DNA Polymerases Have Various Nuclease Activities

KEY CONCEPT
-DNA polymerase I has a unique 5′–3′ exonuclease activity that can be combined with DNA synthesis to perform nick translation.
Replicases often have nuclease activities as well as the ability to synthesize DNA. A 3′–5′ exonuclease activity is typically used to excise bases that have been added to DNA incorrectly. This provides a “proofreading” error-control system .
The first DNA-synthesizing enzyme that researchers characterized was DNA polymerase I, which is a single polypeptide of 103 kD (kilodalton). The chain can be cleaved into two parts by proteolytic treatment. The larger cleavage product (68 kD) is called the Klenow fragment. It is used in synthetic reactions in vitro. It contains the polymerase and the proofreading 3′–5′ exonuclease activities. The active sites are approximately 30 Å apart in the protein, which indicates that there is spatial separation between adding a base and removing one.
The small fragment (35 kD) possesses a 5′–3′ exonucleolytic activity, which excises small groups of nucleotides, up to approximately 10 bases at a time. This activity is coordinated with the synthetic/proofreading activity. It provides DNA polymerase I with a unique ability to start replication in vitro at a nick in DNA. (No other DNA polymerase has this ability.) At a point where a phosphodiester bond has been broken in a double-stranded DNA, the enzyme extends the 3′–OH end. As the new segment of DNA is synthesized, it displaces the existing homologous strand in the duplex. The displaced strand is degraded by the 5′–3′ exonucleolytic activity of the enzyme.
FIGURE 1. illustrates this process of nick translation. The displaced strand is degraded by the 5′–3′ exonuclease activity of the enzyme. The properties of the DNA are unaltered, except that a segment of one strand has been replaced with newly synthesized material, and the position of the nick has been moved along the duplex. This is of great practical use; nick translation has been a major technique for introducing radioactively labeled nucleotides into DNA in vitro.

FIGURE 1. Nick translation replaces part of a preexisting strand of duplex DNA with newly synthesized material.
The coupled 5′–3′ synthetic/3′–5′ exonucleolytic action is used most extensively for filling in short single-stranded regions in doublestranded DNA. These regions arise during lagging strand DNA replication .