Nucleic Acids Research Temperature dependence of the joining by T4 DNA ligase of termini produced by type II restriction endonucleases

The temperature dependence of the T4 DNA ligase-catalyzed joining of plasmid DNA linearized by the action of Haelll, EcoRI and PstI restriction endonucleases has been investigated by electron microscopy analysis. The extent of joining is maxi_ mal at 4° and decreases with increasing temperatures following sigmoid like curves. The temperature at which 50?J of the maximal reaction is still observable increases going from DNA termini without single-stranded overlaps (produced by Haelll) to termini with four nucleotides overlap, composed only by two A and two T (produced by EcoRI) to termini with four nucleotide overlap, composed by A, T, G and C (produced by PstI) INTRODUCTION The physiological substrate for the enzyme DNA ligase (1) is generally believed to be an interruption of a phosphodiester bond in a polynucleotide, with the resulting contiguous 5' P and 3' OH kept in register by an uninterrupted complementary DNA strand. A probably less natural but certainly very useful feature of the DNA ligase is the ability to join two different DNA duplexes, like those produced by most type II restriction endonucleases (2), when held together by hydrogen bonds between complementary sequences as short as two nucleotides (3). A peculiar feature of the T4 DNA ligase (4) is the ability to join end-to-end DNA duplexes possessing no single-stranded overlap, commonly called blunt or flush ended. The optimal temperature for the action of the DNA ligase on physiological © IRL Press Limited, 1 Falconberg Court. London W1V 5FG, U.K. 85 Downloaded from https://academic.oup.com/nar/article-abstract/9/1/85/1043279/Temperature-dependence-of-the-joining-by-T4-DNA by guest on 16 September 2017 Nucleic Acids Research substrates is 37°, but when the enzyme is challenged with separate molecules, the stability of their interaction at the termini becomes also an important parameter in both rate and extent of joining. It has been found (5) that the melting tem perature of the base-paired tetranucleotide (5'-AATT-3') termini produced by the EcoRI restriction endonuclease is 5-6°C, in low ionic strength buffer and 5O% formamide. For the covalent joining by the E.coli DNA ligase of cone sive termini produced by EcoRI Dugaiczyk et al (6) have reported that in the 0-15° interval investigated, the optimal temperature was between 10 and 15°. Using heterogeneous populations of restriction fragments, Sgaramella and Ehrlich (7) found that for the cohesive termini produced by EcoRI, and also for the no-overlap, blunt termini produced by Haelll, the maximal joining by the T4 DNA ligase was around 25°. No study was known to us which dealt with the effect of the base composition of the complementary sequences on the tern perature of joining of restriction endonuclease-generated termini. The data presented here describe the temperature dependence of the joining by the T4 DNA ligase, extracted from induced E.coli cells lysogenized with a lambda phage carrying the T4 gene for the DNA ligase (8,9) of the three different kinds of termini produced by the type II restriction endonucleases EcoRI , PstI and Haelll (1O). MATERIALS AND METHODS DNA. The plasmids pBR322 (11) and pC194 (12) have been isolated from clear lysates, (12,13), followed by CsCl-ethidium bromide equilibrium centrifugation. Enzymes and proteins. The T4 DNA ligase has been extracted from thermally induced E.coli cells lysogenized with a recombinant phage harboring the T4 DNA ligase gene (8), kindly given to us by Drs. K and N.Murray. Details of the purifications procedure