BrownLect7_Revised_2009 - CFIIB PP2A polδ RF-C...

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Unformatted text preview: CFIIB PP2A polδ RF-C Fractionation http://porpax.bio.miami.edu/~cmallery/255/255hist/ecbxp4x3_chrom.jpg tially igen, early uring mase uired and revealed the presence of four polypeptides of 70, 53, 32, and plementation assays with CF 11, T antigen, andtopoisomerase 14 kDa (Fig. 2). To determine whether these polypeptides I (see Wold etal., 1988). Replication protein-A (RP-A), a were associated, samples of the purified material were analyzed by sedimentation in glycerol gradients under sevmulti-subunit protein with single-stranded DNA binding aceral different conditions. Fig. 3 shows the results of such a sedimentation analysis carried out at relatively low ionic tivity, was purified from CF IA. (A preliminary account of the strength (50 mM KCI). It is evident that under these conditions the four polypeptides cosedimented precisely. As t A t purification and properties of RP-A has been described preCa expected, the fraction containing the four polypeptides also contained replication activity as determined by the compleviously (Wold and Kelly, 1988).) A second replication protein, * mentation assay. Under these low-salt conditions this comPCNA, was purified to homogeneity from CF IB. However, plex has a sedimentation coefficient of 6.7 S. The four FIG. 3. Glycerol gradie [salt] SV40 DNA replication when polypeptides also cosedimented in glycerol gradients conwas sedimented on a 4.8-ml we [protein] reconstitute were unable to buffer H containing 50 mM taining 0.5 M KCI or 0.5 M KCI with 1.7 M urea. We conclude CFIA fractions CF for 20 hr at 40C in a Bec from these results that the four polypeptides are tightly purified RP-A and PCNA were substituted for fractionated 2525 associated in a single protein, which Proc.have designated USA 85 (1988) and an aliquot o we Natl. Acad. Sci. Biochemistry: Wold and Kelly column by NaDodSO4/polyacrylamide gel electrophoresis Fractionation and reconstitution: laboc exhe in rigin hese matic sepallular which n (Li ctors ation nding f the . We aracDNA on is lular plica- reat is wo of y. CF move Marc topoisomeraseS.IWold, David H. Weinberg, David M. Virshup, Joachim J. Li, and Thomas J. Kelly CF IB (7 pg), (100 ng). CF IA assays also contained and themaximal signal was 29 pmol of deoxynucleotide incorporated. 11 CF IC assays proteins involved in DNA replication of IAidentify cellular replication proteins, we have made use 6- The To (20 pg) and PCNA (50 ng). Study of the also contained CF a cell-free system capable of replicating plasmid DNA a model system such as SV40 isa first step in understanding eukaryotic chromosomal pmol Using a SV40 and of deoxynucleotide incorporated. maximal signal was 31 replication. plasmid containing thelines of origin of replication (LiDNAKelly, 1984,98- CF ID cell-free system that is capable of replicating 1985). Many evidence indicate that replication molecules containing the SV40 ng), and replication assays DNAwe conducteda series of systematic fractionation- inPCNA 1984, 1985; Stillman DNAGluzman, 1985;IC a(4 pg). contained RP-A origin of replica- vivothis system closely resembles SV40 and CFin (170 ng), (Li and Kelly, tion, From the Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 fractionation of CF IB yielded homogeneous PCNA/cyclin, c 8A 0 a protein that was recently shown to be required for efficient IN 6. 