Interaction of bacteriophage T4 RNA and DNA ligases in joining of duplex DNA at base-paired ends.

A Sugino,H M Goodman,H L Heyneker,J Shine,H W Boyer,N R Cozzarelli

doi:10.1016/s0021-9258(17)40347-4

Abstract

The joining of duplex DNA at base-paired ends by bacteriophage T4 DNA ligase was confirmed using either a synthetic duplex decamer or restriction endonuclease fragments of ColE1 DNA as substrates. The reaction was not linearly dependent on enzyme concentration but increased markedly at high enzyme concentrations. Although T4 RNA ligase did not catalyze this blunt end joining, it makedly stimulated the DNA ligase reaction particularly at low DNA ligase concentrations. The apparent Km for the decamer was 50 micronM in the presence or absence of RNA ligase. In the presence of RNA ligase, T4 DNA ligase had about the same turnover number for blunt end and cohesive end joining. The joining of duplex DNA at base-paired ends was proven by several techniques including restriction endonuclease cleavage of the products. The products of the ligation reaction using restriction enzyme fragments were mostly linear oligomers but included some circular duplexes. Escherichia coli DNA ligase in the presence or absence of RNA ligase did not catalyze blunt end joining. RNA ligase only moderately affected the joining of cohesive ends by T4 DNA ligase or E. coli DNA ligase and did not itself catalyze this reaction.

Highlights

The joining of duplex DNA at base-paired ends by bacteriophage T4 DNA ligase was confirmed using either a synthetic duplex decamer or restriction endonuclease fragments of ColEl DNA as substrates
Ligation of Duplex Decamer by T4 RNA and DNA Ligases -In order to test whether RNA ligase catalyzes the joining of duplex DNA at base-paired ends or influences this a J
As monitored by the production of phosphatase-resistant ligase in contrast to T4 DNA ligase did not catalyze joining of the decamer at 15” (Fig. 2)

Summary

Introduction

The joining of duplex DNA at base-paired ends by bacteriophage T4 DNA ligase was confirmed using either a synthetic duplex decamer or restriction endonuclease fragments of ColEl DNA as substrates. T4 RNA ligase did not catalyze this blunt end joining, it markedly stimulated the DNA ligase reaction ligase concentrations. In the presence of RNA ligase, T4 DNA ligase had about the same turnover number for blunt end and cohesive end joining. The joining of duplex DNA at base-paired ends was proven by several techniques including restriction endonuclease cleavage of the products. The products of the ligation reaction using restriction enzyme fragments gomers but included some circular were mostly linear oliduplexes. Escherichia coli DNA ligase in the presence or absence of RNA ligase did not catalyze blunt end joining. RNA ligase only moderately affected the joining of cohesive ends by T4 DNA ligase or E. coli DNA ligase and did not itself catalyze this reaction. T4 induces the formation of a novel enzyme, RNA ligase, which catalyzes the ATP-dependent ligation of single-stranded

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Results

Conclusion