40
KAI-OLAF NETZER
Subprotocol 5
DNA-Dependent DNA Polymerases - E. Coli DNA Polymerase I and
Klenow Enzyme
Many protocols in molecular biology involve the in vitro synthesis of DNA
or RNA. These reactions are catalyzed by DNA and RNA polymerases, respectively. DNA polymerases synthesize new DNA strands complementary
to template strands which may be either DNA (DNA polymerase I, Klenow
enzyme, T4/T7 polymerase, Taq polymerase) or RNA (reverse transcriptase).
In vivo, both prokaryotic and eukaryotic cells contain multiple DNA
polymerase activities. Three DNA polymerase enzymes have been characterized in E. coli. However, only one enzyme, DNA polymerase III, performs
the replicase function. The others play subsidiary roles in replication or participate in repair of damaged DNA sequences. All bacterial DNA polymerases share the samefundamental type of synthetic activity: DNA is extended in a 5'-----+3' direction by adding nucleotides to the end containing a
free 3' OH. The fidelity of the replication is guaranteed by a postsynthetic
proofreading function performed by a 3' -----+ 5' exonuclease activity. The error
rate in bacteria appears to be w-s to w- 10 •
The most abundant DNA polymerase in E. coli is DNA polymerase I.
Apart from polymerase and proofreading activity, the enzyme possesses
a 5' -----+ 3' exonucleolytic activity. Therefore, the holoenzyme is able to start
DNA replication at nicks, which is of great practical use for labeling DNA
molecules in vitro (nick translation).
Proteolytic cleavage of DNA polymerase I with subtilisin results in two
fragments, the larger of which is called Klenow fragment. The Klenow fragment carries polymerase and proofreading activity but lacks the 5' -----+ 3' exonuclease activity. Therefore, with the use ofKlenow enzyme there is no risk
of template degradation. Klenow enzyme has several applications in molecular biology. One is to generate labeled DNA probes in a random hexamer primed reaction. The random primers bind to denatured, single
stranded DNA and initiate the DNA synthesis. Radiolabeted nucleotides
are incorporated into the newly synthesized strand (random primed labeling). Another application is the filling in of recessed 3' termini created by
digestion ofDNA with certain restriction enzymes (for a protocol, see subprotocoll ). Filling of 3' ends generates blunt ends which can be ligated or to
which synthesis linkers can be added.
KAI-OLAF NETZER
Subprotocol 5
DNA-Dependent DNA Polymerases - E. Coli DNA Polymerase I and
Klenow Enzyme
Many protocols in molecular biology involve the in vitro synthesis of DNA
or RNA. These reactions are catalyzed by DNA and RNA polymerases, respectively. DNA polymerases synthesize new DNA strands complementary
to template strands which may be either DNA (DNA polymerase I, Klenow
enzyme, T4/T7 polymerase, Taq polymerase) or RNA (reverse transcriptase).
In vivo, both prokaryotic and eukaryotic cells contain multiple DNA
polymerase activities. Three DNA polymerase enzymes have been characterized in E. coli. However, only one enzyme, DNA polymerase III, performs
the replicase function. The others play subsidiary roles in replication or participate in repair of damaged DNA sequences. All bacterial DNA polymerases share the samefundamental type of synthetic activity: DNA is extended in a 5'-----+3' direction by adding nucleotides to the end containing a
free 3' OH. The fidelity of the replication is guaranteed by a postsynthetic
proofreading function performed by a 3' -----+ 5' exonuclease activity. The error
rate in bacteria appears to be w-s to w- 10 •
The most abundant DNA polymerase in E. coli is DNA polymerase I.
Apart from polymerase and proofreading activity, the enzyme possesses
a 5' -----+ 3' exonucleolytic activity. Therefore, the holoenzyme is able to start
DNA replication at nicks, which is of great practical use for labeling DNA
molecules in vitro (nick translation).
Proteolytic cleavage of DNA polymerase I with subtilisin results in two
fragments, the larger of which is called Klenow fragment. The Klenow fragment carries polymerase and proofreading activity but lacks the 5' -----+ 3' exonuclease activity. Therefore, with the use ofKlenow enzyme there is no risk
of template degradation. Klenow enzyme has several applications in molecular biology. One is to generate labeled DNA probes in a random hexamer primed reaction. The random primers bind to denatured, single
stranded DNA and initiate the DNA synthesis. Radiolabeted nucleotides
are incorporated into the newly synthesized strand (random primed labeling). Another application is the filling in of recessed 3' termini created by
digestion ofDNA with certain restriction enzymes (for a protocol, see subprotocoll ). Filling of 3' ends generates blunt ends which can be ligated or to
which synthesis linkers can be added.