DNA Polymerase "extendase" activity
ohungnes at bioslave.uio.no
Sat Sep 3 14:17:56 EST 1994
jobling at usuhs.usuhs.mil wrote:
: In Article <346bh8$bm at mars.mahidol.ac.th>
: g3421101 at mucc.mahidol.ac.th (Enrique Jose Labadan Frio - SCBC - 3421101) writes:
: >Dear friends and colleagues,
: > A recent paper in NAR 1994 [22(12):2423] by Costa and Weiner
: >(Stratagene) has some very interesting observations on the "extendase"
: >activity [addition of a single non-template-encoded nuceotide at the 3'
: >end] of DNA-dependent DNA polymerases. Noteworthy are the ff:
: > Klenow (surprised even more! the great blunting enzyme...?)
: > T7 (from which Klenow was derived, by cutting off the 5'-3'
: > exonuclease domain)
: >Dept of Biochemistry
: jsut a thought but someone from a biochemistry dept should know that
: Klenow is derived from DNA polymerase I (Kornberg enzyme!) and not
: T7 polymerase which is a phage enzyme. Klenow like derivatives
: of T7 have been created (sequenase i.e. T7 pol is functionally similar but
: inactivated by chemical treatment I believe; ver 2.0 maybe a genetic deletion
: if I remeber some USB blurb correctly), which may have caused the
: my 2c worth.
: Mike Jobling (JOBLING at USUHS.USUHS.MIL or @USUHS.BITNET)
: Dept Microbiology and Immunology USUHS
: 4301 Jones Bridge Rd Bethesda MD 20814 "Space reserved for something
: Tel (301)295 3405 FAX (301)295 1545 witty - suggestions welcome"
As to the questions, my initial reaction when reading the article was
that it perhaps is a little unfair to operate Klenow at the same dNTP
conc. as Taq (was it 100 microM, I don't have the article with me?). After
all it requires much less dNTPs, and I understand that the balance of
polymerase and proofreading (exonuclease) activity is influenced by this
parameter. My guess is that the "extendase" activity of Klenow is much
less prominent at say 20 - 40 microM of dNTPs. One should keep in mind
that the article is written by people who sell the T7 poly and wouldn't
mind that the T7 comes out best (it has a higher proofreading activity,
probably less inhibited by the high dNTP conc).
my 2 mmol worth,
Olav Hungnes olav.hungnes at embnet.uio.no
National Institute Phone (+47)22042200
of Public Health FAX (+47)22353605
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