DNA Metabolism
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Transcript of DNA Metabolism
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DNA Metabolism
• DNA replication: processes which DNA is being faithfully duplicated.
• DNA recombination: processes which the nucleotide sequence of DNA is being rearranged.
• DNA repair: processes which the structural integrity of DNA is being maintained.
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Objectives of DNA replication• Basic mechanisms: (i) semiconservative, conservative, or rand
om dispersive, (ii) continuous,semidiscontinuous or discontinuous, (iii) unidirectional, bidirectional, or rolling circle.
• Enzymology: (i) identification of genes involved in replication , (ii) biochemical function of the protein products of these genes.
• Replicon: the unit of DNA replication. A single DNA molecule may consist of one replicon (eg., in prokaryotes) or many replicons (eg., in eukaryotes).
• Replication of any replicon may be separated into three phases: initiation, elongation and termination. The methods for identification of replication origin and the enzymes involved in each phase will be discussed.
• Regulation of DNA replication – how cells ensure each DNA is replicated only once per cell cycle.
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Models of DNA replication
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Prediction of experimental outcomes
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DNA replication is semiconservative
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DNA synthesis is catalyzed by DNA polymerases in the presence of (i) primer, (ii) template, (iii) all 4 dNTP, and (iv) a divalent cation such as Mg++, (v) synthesis is from 5’ to 3’.
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Models of DNA chain elongation at replicating fork
Fig. 20.5
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DNA Synthesis Can’t be Continuously on Both Strands (because the DNA duplex is antiparallel and all DNA polymerases synthesize DNA in a 5’ to 3’ direction)
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Semidiscontinuous or discontinuous?
BioEssays 27:633-636 (2005)
Fig. 20.6
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What is the source of primer used for lagging strand synthesis?
Fig. 20.7
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RNA primers 10-12 nt long are used to synthesize Okazaki fragments
Fig. 20.8
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Modes of DNA replication
• Bubbles (eyes) and Y structures.
• Theta mode (circular DNA).
• Displacement loop (D-loop).
• Rolling circle (Lariat or Sigma form)
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Bubble or eyes.
Fig. 20.10
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Theta mode.
Fig. 20.9
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Rolling circle replication
Fig. 20.13
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Fig. 20.14
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Displacement loop
Fig. 15.16
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Fig. 15.5
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Directionality of DNA chain elongation
Fig. 20.11
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Unidirectional replication of colicin E1 DNA
Fig. 20.12
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Enzymology of DNA replication
• Identification of genes involved in replication: (i) isolation of conditional lethal mutants (eg., temperature-sensitive mutations) that affect DNA synthesis, (ii) map and clone the gene of interest.
• Biochemical function of the protein products of these genes.
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Fig. 20.20
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Fig. 20.22
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Proteins involved in the initiation of replication
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Proteins involved in the elongation of DNA
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Initiation of Replication• Start of DNA chains: (i) RNA primer, (ii) terminal
protein primer, (iii) parental strand primer.• Identification of origins: (i) physical mapping: EM,
two-D gel electrophoresis etc., (ii) genetic mapping, (iii) functional mapping by DNA cloning.
• Chromosomal origins: (i) E. coli and other bacterial origins, (ii) origins without an initiator protein (ColE1 and T7), (iii) origins cleaved by initiator endonucleases, (iv) yeast autonomously replicating sequences (ARS).
• Initiation from the E. coli oriC.
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Terminal protein may be used as primer to initiate replication
Fig. 16.2
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Fig. 16.3
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Fig. 16.4
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Parental DNA strand as primer: Nicking by specific endonuclease to produce 3’-OH
Fig. 16.5
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Fig. 16.7
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Fig. 16.11
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Fig. 16.12
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Fig. 16.8
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Two-dimensional gel electrophoresis to identify origin
Fig. 21.5
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Fig. 21.6
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Mapping of SV40 origin by EM
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Fig. 21.3
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Functional cloning of replication origin
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Bacterial replication origins
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The yeast origins of replication are contained within autonomous replicating sequences (ARSs) that are composed of 4 regions (A, B1, B2, and B3). An 11-bp (5-[T/A]TTTAPyPuTTT[T/A]-3’) consensus sequence is highly conserved in ARSs.
Fig. 21.7
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Fig. 21.1
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Elongation at a replication fork
• Replication speed.
• Enzymes involved in DNA elongation at a replication fork and their functions.
• Model of simultaneous synthesis of both DNA strands by PolIII.
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Fig. 21.8
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Fig. 21.9
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Proteins involved in the elongation of DNA
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Elongation
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DNA Polymerases
Processivity of DNA polymerase is determined at low enzyme concentration and in the presence of excessive substrates.
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PolIII* consists of two cores, a clamp-loading complex ( complex) consisting of ’, and two additional proteins and . Holoenzyme is PolIII* plus subunits.
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Fig. 21.17
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Fig. 21.16
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DNA polymerase III
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Model for the synthesis of DNA on the leading and lagging strands by the asymmetric dimer of PolIII
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Fig. 21.19
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Fig. 21.20
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Fig. 21.23
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Fig. 21.24
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Fig. 21.25
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Model for eukaryotic DNA replication
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Termination of Replication
• Circular genomes: (i) termination sequences of E. coli, (ii) production of catenanes, (iii) decatenation by topoisomerase (TopIV in E. coli).
• Linear genomes: (i) end-replication problems of linear DNA, (ii) specialized structure in eukaryotic telomeres, (iii) maintenance of telomere length by telomerase and other mechanisms.
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Fig.21.26
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Fig. 21.27
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TopIV participates in decatenation
Fig. 21.28
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The End Replication Problem of Linear DNA
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End-replication problem of linear DNA
Fig. 21.29
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Formation of t loops in vitro.
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Fig. 21.36
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Regulation of DNA replication
• Control of initiation requires: (i) timing in the cell cycle, (ii) synchrony of initiation at multiple copies of oriC, and (iii) inhibition of immediate reinitiation.
• Processes required for initiation – protein and RNA synthesis, DNA methylation.
• DnaA level and timing of initiation.• DNA mehtylation in the regulation of initiation.• Regulation of ColE1 DNA replication.
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Bacterial replication origins
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Fig. 15.8
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Fig. 15.9
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Fig. 17.18
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Fig. 17.19
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Fig. 17.20
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Fig. 17.21
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A nucleus injected into a Xenopus egg can replicate only once
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Licensing factor controls eukaryotic rereplication
Fig. 15.14
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Licensing factor consists of MCM proteins
Fig. 15.15