Localization of Single-Chain Interruptions in Bacteriophage T5 DNA. II. Electrophoretic Studies

Abstract

Upon denaturation, T5 DNA yields a large number of discrete, single-chain fragments that can be resolved by agarose gel electrophoresis. The positions of the more prominent of these fragments in the T5 duplex were determined by analyzing their sensitivity to digestion with λ exonuclease and their distribution among Eco RI fragments of T5 DNA. These experiments also provide firm evidence concerning the polarity of the strands in T5 DNA. An analogous study was carried out on the fragments produced by treating exonuclease III-degraded T5 DNA with the single-strand-specific SI endonuclease. This procedure yielded over 40 discrete duplex fragments that could be resolved with considerable precision by agarose gel electrophoresis. The positions of most of these fragments were determined by analyzing Eco RI fragments of T5 st (+) and T5 st (0) DNA. Over 20 sites where single-chain interruptions can occur in T5 DNA were identified, and the distribution of interruptions within the terminal repetition was shown to be identical at both ends of the molecule. A precise value for the size of the terminal repetition in T5 DNA was obtained by analyzing SI endonuclease digests of ligase-repaired, circular T5 DNA in agarose gels. The repeated segment represented 8.3% of the T5 st (+) DNA. The results of this study also provide information concerning the properties of λ exonuclease. Hydrolysis by this enzyme was not terminated when single-chain interruptions were encountered either in the strand being degraded or in the complementary strand.