BIOCHEMICAL AND MUTATIONAL STUDIES OF THE 5'-3'-EXONUCLEASE OF DNA-POLYMERASE-I OF ESCHERICHIA-COLI

In order to improve our understanding of the 5'-3' exonuclease reactio n catalyzed by Escherichia coli DNA polymerase I, we have constructed expression plasmids and developed purification methods for whole DNA p olymerase I acid its 5'-3' exonuclease domain that allow the productio n of large quantities of highly purified material suitable for biophys ical and other studies, We have studied the enzymatic properties of th e 5'-3' exonuclease, both as an isolated domain and in the context of the whole polymerase, using a variety of model oligonucleotides to exp lore the enzyme-substrate interaction. The 5'-3' exonuclease is known to be a structure-specific nuclease that cleaves a 5' displaced strand at the junction between single-stranded and duplex regions. Since the isolated domain shows the same structure specificity as the whole pol ymerase, the correct geometry of substrate binding is achieved without the assistance of tl-Le polymerase domain. The 5'-3' exonuclease reac tion has a strict requirement for a free 5' end on the displaced stran d; however, the upstream template and primer strands are dispensable. Site-directed mutagenesis of the ten carboxylate residues that are hig hly conserved among bacterial and bacteriophage 5'-3' exonucleases ind icates that nine of them are important in the reaction. This finding i s discussed in relation to structural and mutational data for related 5' nucleases. (C) 1997 Academic Press Limited.