Alanine substitution mutagenesis of Escherichia coli DNA topoisomerase I, a member of the type IA subfamily of DNA topoisomerases, was carried out to identify amino acid side chains that are involved in transesterification between DNA and the active site tyrosine Tyr-319 of the enzyme. Twelve polar residues that are highly conserved among the type IA enzymes, Glu-9, His-33, Asp-111, Glu-115, Gln-309, Glu-313, Thr-318, Arg-321, Thr-322, Asp-323, His-365, and Thr-496, were selected for alanine substitution. Each of the mutant enzymes was overexpressed, purified, and characterized. Surprisingly, only substitution at Glu-9 and Arg-321 was found to reduce the DNA relaxation activity of the enzyme to an insignificant level. The R321A mutant enzyme, but not the E9A mutant enzyme, was found to retain a reduced level of DNA cleavage activity. Two additional mutant enzymes R321K and E9Q were also constructed and purified. Replacing Arg-321 by lysine has little effect on enzymatic activities; replacing Glu-9 by glutamine greatly reduces the supercoil removal activity but not the DNA cleavage and rejoining activities. From these results and the locations of the amino acids in the crystal structure of the enzyme, it appears that Glu-9 has a critical role in DNA breakage and rejoining, probably through its interaction with the 3' deoxyribosyl oxygen. The positively charged Arg-321 may also participate in these reactions by interacting with the scissile DNA phosphate as a monodentate. Because of the strict conservation of these residues, the findings for the E. coli enzyme are likely to apply to all type IA DNA topoisomerases.