Biologia Genômica. 2º Semestre, Replicação de DNA em Bactérias e no Núcleo Eucariótico. Prof. Marcos Túlio

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1 Biologia Genômica 2º Semestre, 2017 Replicação de DNA em Bactérias e no Núcleo Eucariótico Prof. Marcos Túlio mtoliveria@fcav.unesp.br Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Instituto de Biociências, Letras e Ciências Exatas de S.J.R.P. Universidade Estadual Paulista Júlio de Mesquita Filho

2 DNA Molecules

3 DNA Molecules

4 DNA Molecules

5 11.1 Introduction replicon A unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication. origin A sequence of DNA at which replication is initiated. terminus A segment of DNA at which replication ends. Lewin s Genes X, 2009.

6 Lewin s Genes X, 2009.

7 FIGURE 02: Replicated DNA is seen as a replication bubble flanked by nonreplicated DNA Origin Lewin s Genes X, 2009.

8 FIGURE 11.5 Lewin s Genes X, Robberson & Clayton, PNAS 69:3810-4

9 Lewin s Genes X, 2009.

10 Lewin s Genes X, 2009.

12 11.3 Origins Can Be Mapped by Autoradiography and Electrophoresis Replication forks create Y-shaped structures that change the electrophoretic migration of DNA molecules. FIGURE 07: The position of the origin and the number of replicating forks determine the shape of a replicating restriction fragment Lewin s Genes X, 2009.

13 Principles of Two Dimensional-Neutral Agarose Gel Electrophoresis (2D-NAGE) Priit Joers

14 Principles of 2D-NAGE Priit Joers

15 Priit Joers Principles of 2D-NAGE go for a Southern blot...

16 Principles of 2D-NAGE Priit Joers

17 Priit Joers Origin within fragment -bubble arc

18 Nicking of DNA broken bubbles Priit Joers

19 Passing replication fork Y arc Priit Joers

20 ssdna regions sub-y arc Priit Joers

21 Colliding forks double Y and X Priit Joers

22 Replication Intermediates

23 Replication Intermediates Human 143B Holt et al., Cell 100:515-24

24 Replication Intermediates Mouse Holt et al., Cell 100:515-24

25 Bacteria Lewin s Genes X, 2009.

26 11.4 The Bacterial Genome Is (Usually) a Single Circular Replicon The two replication forks usually meet halfway around the circle, but there are ter sites that cause termination if the replication forks go too far. FIGURE 09: Forks usually meet before terminating

27 Replication Fork Trap

28 Replication Fork Trap

29 Replication Fork Trap ter sites

30 Replication Fork Trap ter sites Tus protein

31 Replication Fork Trap Kamada et al., Nature 383:

32 Replication Fork Trap

33 Replication Fork Trap

34 Initial steps at oric by American Society for Biochemistry and Molecular Biology Carr K M, Kaguni J M J. Biol. Chem. 2001;276:

35 Lewin s Genes X, origin melting

36 HU origin melting

37 14.2 Initiation: Creating the Replication Forks at the Origin oric SSB

38 14.2 Initiation: Creating the Replication Forks at the Origin oric gyrase

40 Initial steps at oric by American Society for Biochemistry and Molecular Biology Carr K M, Kaguni J M J. Biol. Chem. 2001;276:

41 11.5 Methylation of the Bacterial Origin Regulates Initiation oric contains binding sites for DnaA dnaa-boxes. oric also contains eleven GATC/CTAG repeats that are methylated on adenine on both strands.

11.5 Methylation of the Bacterial Origin Regulates Initiation Replication generates hemimethylated DNA, which cannot initiate replication.