205DNA chain elongation but does not appear to be involved in 0 a. 4the initiation of replication (refs. 15 and 20; unpublished 0 data). The replication activity in CF IA was purified by U .5 2chromatography on a column of Affi-Gel Blue. After binding a CF IA, the column was washed with a 11 6- containing 1 M buffer 20 0 5 10 15 KCI and bound proteins were eluted stepwise with the same 98- of potassium Fraction buffer containing increasing concentrations thiocyanate. After dialysis to remove the thiocyanate, the fractions were assayed for replication 66-a in reaction I activity a 4- RP-A I RP-A mixtures containing the complementing fractions CF IB and Stokes radius of -54 A ( CF II, as well as T antigen, topoisomerase I, and a plasmid molecular mass of =160 k I template (pUC.HSO) containing the wild-type SV40-RP-A I' origin of .precise RP-A I' molecular size a DNA replication. Replication activity was eluted at 1.3 M must await more detailed KSCN in a fraction that contained <1%45- total protein. of the We have consistently The overall extent of purification on the Affi-Gel Blue column tide is present in significa was at least 100-fold and the overall purification from crude polypeptides. Moreover, extract was -1600-fold. 4- RP-A II with Staphylococcus aur Analysis of the active fractions from the Affi-Gel Blue II _-RP-A the 53-kDa polypeptide i column by NaDodSO4/polyacrylamide gel electrophoresis 29revealed the presence of four polypeptides of 70, 53, 32, and 70-kDa polypeptide. All 14 kDa (Fig. 2). To determine whether these polypeptides released from the 53-kDa FIG. 2. NaDodSO4/polyacrylwere associated, samples of the purified material were were identical to fragmen amide gel analysis of purified RP-A. analyzed by sedimentation in glycerol gradients under sevnot shown). Thus, the 5 Positions of RP-A subunits are indieral different conditions. Fig. 3 shows the results of such a proteolytic breakdown p cated by arrows. Standards are (in 4 sedimentation analysis carried out at relatively low ionic The --- RP-A IiI 32-kDa polypeptide order of decreasing size, kDa) myosin strength (50 mM KCI). It is evident that under these condiheavy chain, 8-galactosidase, phos70-kDa or 53-kDa polyp tions the four polypeptides cosedimented precisely. As 0 20 40 60 80 100 120 140 160 RP-A fragments were observe BO expected, the fraction containing the four polypeptides also III phorylase b, bovine serum albumin, carbonicAanhydrase. Ch ovalbumin, and Ca Fraction contained replication activity as determined by the comple- Sedimentation statement about the pos * mentation assay. Under these low-salt conditions this complex has a sedimentation coefficient of 6.7 S. The four FIG. 3. Glycerol gradient analysis of RP-A. Pure RP-A (8 ,ug) FIG. 2. Profile of DEAE-Sephacel column. CF I’ was fraction- cosedimented in glycerol gradients conpolypeptides also was sedimented on a 4.8-ml 15-35% (vol/vol) glycerol gradient in ated on DEAE-Sephacel as described under . “Materialsand Methods” 0.5 M KCI with 1.7 M urea. We conclude buffer H containing 50 mM KCl. After centrifugation at 48,000 rpm taining 0.5 M KCI or BIOLOGICAL from these results that the four polypeptides Proc. Natl. Acad. Sci. USA for 20 hr at 40C in a Beckman SW50.1 rotor, the gradient was are tightly section. The protein concentration ( x ) and the conductance in aof protein, which we have Vol. 85, pp. 2523-2527, April fractionated and an aliquot of every other fraction was analyzed by 1988 associated single designated Biochemistry NaDodSO4/8-14% polyacrylamide gel electrophoresis. The sedithe fractions is shown. The activities of RP-A Virus40 DNAPCNA (CF preliminary data indicated that RP-A also Identification of Cellular Proteins Required for Simian (CF IA), RP-A. Futhermore mentation position of protein standards (catalase, Ca; aldolase, A; and had a Replication* bovine serum albumin, of replication chymotrypsinogen, Ch) IB), and CF IC are indicated (0, and 0,respectively). behaved as a complex on a Superose-12 column Purification and characterization B; ovalbumin, 0; protein A, a cellular All complefrom a in vitro replication of on the virus 40 DNA protein required for parallel gradient are shown simianbottom along with the (Received