作者
Vandana Purohit, Nigel DF Grindley, Catherine M Joyce
发表日期
2003/9/2
期刊
Biochemistry
卷号
42
期号
34
页码范围
10200-10211
出版商
American Chemical Society
简介
We have investigated conformational transitions in the Klenow fragment polymerase reaction by stopped-flow fluorescence using DNA substrates containing the fluorescent reporter 2-aminopurine (2-AP) on the template strand, either at the templating position opposite the incoming nucleotide (designated the 0 position) or 5‘ to the templating base (the +1 position). By using both deoxy- and dideoxy-terminated primers, we were able to distinguish steps that accompany ternary complex formation from those that occur during nucleotide incorporation. The fluorescence changes revealed two extremely rapid steps that occur early in the pathway for correct nucleotide incorporation. The first, detectable with the 2-AP reporter at the 0 position, occurs within the first few milliseconds and is associated with dNTP binding. This is followed by a rapid step involving relative movement of the +1 base, detectable when the 2-AP …
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