42 11.5 Methylation of the Bacterial Origin Regulates Initiation Replication generates hemimethylated DNA, which cannot initiate replication. There is a 13-minute delay before the GATC/CTAG repeats are remethylated. FIGURE 11: Only fully methylated origins can initiate replication

44 SeqA protein

45 SeqA protein Kaguni, ARM 60:

46 DnaA (ATP)

47 dnaa dnaa

48 Initial steps at oric by American Society for Biochemistry and Molecular Biology Carr K M, Kaguni J M J. Biol. Chem. 2001;276:

49 Hda Regulatory Inactivation of DnaA (RIDA)

50 Hansen et al., JMB 367:

51 Regulation of Initiation of DNA Replication in Bacteria (E. coli) All About DnaA Hemimethylation of oric Sequestration of oric by SeqA. Hemimethylation of dnaa gene promoter Hydrolysis of ATP by DnaA + Hda Titration of DnaA by data locus

52 Helicase + Helicase Loader

53 DnaB Structure Bailey et al., Science 318:

54 The Prepriming Complex of E. coli Mott et al., Cell 135:

55 Transition from Initiation to Elongation Makowska-Grzyska & Kaguni, Mol Cell 37:

56 Transition from Initiation to Elongation DnaB + DnaG (model) DnaG primase Bailey et al., Science 318: Corn et al., NSMB 15:163-9.

57 Transition from Initiation to Elongation

58 E. coli pol III holoenzyme Subunits Catalytic core: α (pol activity), ε (exo activity), θ (?) Processivity factor: β 2 (sliding clamp) Clamp Loader (DnaX/γ complex): γ, τ 2, δ, δ, χ, ψ. Lewin s Genes X, 2009.

59 E. coli pol III core Subunits α 5-3 polymerase activity ε 3-5 exonuclease activity θ stimulate ε

60 14.5 DNA Polymerases Control the Fidelity of Replication DNA polymerases often have a 3 5 exonuclease activity that is used to excise incorrectly paired bases. The fidelity of replication is improved by proofreading by a factor of ~100. Lewin s Genes X, 2009.

61 The Processivity Factor (Sliding Clamp)

62 The Clamp Loader Jeruzalmi et al, Cell 106: Kelch et al, Science 334:

63 The Clamp Loader Jeruzalmi et al, Cell 106:

64 The Clamp Loader Jeruzalmi et al, Cell 106:

65 E. coli pol III holoenzyme

66 Loading the Polymerase

67 Loading the Polymerase

68 Putting the pieces together: The E. coli Replisome Leading strand Lagging strand McHenry, COCB 15:

69 Putting the pieces together: The E. coli Replisome Leading strand Lagging strand McHenry, COCB 15: τ links Pol III HE to DnaB/DnaG

70 Putting the pieces together: The E. coli Replisome SSB + ssdna DnaG binds SSB (ssdna) χψ link Pol III HE to SSB McHenry, COCB 15:

14.12 The Clamp Controls Association of Core Enzyme with DNA The helicase DnaB is responsible for interacting with the primase DnaG to initiate each Okazaki

71 14.12 The Clamp Controls Association of Core Enzyme with DNA The helicase DnaB is responsible for interacting with the primase DnaG to initiate each Okazaki fragment. FIGURE 21: Each catalytic core of Pol III synthesizes a daughter strand. DnaB is responsible for forward movement at the replication fork Lewin s Genes X, 2009.

72 14.12 The Clamp Controls Association of Core Enzyme with DNA

73 E. coli DNA replication

74 The E. coli Replisome Trimeric polymerase? Reyes-Lamothe et al., Science 328:

75 The E. coli Replisome Trimeric polymerase? Georgescu et al., NSMB 19:113-6.

76 Coordination of leading and lagging strand syntheses Graham et al., Cell 169:

77 Coordination of leading and lagging strand syntheses Graham et al., Cell 169:

78 14.13 Okazaki Fragments Are Linked by Ligase Each Okazaki fragment starts with a primer and stops before the next fragment. RNase H + DNA polymerase I removes the primer and replaces it with DNA. Lewin s Genes X, 2009.

79 14.13 Okazaki Fragments Are Linked by Ligase DNA ligase makes the bond that connects the 3 end of one Okazaki fragment to the 5 beginning of the next fragment. FIGURE 25: DNA ligase seals nicks between adjacent nucleotides by employing an enzyme-amp intermediate Lewin s Genes X, 2009.

