Studies on polynucleotides. LXXXVII. The joining of short deoxyribopolynucleotides by DNA-joining enzymes.

Abstract

Several groups of workers have reported on the discoveries of enzymes that repair single-stranded breaks in double-stranded DNA.1-5 The presence of these enzymes has been demonstrated in E. coli1' , 4 and in T4 bacteriophageinfected E. coli cells.2' 5 The action of the enzymes involves the formation of a phosphodiester linkage between a 5'-phosphomonoester group and a 3'-hydroxyl group when these are present at the site of a single-stranded break. These enzymes could clearly be a boon to the problem of the total synthesis of biologically specific high-molecular-weight DNA in that properly aligned, relatively short, chemically synthesized deoxyribopolynucleotides could be joined to form continuous bihelical structures. To explore this possibility, we have studied both the E. coli and T4-induced enzymes for the minimum polynucleotide chain length they require in order to bring about the joining reaction. In the present initial study, several selected series of polynucleotides with repeating nucleotide sequences were used. Two types of experiments were carried out. In the first, a short deoxyribopolynucleotide carrying a 5'-phosphate group at one end and a 3'-hydroxyl group at the opposite end was tested for end-to-end joining in the presence of a long complementary deoxyribopolynucleotide. These experiments showed that oligonucleotides as short as hexaor heptanucleotides could be joined. In the second type of experiments, both of the complementary polynucleotides were short. Thus, a combination studied most in the present work was that of the hexadecanucleotide, d(TTAC)4,6 with the octanucleotide, d-P82(TAAG)2.6 Both enzymes brought about the joining of two molecules of the octanucleotide to form the expected hexadecanucleotide. Finally, a study of the temperature-absorbance profiles of the two typical sets of complementary polynucleotides, d-P32(TG)36 + poly d-CA and d(TTAC)4 + d-P32(TAAG)2, showed that under the conditions used successfully for the enzyme-catalyzed reactions, the polynucleotides are able to form (largely or entirely) double-stranded structures without the agency of the enzymes. Materials and Methods.-Deoxyribo-, oligo-, and polynucleotides: Poly d-A6 and poly d-T were prepared from poly dA:dT6, and, similarly, poly d-CA6 and poly d-TG6 were prepared from poly d-CA: d-TG6 by separation of the strands in alkaline cesium chloride density gradient.7 8 Samples of poly d-CA and poly d-TG, and also of poly r-CA and poly r-UG,9 were generously donated by Dr. A. R. Morgan. Short-chain poly-deoxyriboadenylates (d-A7-d-Ai5)6 were prepared by controlled micrococcal nuclease degradation of poly d-A followed by treatment with bacterial alkaline phosphatase and chromatography on a DEAE-cellulose column. The following series of deoxyribopolynucleotides were chemically prepared samples, syntheses of which have been previously described: dT6,6 dT7,6 and higher homologues up to dT15; 10 11 short polynucleotides with repeating dinucleotide sequences, d(TG)2_5,12 d(CA)2_5,2 d(TC)8, and d(AG)4;12 deoxyribopolynucleotides with repeating tetranucleotide sequences, d(TAAG)213 and d(TTAC)4.'4