for publication, 1988) mentation assays contained T antigen (0.8 pg), CFAugust 17,(20 pg), and direction of sedimentation (long arrow). Positions unwinding) (eukaryotic DNA replication/initiation of replication/single-stranded-DNA-binding proteins/origin-specific of RP-A subunits 2051. RP-A. Futhermore preliminary data indicated that RP-A also 10 reaction (6). complex for the origin-dependent unwinding behaved as a Further on a Superose-12 column and had a 1CFIB oaded from www.jbc.org at University of Toronto on February 24, 2009 mentation position of protei bovine serum albumin, B; from a parallel gradient are direction of sedimentation ( are indicated at right. The RP-A I and RP-A I' in every Aliquots of each fraction (1 KCI) were also assayed for containing 0.8 ,ug of T antige of topoisomerase I, and 50 ng wild-type origin of SV40 D total incorporation (pmol) o NaDodSO4/8-14% polyacr p ” t t tt t THE JOURNAL OF CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 264, No. 5, Issue of‘February 15, pp. 2801-2809,1989 Printed in U.S.A. m, MARC S. WOLD of are indicated at right. The bands visible between the positions of RP-A I and RP-A I' in every lane of the gel are an artifact of staining. Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205 Aliquots of each fraction (10 ,ul dialyzed into buffer H with 15 mM Communicated by M. Daniel KCI)December also assayed for replication 1987) Lane, were 23, 1987 (received for review November 25, activity in reaction mixtures containing 0.8 ,ug40of T antigen, 5ug ofDNA replication,tg of originally described by CF IB, 20 system CF II, 100 ng The replication of simian virus (SV40) ABSTRACT of cellular replication proteins. To in vitro SV40 (12, (pUC.HSO) containing the Li and Kelly DNA is largely dependent upon topoisomerase I, and 50 ng of a plasmid13). Analysis of the system indicates that define these proteins we have made use of a cell-free systemSV40 DNAare a minimum of six cellular proteins in addition to T wild-type origin of that there replication; for the are of SV40 DNA. In results shown as replication antigen that are required is capable of replicating plasmid DNA molecules containing the total incorporation (pmol) of deoxynucleotides.purification and characterization this report we describe the SV40 origin of replication. Systematic fractionation-reconstiAND THOMAS KELLY reconstitution experiments for the purpose of identi- Li Wobbe et al., 1985; et al., 1986; Decker et al., 1986). 66D RP-A I tution experiments indicate that there are a minimum of six cellular proteins that are required for efficient viral DNA of a previously undescribed cellular replication protein, RP-A. RP-A is a multisubunit protein that binds to single- replication in vitro with 32P-dCTP separate products on alkaline gel replication in vitro with 32P-dCTP separate products on alkaline gel Complete reconstitution of SV40 DNA replication: Waga and Stillman. Science 369 (1994) 207-212 Eukaryotes: Processing Okazaki fragments: Figure1 Models for Okazaki fragment maturation. (A) RNase H/FEN1 pathway. (B) FEN1-only pathway. (C) Dna2p/RPA/FEN1 pathway. Annual Review of Biochemistry Vol. 73: 589-615 (Volume publication date July 2004) DNA ligase: • during replication of the human genome, approximately 2 × 107 Okazaki fragments are generated by discontinuous lagging strand DNA synthesis PPi Adapted from: http://bitesizebio.com/2007/10/31/the-basics-how-does-dna-ligation-work/ DNA ligase: • during replication of the human genome, approximately 2 × 107 Okazaki fragments are generated by discontinuous lagging strand DNA synthesis PPi Adapted from: http://bitesizebio.com/2007/10/31/the-basics-how-does-dna-ligation-work/ Nature 432, 473-478 (25 November 2004) DNA Replication, part IV Initiation of DNA replication • replication origins • cloning yeast origins • the critical