentrAtion, presumAbly when All structured single-strAnded DNA wAs melted. Results suggest thAt displAced flAps with secondAry structure formed during OkAzAki frAgment mAturAtion cAn be melted by hRPA And subsequently AnneAled to A complementAry ectopic DNA site, forming recombinAtion intermediAtes thAt cAn leAd to genomic instAbility.

  • MechAnism underlying ReplicAtion Protein A stimulAtion of DNA ligAse I.
    Journal of Biological Chemistry, 2001
    Co-Authors: Tamara A. Ranalli, Michael S. Demott, Robert A. Bambara
    Abstract:

    AbstrAct ReplicAtion Protein A (RPA) is A heterotrimeric single-strAnded DNA-binding Protein thAt pArticipAtes in multiple DNA trAnsActions thAt include ReplicAtion And repAir. BAse excision repAir is A centrAl DNA repAir pAthwAy, responsible for the removAl of dAmAged bAses. We hAve shown previously thAt RPA wAs Able to stimulAte long pAtch bAse excision repAir reconstituted in vitro. Herein we show thAt humAn RPA stimulAtes the Activity of the bAse excision repAir component humAn DNA ligAse I by ApproximAtely 15-fold. Other AnAlyzed single-strAnded binding Proteins would not substitute, Attesting to the specificity of the stimulAtion. Conversely, RPA wAs unAble to stimulAte the functionAlly homologous ATP-dependent ligAse from T4 bActeriophAge. Kinetic AnAlyses suggest thAt cAtAlysis of ligAtion is enhAnced by RPA, As A 4-fold increAse in k cAt is observed, whereAsK m is not significAntly chAnged. SubstrAte competition experiments further support the conclusion thAt RPA does not Alter the specificity or rAte of substrAte binding by DNA ligAse I. AdditionAlly, RPA is unAble to significAntly enhAnce ligAtion on substrAtes contAining An unAnneAled 3′-upstreAm primer terminus, suggesting thAt RPA does not stAbilize the nick site to enhAnce ligAse recognition. Furthermore when DNA ligAse I is pre-bound to the substrAte And limited to A single turnover, RPA is still Able to stimulAte ligAtion. OverAll, the results support A mechAnism of stimulAtion thAt involves increAsing the rAte of cAtAlysis of ligAtion.

  • ReplicAtion Protein A StimulAtes Long PAtch DNA BAse Excision RepAir
    The Journal of biological chemistry, 1998
    Co-Authors: Michael S. Demott, Seymour Zigman, Robert A. Bambara
    Abstract:

    AbstrAct Two pAthwAys for completion of DNA bAse excision repAir (BER) hAve recently emerged. In one, cAlled short pAtch BER, only the dAmAged nucleotide is replAced, whereAs in the second, known As long pAtch BER, the monobAsic lesion is removed Along with AdditionAl downstreAm nucleotides. FlAp endonucleAse 1, which preferentiAlly cleAves unAnneAled 5′-flAp structures in DNA, hAs been shown to plAy A cruciAl role in the long pAtch mode of repAir. This nucleAse will efficiently releAse 5′-terminAl AbAsic lesions As pArt of An intAct oligonucleotide when cleAvAge is combined with strAnd displAcement synthesis. Further gAp filling And ligAtion complete repAir. We reconstituted the finAl steps of long pAtch bAse excision repAir in vitro using cAlf DNA polymerAse e to provide strAnd displAcement synthesis, humAn flAp endonucleAse 1, And humAn DNA ligAse I. ReplicAtion Protein A is An importAnt constituent of the DNA ReplicAtion mAchinery. It Also hAs been shown to interAct with An eArly component of bAse excision repAir: urAcil glycosylAse. Here we show thAt humAn ReplicAtion Protein A greAtly stimulAtes long pAtch bAse excision repAir.

Aziz Sancar - One of the best experts on this subject based on the ideXlab platform.

  • in vitro AnAlysis of the role of ReplicAtion Protein A rpA And rpA phosphorylAtion in Atr mediAted checkpoint signAling
    Journal of Biological Chemistry, 2012
    Co-Authors: Laura A Lindseyboltz, Marc S Wold, Joyce T Reardon, Aziz Sancar
    Abstract:

    AbstrAct ReplicAtion Protein A (RPA) plAys essentiAl roles in DNA metAbolism, including ReplicAtion, checkpoint, And repAir. Recently, we described An in vitro system in which the phosphorylAtion of humAn Chk1 kinAse by ATR (AtAxiA telAngiectAsiA mutAted- And RAd3-relAted) is dependent on RPA bound to single-strAnded DNA. Here, we report thAt phosphorylAtion of other ATR tArgets, p53 And RAd17, hAs the sAme requirements And thAt RPA is Also phosphorylAted in this system. At high p53 or RAd17-RFC concentrAtions, RPA phosphorylAtion is inhibited And, in this system, RPA with phosphomimetic mutAtions cAnnot support ATR kinAse function while A non-phosphorylAtAble RPA mutAnt exhibits full Activity. PhosphorylAtion of these ATR substrAtes depends on the recruitment of ATR And the substrAtes by RPA to the RPA-ssDNA complex. FinAlly, mutAnt RPAs lAcking checkpoint function exhibit essentiAlly normAl Activity in nucleotide excision repAir, reveAling RPA sepArAtion of function for checkpoint And excision repAir.

  • tipin ReplicAtion Protein A interAction mediAtes chk1 phosphorylAtion by Atr in response to genotoxic stress
    Journal of Biological Chemistry, 2010
    Co-Authors: Michael G Kemp, Zafer Akan, Secil Yilmaz, Mary Grillo, Stephanie L Smithroe, Taehong Kang, Marila Cordeirostone, William K Kaufmann, Robert T Abraham, Aziz Sancar
    Abstract:

    MAmmAliAn Timeless is A multifunctionAl Protein thAt performs essentiAl roles in the circAdiAn clock, chromosome cohesion, DNA ReplicAtion fork protection, And DNA ReplicAtion/DNA dAmAge checkpoint pAthwAys. The humAn Timeless exists in A tight complex with A smAller Protein cAlled Tipin (Timeless-interActing Protein). Here we investigAted the mechAnism by which the Timeless-Tipin complex functions As A mediAtor in the ATR-Chk1 DNA dAmAge checkpoint pAthwAy. We find thAt the Timeless-Tipin complex specificAlly mediAtes Chk1 phosphorylAtion by ATR in response to DNA dAmAge And ReplicAtion stress through interAction of Tipin with the 34-kDA subunit of ReplicAtion Protein A (RPA). The Tipin-RPA interAction stAbilizes Timeless-Tipin And Tipin-ClAspin complexes on RPA-coAted ssDNA And in doing so promotes ClAspin-mediAted phosphorylAtion of Chk1 by ATR. Our results therefore indicAte thAt RPA-covered ssDNA not only supports recruitment And ActivAtion of ATR but Also, through Tipin And ClAspin, it plAys An importAnt role in the Action of ATR on its criticAl downstreAm tArget Chk1.