MCB3_6 - Biological synthesis of oligonucleotides! In the...

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Biological synthesis of oligonucleotides In the cell ( in vivo ), nucleotide 5’ triphosphates are joined in a 5’-3’ direction of the growing chain. DNA is synthesized in a template-dependent manner: Semi-conservative, meaning that each daughter cell inherits one old and one new strand ! of dsDNA. 1
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Arthur Kornberg discovered (the first) DNA polymerase Enzyme requirements were unknown, so many variables had to be tested to develop an in vitro DNA polymerase assay. The biochemical strategy: 1. Grow E. coli bacteria. 2. Break open cells. 3. Prepare soluble enzyme extract. 4. Fractionate extract to resolve different cellular components from each other; repeat; repeat. 5. Search for DNA polymerase activity using a biochemical assay: incorporate radioactive nucleotide monomers into oligomer DNA chains. 2
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Fig 4-2 Four requirements of DNA-templated (DNA-dependent) DNA polymerases • single-stranded template • deoxyribonucleotides with 5’ triphosphate (dNTPs) magnesium ions annealed primer with 3’ OH Synthesis ONLY occurs in the 5’-3’ direction 3 Inorganic pyrophosphate (PPi) is lost when a dNMP (monophosphate) adds to the chain
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DNA polymerase: 5’-3’ polymerase activity Polymerase domains can be modeled after a right hand palm, thumb, and fingers 4
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5’-3’ polymerase and 3’-5’ exonuclease (Klenow fragment) 5’-3’ exonuclease E. coli DNA polymerase I has 3 distinct activities in 3 distinct protein domains. Nuclease = enzyme that disrupts phosphodiester backbone Exo = cleaves from 5’ or 3’ end; Endo = cleaves internally 5
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5’-3’ exonuclease Converts duplex DNA or DNA-RNA hybrid to ssDNA, beginning at the site of a nick (discontinuous backbone on one strand). For this class, assume that it can NOT begin at a dsDNA break. Moves 5’-3’, ACTS ONLY when the 5’ end of a chain is free. E. coli DNA polymerase I 6
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OH OH E. coli DNA polymerase I combination of 5’-3’ exo + 5’-3’ pol allow “nick translation” Replacing RNA or DNA in a nicked duplex with new DNA has important roles in DNA replication and DNA repair. 5’ 3’ 5’ 3’ 5’ 3’ 7
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Proof-reading (editing) of the 3’ dNMP by the 3’-5’ exonuclease activity: 8 This exonuclease activity moves 3’-5’ on dsDNA, so it acts ONLY at the 3’ end of a chain. (For class, assume that 3’-5’ exo requires a ds/ssDNA junction with a 5’ overhang.) This is opposite the polarity of 5’-3’ polymerase activity: polymerase activity must HALT to allow 3’-5’ exonuclease activity. dNMP is released as product, leaving a 3’ OH group on the polynucleotide. E. coli DNA polymerase I 3’-5’ exonuclease 5’ 5’ 3’
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The 3’ end of the growing chain must switch between synthesis and editing active sites. 9
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Incorrect nucleotide addition disfavors polymerase activity and favors proofreading activity.
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MCB3_6 - Biological synthesis of oligonucleotides! In the...

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