28] Structure-function analysis of 3′ → 5′-exonuclease of DNA polymerases

Abstract

This chapter first describes the mutational studies that were conducted on the Klenow fragment 3' →9 5'-exonuclease, and then summarize similar studies on other DNA polymerases. The 3' →0 5'-exonuclease activity of DNA polymerases acts in opposition to the polymerase activity and serves as a proofreader, by removing polymerase errors. This activity is present in the majority of DNA-dependent DNA polymerases, but is absent in the reverse transcriptase (RT) family. The 3' → 5'-exonuclease is usually part of the same polypeptide chain as the DNA polymerase; an exception is the multi subunit DNA polymerase III of Escherichia coli , where the editing function is present on a separate subunit (e) within the core polymerase. In the structure of the Klenow fragment of DNA polymerase I, the 3' → 5'-exonuclease is located on a discrete structural domain, 3 and it seems likely that other DNA polymerases are arranged in a similar modular fashion. The preferred substrate for the exonuclease is single-stranded DNA, and a variety of data are consistent with the idea that the primer terminus of a duplex DNA substrate is bound as a frayed or single-stranded end at the exonuclease active site.