BrownLect6_Revised_2009 - 2. The single-stranded template...

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Unformatted text preview: 2. The single-stranded template is unstable Solution: SSBs What happens to single-stranded DNA? The EMBO Journal (2001) 20, 612–618 The EMBO Journal (2002) 21, 1855–1863 2. The single-stranded template is unstable Solution: SSBs What happens to single-stranded DNA? The EMBO Journal (2001) 20, 612–618 Journal of Molecular Biology Volume 374, Issue 4, 7 December 2007, Pages 865-876 The EMBO Journal (2002) 21, 1855–1863 3. Can’t start DNA chains Solution: DNA primase http://herkules.oulu.fi/isbn9514259688/html/x241.html http://www.genomeknowledge.org/cgi-bin/eventbrowser_st_id?ST_ID=REACT_2244.2 4. Too slow and too distributive Solution: the sliding clamp Annual Review of Biochemistry Vol. 74: 283-315 (Volume publication date July 2005) Loading the clamp onto DNA Annual Review of Biochemistry Vol. 74: 283-315 (Volume publication date July 2005) J. Biol. Chem., Vol. 281, Issue 25, 17528-17539 • products are longer when PCNA is present • polymerase activity is stimulated 184 pmol 51 pmol • products are longer when PCNA is present • polymerase activity is stimulated lane 1: 184 pmol/51 units = 3.6 pmol/unit lane 3: 51 pmol/0.12 units = 425 pmol/unit 184 pmol 51 pmol Specialized DNA polymerases at the eukaryotic replication fork Adapted from: R. A. Bambara, R. S. Murante, and L. A. Henricksen (1997) Enzymes and reactions at the eukaryotic DNA replication fork. J. Biol. Chem. 272: 4647-4650. 5. No 3’ to 5’ polymerase Solution: the replisome DNA helicase DNA polymerase DNA primase clamp loader single-stranded DNA binding protein clamp (processivity factor) E. coli DnaB Pol III DnaG SSB gamma complex beta clamp Pol III Eukaryotes: Short Review 159 Table 1. Key Components of the Replisome Function Fork unwinding Primase DNA polymerase Sliding clamp Clamp loader SSB Uncertain a b E. coli Complex DnaB DnaG Pol III core b g complex SSB Subunit Organization Homohexamer Monomer Heterotrimer Homodimer gt2dd0 ccb Homotetramer Eukaryotic Complex Mcm2-7 Pol a/Primase Pol d and Pol 3 PCNA RFC RPA GINS, Cdc45, Dpb11, Mcm10, Sld2, Sld3, others Subunit Organization Heterohexamer Heterotetramer Heterotetramersa Homotrimer Heteropentamer Heterotrimer Pol d is a heterotrimer in S. cerevisiae. In E. coli, the clamp loader consists of five subunits (gt2dd0 ) plus c and c, which have auxiliary roles. and their ligation to the upstream fragment (reviewed in Liu et al. [2004]). Pol d initiates the process by balancing its polymerase and 30 –50 proofreading exonuclease functions to regulate displacement of the 50 end of the downstream Okazaki fragment, thereby converting the downstream RNA primer into an incremental 50 flap substrate for the Dna2 and Fen1 nucleases (Garg et al., 2004). The crystal structure of human DNA ligase I reveals that, like the PCNA clamp to which it binds, ligase encircles the duplex, but unlike PCNA, it makes extensive contact with the DNA to carry out the ligation reaction (Pascal et al., 2004). Fen1 also binds PCNA, suggesting that the clamp may mediate yet another molecular switch similar to the primase-polymerase switch described above. Corn, J.E., Pease, P.J., Hura, G.L., and Berger, J.M. (2005). Crosstalk between primase subunits can act to regulate primer synthesis in trans. Mol. Cell 20, 391–401. Fukui, T., Yamauchi, K., Muroya, T., Akiyama, M., Maki, H., Sugino, A., and Waga, S. (2004). Distinct roles of DNA polymerases delta and epsilon at the replication fork in Xenopus egg extracts. Genes Cells 9, 179–191. Molecular Cell 23, 155–160, July 21, 2006 ª2006 E Garg, P., Stith, C.M., Sabouri, N., Johansson, E., and Burgers, P.M. (2004). Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication. Genes Dev. 18, 2764–2773. Hacker, K.J., and Alberts, B.M. (1994). The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for polymerase release following the termination of each Okazaki fragment. J. Biol. Chem. 269, 24221–24228. DNA Replication: K and Dont Mind the Heller, R.C., and Marians, K.J. (2006). Replication fork reactivation downstream of a blocked nascent leading strand. Nature 439, 557–562. Lance D. Langston1 and Mike O’Donnel Higuchi, K., Katayama, T., Iwai, S., 1 Howard Hughes Medical Institute Hidaka, M., Horiuchi, T., and DNA Replication, part III • How SV40 replication in vitro identified eukaryotic replication proteins • Processing and sealing Okazaki fragments 30 http://www.pdb.org/pdb/explore.do?structureId=1SVA 31 helicase domains http://www.pdb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb47_2.html ble extracts of SV40-infected monkey cells to support DNA synthesis dependent on the viral origin of replication. The extracts were prepared from COS-1 or BSC-40 cells 39 hr after infection with SV40 cslO85, a viral mutant that overproduces wild-type T antigen (19). Two DNA templates were employed. Plasmid pJLO consists of nucleotides 5171128 of the SV40 genome cloned in the vector pKP45, a derivative of pBR322. The viral DNA segment in this construct includes the complete wild-type SV40 origin of DNA replication (14). Plasmid pJLO-d4 is identical to pJLO except for the deletion of 4 base pairs at map positions 5239-5242 within the origin. This deletion is known to cause a severe defect COS-1 SV40 DNA replication in vitro this corresponds to the synthesis of 2000-4000 pJLO molecules per cell equivalent of extract. Similar results were obtained with an extract from COS-1 cells infected with wildtype SV40 except that the extent of incorporation was lower (data not shown). T-Antigen Dependence of SV40 DNA Replication in Vitro. The only SV40 gene product known to be directly required for viral DNA replication is the viral initiation protein, T antigen. Therefore, we tested the ability of extracts from uninfected monkey cells to support DNA replication in the presence of purified T antigen. The extracts were prepared from subconfluent COS-1 or BSC-40 cells; T antigen was puBSC-40 I - I 1 - DNA 2 pJ LO 4 1 2 pJ LOd4 4 -1 4 DNA 2 pJ LO pJ LOd4 I 1 2 1 41 4 1 2 4 .a". .13 . I~~~~~~~~~~~~~~1 ..... M SV4() * I I I ' qg.lj-`7f :f:f" Plasmid [ i. .0 .. FIG. 1. In vitro replication of plasmid DNA containing the SV40 origin of replication. DNA was synthesized in standard reaction mixtures containing extract from COS-1 or BSC-40 cells infected with SV40 cs1085. As indicated above each lane, the reaction mixtures incubated for 1, 2, or 4 hr at 37° C in the absence of added DNA or in the presence of pJLO DNA (wild-type SV40 origin) pJLO-d4 DNA (mutant SV40 origin). The radioactive reaction products were electrophoresed in a 1.5% agarose gel, and the gels then autoradiographed. The marks the position of the endogenous SV40 form I DNA. The bracket indicates the region of the gel occupied by the topological isomers of the plasmid templates pJLO and pJLO-d4. were or were arrow ble extracts of SV40-infected monkey cells to support DNA synthesis dependent on the viral origin of replication. The extracts were prepared from COS-1 or BSC-40 cells 39 hr after infection with SV40 cslO85, a viral mutant that overproduces wild-type T antigen (19). Two DNA templates were employed. Plasmid pJLO consists of nucleotides 5171128 of the SV40 genome cloned in the vector pKP45, a derivative of pBR322. The viral DNA segment in this construct includes the complete wild-type SV40 origin of DNA replication (14). Plasmid pJLO-d4 is identical to pJLO except for the deletion of 4 base pairs at map positions 5239-5242 within the origin. This deletion is known to cause a severe defect COS-1 SV40 DNA replication in vitro this corresponds to the synthesis of 2000-4000 pJLO molecules per cell equivalent of extract. Similar results were obtained with an extract from COS-1 cells infected with