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NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I), d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) INTRODUCTION [1,2]The role of DNA ligases is well established in DNA replication, in DNA repair and in genetic recombination in prokaryotic and in eukaryotic cells [3]. These enzymes catalyse the restoration of an interruption of a single strand in double-helical DNA. For the joining activity the enzyme requires juxtaposed 3'-hydroxyl and 5'-phosphoryl ends aligned in a duplex structure [4]. The enzymes obtained from unaffected and from T4-infected E. coli have been investigated most thoroughly [4]. However, until now little information is available concerning structural details of the nicked duplex structure.The present work describes an NMR study, augmented with biochemical experiments, of a synthetic nicked duplex structure. The compound consists of a 12 base-paired duplex that features an interruption in the centre of one strand of the double helix. The conformational properties of the nicked duplex are compared with those of the intact doublehelical fragment. Furthermore, the influence of removal of the phosphate at the interruption is demonstrated. Thermodynamic analysis of duplex formation of the nicked as well as of the intact duplex structure is used to study the amount of cooperativity of the two hexamer strands in the melting behaviour of the nicked duplex. Finally, the T4 polynucleotide ligase activity upon this synthetic nicked duplex fragment is reported.

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In a previous study it was shown that RNase P from E. coli cleaves the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA in vitro (Guerrier-Takada et al. (1988) Cell, 53, 267-272). Cleavage takes place at the 3' side of the loop that crosses the deep groove of the pseudoknot structure present in the aminoacyl acceptor domain. In the present study fragments of TYMV RNA with mutations in the pseudoknot, generated by transcription in vitro, were tested for susceptibility to cleavage by RNase P. Changes in the specificity with respect to the site of cleavage and decreases in the rate of cleavage were observed with most of these substrates. The behaviour of various mutants in the reaction catalyzed by RNase P is in agreement with the present model of the TYMV RNA pseudoknot (Dumas et al. (1987), J. Biomol. Struct. Dyn. 263, 652-657). Base substitutions in the loop that crosses the shallow groove of the pseudoknot structure resulted, however, in an unexpected decrease in the rate of cleavage, probably due to conformational changes in the substrates. Studies on other tRNA-like structures revealed an important role in the reaction with RNase P for both the nucleotide at the 3' side of the loop that spans the deep groove and the nucleotide at position 4, which correspond to positions--1 and 73, respectively, in tRNA precursors.