Unformatted text preview: 3'OH attacks and the pyrophosphates leave (gamma and beta) Chemistry of DNA synthesis Mechanism of DNA polymerase
Only correctly paired bases are positioned such that a phosphodiester bond can form Incorrect base pairing Dramatically lower rates of nucleotide addition
kinetic proofreading, the rate is more conservative Catalytically unfavorable alignment of substrates DNA polymerase looks at the confirmation of the incoming nucleotide if it fits in If base pairing is correct, the components is aligned if not, then it doesn't align correctly and reactions doesn't take place. side chain is not favorable DNA polymerase discriminate dNTP from rNTP Only deoxyribonucleotides fit properly Active site of DNA polymerase has 2 divalent metal ions bound
Metal ion A interacts with the 3'OH Metal ion B interacts with the triphosphate of the incoming dNTP
EDTA removes metal ions DNA polymerase grips the template and the incoming nucleotide when a correct base pair is made tyrosine can do stacking interactions arginine and lysine is positively charged and itneract with the phosphate To study the role of the Lys of the O-helix, whether the positive charge on the side chain plays a role in substrate binding and catalysis -mutate residue to alanine and arginine The structure of T7 DNA polymerase bound to its substrate in the closed conformation DNA polymerases cannot synthesize DNA s...
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