080818.PMCB.lecture.4

080818.PMCB.lecture.4 - Plant Molecular and Cellular...

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Plant Molecular and Cellular Biology Lecture 4: E. coli DNA Replicase Structure & Function Gary Peter
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Learning Objectives 1. List and explain the mechanisms by which E. coli DNA is replicated 2. Describe and explain the structure and functions of the enzymes and their subunits that replicate DNA in E. coli
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Processivity z The number of nucleotides added during each binding and release from the primed template z The ability of the DNA polymerase to remain associated with the DNA template z Typical processivity of enzymes in vitro z Klenow 50-60 nt z T7 300 nt z Taq 22 nt z Pfu 6.4 nt Wang et al., Nucleic Acids Res. 2004; 32(3): 1197–1207
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Strand Displacement Activity z Strand Displacement: z The ability to displace downstream DNA encountered during synthesis. z Protocols such as the isothermal ampli cation method Strand Displacement Ampli cation (SDA) exploit this activity. z When new synthesis starts at a nick it displaces a strand. The displaced strand then itself becomes a template for the synthesis of a new strand.
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Strand Displacement & Processivity of Bacteriophage Phi29 DNA Polymerase z This polymerase has excellent strand displacement activity and high processivity and is used in strand displacement amplification (SDA) z High displacement is likely due to a tunnel that is too small for dsDNA to enter and requires/induces strand separation z The high processivity is likely due to topological encirclement of both the downstream template and the upstream dsDNA z This structure abrogates the need for ancillary factors such as helicase and the clamp Kamtekar et al., 2004 Mol. Cell 16 (4): 609-618
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Functionality of Various DNA Polymerases 3'->5' Proofreading Strand Displacement Primary Applications Mesophilic DNA Polymerases phi29 DNA Polymerase ++++ +++++ Strand Displacement Applications T4 DNA Polymerase +++++ - Polishing Ends, 2nd Strand Synthesis DNA Polymerase I ++ -* Nick Translation DNA Polymerase I, Klenow Fragment ++ ++ Polishing Ends Klenow Fragment (3' -> 5' exo-) - +++ Labeling T7 DNA Polymerase (unmodified) ++++ - Site Directed Mutagenesis Terminal Transferase -N A 3' terminal Tailing *Degrades displaced strand http://www.neb.com/nebecomm/tech_referenc e/polymerases/polymerases_from_neb.asp
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3'->5' Proofreading Strand Displacement Primary Applications Mesophilic DNA Polymerases 3'->5' Proofreading Strand Displacement Primary Applications Mesophilic DNA Polymerases hermophilic DNA Polymerases Phusion™ High Fidelity DNA Polymerase +++++ - PCR (high fidelity) Phusion™ Hot Start High Fidelity DNA Polymerase +++++ - Hot Start PCR (high fidelity) DyNAzyme™ EXT DNA Polymerase + + PCR (difficult or long) DyNAzyme™ II Hot Start DNA Polymerase - - PCR (hot start) Taq DNA Polymerase - -* PCR (routine), Primer Extension Vent R DNA Polymerase +++ ++ PCR (high fidelity), Primer Extension Vent R (exo-) DNA Polymerase - +++ PCR, Sequencing Deep Vent R DNA Polymerase +++ ++ PCR (high fidelity), Primer Extension Deep Vent R (exo-) DNA Polymerase - +++ PCR (long), Primer Extension 9°N m DNA Polymerase + +++ Primer Extension Therminator DNA Polymerase - + Chain Terminator Applications Bst DNA Polymerase, Large Fragment - +++ Strand Displacement Applications
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080818.PMCB.lecture.4 - Plant Molecular and Cellular...

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