elements of yeast origins • the replicator • other eukaryotes complex with heterochromatin and H high-resolution electron microscopy of the different initiator 1997;91:311–323. proteins, ide ally complexed with e ach other or with the D N A. 21. Ritzi M, Baack M, Musahl C, Romano Independently, more emphasis should be placed in D N AHuman minichromosome maintenanc recognition complex 2 protein on ch binding and unwinding assays with initiator proteins, until the 24543–24549. initiation re action can be reconstituted in vitro with purified ´ 22. Mendez J, Stillman B. Chromatin asso components. This reconstitution presents a phenomenal exThe replicon model (Brenner & Jacob, 1963): tion complex, cdc6, and minichromoso perimental challenge, but if achieved, it the cell cycle: assembly Every autonomous unit of e ach one of the many proteinswould help define the determinant(s) of pre-replic of replication carries some genetic Mol Cell Biol 2000;20:8602–8612. role assembled at D N A 23. Austin RJ, that control(s) its own replication: a gene controlling the synthesis Orr-Weaver DNABell SP. Dro of aof TL, replication replication origins. ACE3, an origin 1999;13:2639–2649. diffusable protein (the initiator) and a DNA element that would be 24. Bielinsky AK, Blitzblau H, Beall EL, Ez recognized by it (theAcknowledgments replicator). Gerbi SA. Origin recognition complex b We are grateful to all members of B.S. laboratory for stimulating discussions, Y.J. Sheu for critically re ading the manuscript, H. Araki and H. Takisawa for sharing results ahe ad of publication, and J. Duffy for the artwork in F igures 1 – 3. References 1. Watson JD, Crick FH. A structure for deoxyribose nucleic acid. Nature 1953;171:737–738. 2. Wilkins MH, Stokes AR, Wilson HR. Molecular structure of deoxypentose nucleic acids. Nature 1953;171:738–740. 3. Franklyn RE, Gosling RG. Molecular configuration in sodium thymonucleate. Nature 1953;171:740–741. 4. Watson JD, Crick FH. Genetic implications of the structure of deoxyribonucleic acid. Nature 1953;171:964–967. 5. Kornberg A, Lehman IR, Bessman MJ, Simms ES. Enzymic synthesis of deoxyribonucleic acid. Biochim Biophys Acta 1956;21:197. 6. Friedberg EC, Feaver WJ, Gerlach VL. The many faces of DNA polymerases: strategies for mutagenesis and for mutational avoidance. Proc Natl Acad Sci USA 2000;97:5681–5683. ¨ 7. Hubscher U, Maga G, Spadari S. Eukaryotic DNA polymerases. Annu Rev Biochem 2002;71:133–163. 8. Waga S, Stillman B. The DNA replication fork in eukaryotic cells. Annu Rev Biochem 1998;67:721–751. 9. Jacob F, Brenner S, Cuzin F. On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp Quant Biol 1963;28:329–348. 10. Neuwald AF, Aravind L, Spouge JL, Koonin EV. AAAþ: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res 1999;9:27–43. 11. Diffley JF, Cocker JH, Dowell SJ, Rowley A. Two steps in the assembly 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. origin. Curr Biol 2001;11:1427–1431. Ladenburger EM, Keller C, Knippers R for human origin recognition complex p an origin of DNA replication. Mol Cell B Keller C, Ladenburger EM, Knippers R marks a replication origin in the hum Chem 2002;277:31430–31440. Hartwell LH. Sequential function of synthesis in the yeast cell cycle. J Mo Kelly TJ, Martin GS, Forsburg SL, S The fission yeast cdc18þ gene produc mitosis. Cell 1993;74:371–382. Liang C, Weinreich M, Stillman B. determine the frequency of initiation o Cell 1995;81:667–676. Bell SP, Mitchell J, Leber J, Kobayash structure of Orc1p reveals similarity t and transcriptional silencing. Cell 199 Piatti S, Lengauer C, Nasmyth K. Cdc6 novo synthesis in G1 is important fo preventing a ‘reductional’ anaphase omyces cerevisiae. EMBO J 1995;14:3 Cocker JH, Piatti S, Santocanale C, Na role for the Cdc6 protein in forming t budding yeast. Nature 1996;379:180– Tanaka T, Knapp