80 E. coli DNA replication Summary DnaA melts oric and recruits DnaB helicase/dnac helicase loader. DnaB helicase recruits DnaG primase. Priming releases DnaC from prepriming complex. DnaB helicase keeps interacting with DnaG primase transiently throughout lagging-strand synthesis. DnaX clamp loader loads β 2 clamp on primer-template (via interactions with δ subunit). Pol III core (α subunit) interacts with β 2 clamp and primer-template.

81 E. coli DNA replication Summary Two (Three!) Pol III cores are kept together in the replisome through the τ subunits of the DnaX clamp loader. In the lagging strand, DnaX clamp loader is constantly loading β 2 clamps onto new primer-templates; it also promotes Pol III core hopping from the old Okazaki fragment to the new primer. The τ subunits of DnaX clamp loader are also important for interacting with DnaB helicase (τ is the guy!)

82 E. coli DNA replication Summary The χψ subunits of DnaX clamp loader (τ attaches them to the ring) interact with SSB transiently, which interact with DnaG primase transiently. RNase H, DNA pol I and DNA ligase are responsible for the maturation of the Okazaki fragments.

83 Eukaryotes FIGURE 13: The eukaryote cell cycle

11.7 Each Eukaryotic Chromosome Contains Many Replicons Eukaryotic replicons are 40 to 100 kb in length. Individual replicons are activated at characteristic times during S phase.

84 11.7 Each Eukaryotic Chromosome Contains Many Replicons Eukaryotic replicons are 40 to 100 kb in length. Individual replicons are activated at characteristic times during S phase. Regional activation patterns suggest that replicons near one another are activated at the same time. FIGURE 14: A eukaryotic chromosome contains multiple origins of replication that ultimately merge during replication

85 FIGURE 15: Replication forks are organized into foci in the nucleus Photos courtesy of Anthony D. Mills and Ron Laskey, Hutchinson/MRC Research Center, University of Cambridge.

86 11.8 Replication Origins Can Be Isolated in Yeast Origins in S. cerevisiae are short A-T sequences that have an essential 11 bp sequence. The ORC is a complex of six proteins that binds to an ARS. FIGURE 16: An ARS extends for ~50 bp and includes a consensus sequence (A) and additional elements (B1 B3)

89 MCM 7 MCM 2 MCM 3 MCM 7 MCM 2 MCM 3 ORC1 ORC2 ORC3 MCM 6 MCM 5 MCM 4 MCM 6 MCM 5 MCM 4 Cdt1 Cdc6 ORC6 ORC5 ORC4

90 11.9 Licensing Factor Controls Eukaryotic Rereplication Licensing factor is necessary for initiation of replication at each origin. Licensing factor is present in the nucleus prior to replication, but is removed, inactivated, or destroyed by replication.

91 11.9 Licensing Factor Controls Eukaryotic Rereplication Initiation of another replication cycle becomes possible only after licensing factor reenters the nucleus after mitosis. FIGURE 18: Licensing factor in the nucleus is inactivated after replication

92 The ORC is a protein complex that is associated with yeast origins throughout the cell cycle. Cdc6 protein is an unstable protein that is synthesized only in G1. MCM 7 MCM 2 MCM 3 MCM 7 MCM 2 MCM 3 ORC1 ORC2 ORC3 MCM 6 MCM 5 MCM 4 MCM 6 MCM 5 MCM 4 Cdt1 Cdc6 ORC6 ORC5 ORC4 Cdc6 binds to ORC and allows MCM proteins to bind. Cdt1 facilitates MCM loading on origins.

93 11.10 Licensing Factor Consists of MCM Proteins When replication is initiated, Cdc6, Cdt1, and MCM proteins are displaced. The degradation of Cdc6 prevents reinitiation. MCM 7 MCM 2 MCM 3 MCM 7 MCM 2 MCM 3 ORC1 ORC2 ORC3 MCM 6 MCM 5 MCM 4 MCM 6 MCM 5 MCM 4 Cdt1 Cdc6 ORC6 ORC5 ORC4

94 Some MCM proteins are in the nucleus throughout the cell cycle, but others may enter only after mitosis. FIGURE 19: Proteins at the origin control susceptibility to initiation

95 Regulation of Initiation of DNA Replication in Eukaryotes (yeast) ORC recognizes the origin Cdc6 is rapidly degraded Some MCM proteins are licensing factors (only enter the nucleus when the envelope is disrupted during mitosis)

