Covalent Attachment of Polyribonucleotides to Polydeoxyribonucleotides Catalyzed by Deoxyribonucleic Acid Ligase

Kamalendu Nath,Jerard Hurwitz

doi:10.1016/s0021-9258(19)42528-3

Kamalendu Nath, Jerard Hurwitz

Open Access

https://doi.org/10.1016/s0021-9258(19)42528-3

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Abstract

Abstract DNA ligase isolated from Escherichia coli or phage T4-infected E. coli catalyzes the covalent joining of the 5'-phosphate termini of polydeoxyribonucleotides and 3'-hydroxyl termini of polyribonucleotides. This reaction occurs with poly(dA) and poly(A) in the presence of poly(dT). The 5'-phosphate terminus of poly[d(A-T)] can be linked by an intramolecular reaction to its 3'-hydroxyl terminus substituted with a ribonucleotide. Phage T4-induced DNA ligase also joins poly(dT) to poly(U) in the presence of poly(dA). The joining of 5'-phosphate termini of polyribonucleotides to 3'-hydroxyl termini of polydeoxyribonucleotides was detected only with phage T4-induced DNA ligase. This reaction, however, occurred to a limited extent with poly(dA) substrates containing AMP residues at the 5' terminus in the presence of poly(dT). These results suggest that although DNA ligase from E. coli is incapable of joining RNA to RNA, it is capable of joining 5'-phosphate terminus of a DNA to the 3'-hydroxyl terminus of RNA in addition to the well known joining reaction between DNA to DNA. The phage T4-induced DNA ligase on the other hand joins DNA to DNA, RNA to RNA, and RNA to DNA in all combinations.

Highlights

5’-phosphate terminus of poly[d(A-T)] can be linked by an intramolecular reaction to its 3’-hydroxyl terminus substituted with a ribonucleotide
The joining of 5’-phosphate termini of polyribonucleotides to 3’-hydroxyl termini of polydeoxyribonucleotides was detected only with phage T4-induced DNA ligase. This reaction, occurred to a limited extent with poly(dA) substrates containing AMP residues at the 5’ terminus in the presence of poly(dT). These results suggest that DNA ligase from E
It carries out the linkage of polydeoxyribonucleotides possessing a mispaired base [14] and utilizes polyribonucleotides as template for the joining of polydeosyribonucleotides (10, II, 15). None of these reactions were catalyzed by DNA ligase from E. coli. Both E. coli and T4-DNA ligase join the 5’.phosphate termini of polydeoxyribonucleotides to the 3’-hydroxyl cuds of polyribonucleotides

Summary

SUMMARY

DNA ligase isolated from Escherichia coli or phage T4-infected E. coli catalyzes the covalent joining of the 5’-phosphate termini of polydeoxyribonucleotides and 3’-hydroxyl termini of polyribonucleotides. The joining of 5’-phosphate termini of polyribonucleotides to 3’-hydroxyl termini of polydeoxyribonucleotides was detected only with phage T4-induced DNA ligase. This reaction, occurred to a limited extent with poly(dA) substrates containing AMP residues at the 5’ terminus in the presence of poly(dT). T4-induced DNA ligase catalyzes the joining of polyribonuclrotides [10, 11] and DNA duplex structures at their base-paired ends [12, 13] It carries out the linkage of polydeoxyribonucleotides possessing a mispaired base [14] and utilizes polyribonucleotides as template for the joining of polydeosyribonucleotides (10, II, 15). Phosphate termini of polyribonucleotides to the 3’-hydroxyl ends of polydeoxyribonucleotides

PROCEDURE

Methods

RESULTS

The discrepancy between Experiments

The polynucleotide concentrations were

II 0 20 40 60