SummaryTwo mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of in¯icting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at noncognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.

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The gene encoding the EcoRI endonuclease was altered by site-directed mutagenesis to introduce multiple substitutions of M137 and 1197, two amino acids which were suggested by the revised crystal structure to mediate recognition of the cytosines in the 5'-GAATTC-3' target sequence. Eight substitutions of M137 and ten substitutions of 1197 were isolated. With the exception of M137W, M137P and M137K, all mutant enzymes retained enough activity to damage cellular DNA in the absence of the EcoRI methyltransferase. All M137 replacements abolished the ability of the enzyme to restrict phage growth. Conservative replacements at 1197 (L, V) did not impair phage restriction, whereas non-conservative changes reduced (G, W) or abolished (D, P) restriction. In general, substitutions at M137 were more deleterious than substitutions at I197. Double mutants with combinations of M137G/A and I197G/A mutations exhibited a phenotype characteristic for the respective single M137 mutant. Double mutants carrying combinations of the M137G/A replacements and substitutions at R200 were viable even in the absence of the methyltransferase, suggesting that disrupting contacts to both bases of the GC base pair inactivates the enzyme. None of the replacements resulted in relaxed recognition specificity. In summary, our findings are consistent with a role for M137 but do not support such a role for I197 in substrate recognition by the EcoRI endonuclease.

11-Nitrates of 9a,11b-dihydroxy derivatives of estrone, estradiol, and ethinylestradiol were synthesized by oxidative nitration of the corresponding estratriene 3-acetates with cerium ammonium nitrate. Three methods are given for this reaction. Compounds had high antifertility and estrogen activity. Antifertility actions were much greater than their estrogen activities, as compared with the similar levels seen with ethinylestradiol.