96 FIGURE 27: Similar functions are required at all replication forks Lewin s Genes X, 2009.

97 Eukaryotic Nucleus (Archea)

98 Eukaryotic Nucleus (Archea) The MCM2-7 helicase MCM 7 MCM 2 MCM 3 MCM 7 MCM 2 MCM 3 ORC1 ORC2 ORC3 MCM 6 MCM 5 MCM 4 MCM 6 MCM 5 MCM 4 Cdt1 Cdc6 ORC6 ORC5 ORC4

99 Eukaryotic Nucleus (Archea) Pol α/primase RNA stretch of 11 nt + DNA stretch of variable sizes

100 Eukaryotic Nucleus (Archea) Replication Protein A (RPA) the SSB E. coli SSB

101 Eukaryotic Nucleus (Archea) Proliferating Cell Nuclear Antigen (PCNA) the Sliding Clamp

102 Eukaryotic Nucleus (Archea) Replication Factor C (RFC) the Clamp Loader

103 14.14 Separate Eukaryotic DNA Polymerases Undertake Initiation and Elongation DNA polymerase ε elongates the leading strand and a second DNA polymerase δ elongates the lagging strand.

104 Eukaryotic Nucleus (Archea) Primer Removal and Maturation of Okazaki fragments

105 12.2 The Ends of Linear DNA Are a Problem for Replication Special arrangements must be made to replicate the DNA strand with a 5 end. FIGURE 01: Replication could run off the 3 end of a newly synthesized linear strand, but could it initiate at a 5 end?

106 9.16 Telomeres Have Simple Repeating Sequences The telomere is required for the stability of the chromosome end. A telomere consists of a simple repeat where a C+A-rich strand has the sequence C >1 (A/T) 1 4. FIGURE 27: A typical telomere has a simple repeating structure with a G-Trich strand that extends beyond the C-A-rich strand

displacing its homolog in an upstream region of the telomere.

107 9.17 Telomeres Seal the Chromosome Ends and Function in Meiotic Chromosome Pairing The protein TRF2 catalyzes a reaction in which the 3 repeating unit of the G+T-rich strand forms a loop by displacing its homolog in an upstream region of the telomere. FIGURE 29a: A loop forms at the end of chromosomal DNA Photo courtesy of Jack Griffith, University of North Carolina at Chapel Hill.

108 9.18 Telomeres Are Synthesized by a Ribonucleoprotein Enzyme FIGURE 32: Telomerase positions itself by base pairing between the RNA template and the protruding single-stranded DNA primer

109 9.19 Telomeres Are Essential for Survival Telomerase is expressed in actively dividing cells and is not expressed in quiescent cells. Loss of telomeres results in senescence. Escape from senescence can occur if telomerase is reactivated, or via unequal homologous recombination to restore telomeres. FIGURE 33: Mutation in telomerase causes telomeres to shorten in each cell division

110 Systems other than E. coli DNA replication Summary Coordination of leading- and lagging-strand synthesis in the eukaryotic nucleus is obscure. Primers are synthesized by the heterotetrameric Pol α/primase. They are ~half RNA, ~half DNA. Although no sequence homology is found among nuclear, bacterial and T4 sliding clamps and clamp loaders, their general structure is very similar (donutshape, 3/6fold symmetry).

111 Systems other than E. coli DNA replication Summary The heterotrimeric nuclear RPA has no homology with the homotetrameric bacterial SSB, despite possessing similar structural folding domains for binding ssdna. Two distinct polymerases (ε and δ) are required to leading and lagging strand synthesis, respectively, in the nucleus. Okazaki fragments maturation is accomplished by a complex with PCNA, Pol δ/β, Fen1 and DNA ligase I. A specialized polymerase (telomerase) is responsible for replication of the chromosomal ends.

DNA Replication II Biochemistry 302. Bob Kelm January 28, 2004

DNA Replication II Biochemistry 302 Bob Kelm January 28, 2004 Conceptual model for proofreading based on kinetic considerations Fig. 24.44 stalling transient melting exonuclease site occupancy Following