The primer strand is one nucleotide shorter than the

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The primer strand is one nucleotide shorter than the template, although in reality the template strand would extend out of the complex above, and both strands would extend as a duplex below. The incoming nucleoside triphosphate (a C) is base paired to the next G in the template strand. The magnesium to the rear is stabilizing the beta and gamma phosphates of the dNTP The magnesium to the front is most likely tickling the 3'OH to stimulate its nucleophilic attack on the alpha phosphate. *In order to prevent DNA synthesis from occuring in the crystal, the 3' nucleotide of the template strand was made by incorporating dideoxycytidine triphosphate (ddCTP). The next nucleotide complementary to the template is also a C. Thus the primer template complex is rendered unreactive by the lack of a 3'OH. A molecule of ddCTP can still enter the active site and is positioned for a reaction that does not occur.
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Clickerz: Where does most of the specificity for selecting the next nucleotide come from? A. H-bonds between the dNTP & the enzyme B. Stacking between the dNTP & the last base of the primer C. Inner sphere coordination between the magnesium & the dNTP beta phosphate D. Van der Waals interactions between the enzyme & the magnesium E. H-bonding between the template & the dNTP
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* DNA polymerases often carry multiple activities. Example: E coli DNA polymerase I (pol I) o In addition to its 5'->3' polymerase, pol I has a 3'->5' exonuclease activity, and a 5'->3' exonuclease activity. o The 3'->5' exonuclease activity is a proof reading activity. o The 5'->3' exonuclease is a kind of "cowcatcher" nuclease that can chew a strand away in front of the enzyme as a new strand is being synthesized. o All three activities are part of a single polypeptide chain. o The amino-terminal (N-terminal) third carries the 5'->3' nuclease whereas the carboxy-terminal (C-terminal) two thirds contains the polymerase and 3'->5' exonuclease activity. o The two parts of the protein can be cleaved apart using a mild protease treatment. The C-terminal fragment of pol I is called the large fragment or the Klenow fragment. o These three activities work in concert with each other.
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* The 3'->5' proofreading exonuclease of pol I corrects misincorporated nucleotides. o The active sites for the polymerase activity and the exonuclease activity are about 35 angstroms apart. o The enzyme does not apparently need to dissociate from the primer template complex in order to fit the 3' nucleotide of the primer into either active site. o When incorporation of the wrong nucleotide occurs (about 1E-4), the 3' nucleotide on the primer strand is not in a Watson-Crick base pair with the template. (They are said to be mispaired). o A mispaired primer end strongly inhibits incorporation of the next nucleotide, probably because the 3'OH of the primer cannot be easily positioned.
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