wildtype SV40 except that the extent of incorporation was lower (data not shown). T-Antigen Dependence of SV40 DNA Replication in Vitro. The only SV40 gene product known to be directly required for viral DNA replication is the viral initiation protein, T antigen. Therefore, we tested the ability of extracts from uninfected monkey cells to support DNA replication in the presence of purified T antigen. The extracts were prepared from subconfluent COS-1 or BSC-40 cells; T antigen was puBSC-40 I - I 1 - DNA 2 pJ LO 4 1 2 pJ LOd4 4 -1 4 DNA 2 pJ LO pJ LOd4 I 1 2 1 41 4 1 2 4 .a". .13 . I~~~~~~~~~~~~~~1 ..... M 2.3 kb SV4() * I I I ' qg.lj-`7f :f:f" Plasmid [ i. .0 .. FIG. 1. In vitro replication of plasmid DNA containing the SV40 origin of replication. DNA was synthesized in standard reaction mixtures containing extract from COS-1 or BSC-40 cells infected with SV40 cs1085. As indicated above each lane, the reaction mixtures incubated for 1, 2, or 4 hr at 37° C in the absence of added DNA or in the presence of pJLO DNA (wild-type SV40 origin) pJLO-d4 DNA (mutant SV40 origin). The radioactive reaction products were electrophoresed in a 1.5% agarose gel, and the gels then autoradiographed. The marks the position of the endogenous SV40 form I DNA. The bracket indicates the region of the gel occupied by the topological isomers of the plasmid templates pJLO and pJLO-d4. were or were arrow ble extracts of SV40-infected monkey cells to support DNA synthesis dependent on the viral origin of replication. The extracts were prepared from COS-1 or BSC-40 cells 39 hr after infection with SV40 cslO85, a viral mutant that overproduces wild-type T antigen (19). Two DNA templates were employed. Plasmid pJLO consists of nucleotides 5171128 of the SV40 genome cloned in the vector pKP45, a derivative of pBR322. The viral DNA segment in this construct includes the complete wild-type SV40 origin of DNA replication (14). Plasmid pJLO-d4 is identical to pJLO except for the deletion of 4 base pairs at map positions 5239-5242 within the origin. This deletion is known to cause a severe defect COS-1 SV40 DNA replication in vitro this corresponds to the synthesis of 2000-4000 pJLO molecules per cell equivalent of extract. Similar results were obtained with an extract from COS-1 cells infected with wildtype SV40 except that the extent of incorporation was lower (data not shown). T-Antigen Dependence of SV40 DNA Replication in Vitro. The only SV40 gene product known to be directly required for viral DNA replication is the viral initiation protein, T antigen. Therefore, we tested the ability of extracts from uninfected monkey cells to support DNA replication in the presence of purified T antigen. The extracts were prepared from subconfluent COS-1 or BSC-40 cells; T antigen was puBSC-40 I - I 1 - DNA 2 pJ LO 4 1 2 pJ LOd4 4 -1 4 DNA 2 pJ LO pJ LOd4 I 1 2 1 41 4 1 2 4 .a". .13 . I~~~~~~~~~~~~~~1 ..... M 2.3 kb SV4() * I I I ' qg.lj-`7f :f:f" Plasmid [ i. .0 .. FIG. 1. In vitro replication of plasmid DNA containing the SV40 origin of replication. DNA was synthesized in standard reaction mixtures containing extract from COS-1 or BSC-40 cells infected with SV40 cs1085. As indicated above each lane, the reaction mixtures incubated for 1, 2, or 4 hr at 37° C in the absence of added DNA or in the presence of pJLO DNA (wild-type SV40 origin) pJLO-d4 DNA (mutant SV40 origin). The radioactive reaction products were electrophoresed in a 1.5% agarose gel, and the gels then autoradiographed. The marks the position of the endogenous SV40 form I DNA. The bracket indicates the region of the gel occupied by the topological isomers of the plasmid templates pJLO and pJLO-d4. were or were arrow replicated DNA unreplicated marker DNA CFIIB PP2A polδ RF-C ...
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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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