D, Nasmyth K. Load replication origins is regulated by Cdc6 660. Donovan S, Harwood J, Drury LS, Diffle 45 of Mcm proteins onto pre-replicative c Natl Acad Sci USA 1997;94:5611–561 Liang C, Stillman B. Persistent init Replication origins Initiation of DNA replication occurs at a specific site in the E. coli chromosome, oriC E. coli genome = ~5 x 106 bp; 1 circular chromosome yeast genome = 12.5 x 106 bp; 16 linear chromosomes human genome = 3.3 x 109 bp; 23 linear chromosomes • clearly, replication in organisms with multiple chromosomes could not proceed from a single origin. Nor could replication of a genome that is 1000x larger occur efficiently from a single origin. Multiple origins in eukaryotic chromosomes: Assays for replicator sequences: Nature Reviews Molecular Cell Biology 5, 848-855 (October 2004) Dissecting the functional elements of ARS1: ARS1 • ARS1 was inserted into a vector for yeast transformation • CEN is a yeast centromere • URA3 is a yeast selectable marker CEN This plasmid transforms ura- yeast to URA+ at high efficiency, and replicated autonomously URA3 Marahrens and Stillman, Science 255 (1992) 817-823. Dissecting the functional elements of ARS1: High frequency transformation assay: ARS1 • ARS1 was inserted into a vector for yeast transformation • CEN is a yeast centromere • URA3 is a yeast selectable marker CEN This plasmid transforms ura- yeast to URA+ at high efficiency, and replicated autonomously URA3 Marahrens and Stillman, Science 255 (1992) 817-823. Dissecting the functional elements of ARS1: High frequency transformation assay: Plasmid stability assay: ARS1 • ARS1 was inserted into a vector for yeast transformation • CEN is a yeast centromere plasmid • transform each • URA3 is ainto yeast yeast selectable marker • isolate a transformant This plasmid transforms ura- yeast • grow for 14 generations to URA+ at high efficiency, and in non-selective medium replicated• measure the fraction of autonomously cells that retain the plasmid CEN URA3 Marahrens and Stillman, Science 255 (1992) 817-823. Dissecting the functional elements of ARS1: Marahrens and Stillman, Science 255 (1992) 817-823. Autonomous Replication Sequences: Autonomous Replication Sequences: • chromosomal sequences • confer high frequency of transformation on plasmid • some are chromosomal origins • all share an 11-bp element (ACS) • mutational analysis identified additional elements B3 B2 B1 A ABF1 site A unwinding element? ACS, essential ORC binding Eukaryotic replication origins: Budding yeast Fission yeast Human Frog Short, well-defined replicator (ARS); 11 bp consensus recognized by initiator protein Large replicator (500-1000 bp); AT-rich but no consensus Large replicator (4 kb) or initiation zones (50 kb); no consensus Any DNA will replicate during early embryonic cycles; no specific replication origins Identification and Purification of ORC: • assay = DNase I footprinting • nuclear extract (only ABF1 evident) cation exchange anion exchange dsDNA ARS affinity glycerol gradient Bell & Stillman, Nature 357: 128–134 (1992) Identification and Purification of ORC: • assay = DNase I footprinting • nuclear extract (only ABF1 evident) cation exchange anion exchange dsDNA ARS affinity glycerol gradient Bell & Stillman, Nature 357: 128–134 (1992) The origin recognition complex: Budding yeast Fission yeast Human Frog Contacts box A and B1 via Orc1, 2, 4, and 5 Binds asymmetric AT tracts via unique domain on Orc4 Binds DNA without sequence specificity Binds DNA without sequence specificity How are initiation sites selected on human chromosomes? ...
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This note was uploaded on 01/23/2012 for the course BCHM 311 taught by Professor Kelley during the Spring '09 term at University of Toronto- Toronto.

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