101309-BCH311

101309-BCH311 - 3OH nucleophilic attack on the 5 P on an...

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Fig. 10-6, p.245 3’,5’ Phosphodiester bond 3’OH nucleophilic attack on the 5’ P on an incoming deoxyribonucleoside triphosphate
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E. coli DNA Polymerases There are five DNA polymerases in i. DNA Pol I * Single-subunit/DNA replication ii. DNA Pol II Multi-subunit/DNA repair iii. DNA Pol III * Multi-subunit/DNA replication iv. DNA Pol IV DNA repair/SOS response v. DNA Pol V DNA repair/SOS response Two very important properties of DNA polymerases (in general) are; i. Speed (turnover number) - how many nucleotides can be added per minute ii. Processivity - number of nucleotides joined before the polymerase dissociates from the template
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Table 10-1, p.246 Properties of E. coli DNA Polymerases Turnover number (min -1 ) = speed Processivity = number of nucleotides joined before the polymerase dissociates from the template Note the additional functions of the polymerases *polymerization subunit only
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Table 10-2, p.242 E. coli DNA Pol III Holoenzyme The E. coli DNA Pol III holoenzyme is a multisubunit protein complex DNA Pol III consists of a core enzyme responsible for the polymerization and 3’ exonuclease - consisting of α -, ε -, and θ - subunits DNA Pol III also consists of a dimer of α - subunits responsible for DNA binding, and the γ - complex - consisting of γ -, δ -, δ ’-, χ -, and ψ - subunits - which allows the β - subunit to form a clamp that surrounds the DNA and slide along as polymerization proceeds
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Fig. 10-7, p.246 The β - subunit of E. coli DNA Pol III The β subunit exists in a dimer ( homodimer ) that encircles the DNA in a closed loop The β subunit acts as a processivity factor (or sliding clamp) for the Pol α ( catalytic ) subunit
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Fig. 10-20b, p.260 PCNA is the eukaryotic functional homologue of the β subunit PCNA, for P roliferating C ell N uclear A ntigen, is the eukaryotic homologue of the E. coli - subunit PCNA functions as a processivity factor for the major human replicative DNA polymerases, Pol δ and Pol ε
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Fig. 10-8, p.247 Components of the DNA Replication Reaction All four deoxyribonucleoside triphosphates, dATP, dTTP, dCTP, and dGTP Mg 2+ - DNA polymerase co-factor, facilitates the 3’-OH attack on 5’-PO 4 2- A single-stranded DNA template Primer, a short oligonucleotide strand to which the growing polynucleotide chain is covalently attached - this primer is RNA All four ribonucleoside triphosphates, ATP, UTP, CTP, and GTP
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DNA Replication - Priming DNA polymerases need a 3’ OH group (the reactive nucleophile) to which the incoming deoxyribonucleotide can be added RNA serves as a primer for DNA synthesis In order to get DNA replication started ( or primed ), an enzyme called primase (a DNA-dependent RNA polymerase) copies a short stretch of the DNA template to produce the RNA primer The primer provides the 3’ OH group for deoxyribonucleotide addition, however, this primer must be subsequently removed 3’-TAGTTCTAGTCTGACNNNNNNNNNNNNN-5’ 5’- AUCA C GAUC G GACUG -3’ How is this RNA primer recognized to facilitate its removal?
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This note was uploaded on 12/05/2009 for the course BCH 311 taught by Professor Howlett during the Fall '08 term at Rhode Island.

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101309-BCH311 - 3OH nucleophilic attack on the 5 P on an...

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