Kinase 345
The associated 3'-phosphatase activity causes the hydrolysis of the
3'-phosphate group of a variety of substrate molecules, including
deoxynucleoside 3'-monophosphates, deoxynucleoside 3',5'-diphos-
phates, and 3'-phosphorylated polynucleotides, to form a 3'-hydroxyl
group and release inorganic phosphate.
3.2. Substrate
3.2.1. Acceptor
The 5'-hydroxylated nucleoside moiety in the reaction may com-
prise single- or double-stranded DNA, RNA, a synthetic oligonucleotide,
a nucleoside-3'-monophosphate, or a deoxynucleoside-3'-monophos-
phate. The enzyme can act on any molecule terminating in a naturally
occurring nucleoside. By contrast, rat liver polynucleotide kinase cannot
act on RNA or oligonucleotides <10 bases in length.
When the molecule is double-stranded DNA, a protruding 5' termi-
nus is a better substrate than a blunt or recessed end. However, by
increasing the concentration of the phosphate donor (usually ATP), all
5' termini can be completely phosphorylated. Increased enzyme con-
centrations also cause more efficient kinasing of recessed termini.
Nicks in duplex DNA will act as substrates for reaction, but the rate
of kinasing is 10- to 30-fold slower than for single-stranded DNA, or
protruding 5' termini (19,20), and phosphorylation is incomplete, with
only 70% being achievable even after long reaction times (8). Raising
the ATP concentration will not promote complete phosphorylation in
this case.
The size of the acceptor molecule has little effect on the rate of
reaction, within the range 150-50,000 nucleotides. The K m value for
large DNA fragments released by nuclease treatment is 7.6 jxM (21).
K m for nucleoside-3'-phosphates and oligonucleotides is 22.2-143.0
jxM depending on the 5' base and the length of the oligonucleotide (22).
3.2.2. Donor
The phosphate group donor for the kinase reaction may be any nucleo-
side triphosphate. Although ATP is used routinely for experimental or
assay purposes, CTP, UTP, GTP, dATP, and TTP perform equally well
(21). The ATP concentration should be at least 1 ~/for the reaction
to proceed, and excess ATP is required for optimal kinasing (23).