Linked Life Data

8652560

Source:http://linkedlifedata.com/resource/pubmed/id/8652560

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
1996-7-30
pubmed:abstractText
Taq and T7 DNA polymerases have become basic molecular biology "tools" for DNA sequence analysis. However, Taq, unlike T7 DNA polymerase, is strongly biased against the incorporation of 2',3'-dideoxynucleotide triphosphates (ddNTPs) indicating very different substrate selectivities. Equilibrium binding and rate constants were measured for 2',3'-ddNTPs as well as for several other 3'-substituted terminators and compared to 2'-deoxynucleotide substrates (dNTPs). In steady-state experiments, Taq Pol I was strongly biased in favor of dATP1 over ddATP incorporation by about 700 to 1, in contrast to T7 DNA polymerase which showed a preference of only about 4 to 1. Manganese reduced but did not eliminate selectivity against 2',3'-ddNTPs. Transient kinetic traces indicated different rate-limiting steps for substrate and terminator incorporation. Further mechanistic studies showed that the binding constants for substrates and terminators were equivalent. However, the rate constants for phosphodiester bond formation for 2',3'-ddNTPs were 200-3000-fold lower than for dNTPs. Alternative terminators showed only slight improvements. The data were consistent with a model in which both substrates and terminators undergo ground-state binding followed by formation of a tight-binding Enz.DNA.Nucleotide complex. Immediately after complex formation, substrates undergo a rapid nucleoside phosphoryl transfer reaction. However, the reaction rates for terminators were slower presumably due to misalignment of reactive groups in the active site. Thus, the strong bias that Taq DNA polymerase shows against terminators is due to a very slow "chemistry" step. Such a strong bias has several kinetic consequences for DNA sequence patterns. These consequences are discussed in the text.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2189-200
pubmed:dateRevised
2000-12-18
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Slow rate of phosphodiester bond formation accounts for the strong bias that Taq DNA polymerase shows against 2',3'-dideoxynucleotide terminators.
pubmed:affiliation
Genetic Analysis Group, Applied Biosystem Division, Perkin Elmer Corporation, Foster City, California 94401, USA.
pubmed:publicationType
Journal Article