Unstable high molecular weight inverted repetitive DNA in human lymphocytes.

Abstract

About 1% of newly synthesized DNA from PHA-stimulated human lymphocytes can be isolated as large (up to 90 kilobase pairs) double stranded fragments that resist sequential alkali and heat denaturation steps but are not closed circular. By electron microscopy about 1% have single-strand hairpin loops at one end and therefore present inverted repetitive sequences (IR-DNA). Most of the remainder have a blunt-appearing double-strand terminus at both ends (78%) or one end (18%). Indirect evidence indicates that these also are inverted complementary structures with terminal hairpin loops too small to be visualized: (1) Treatment with either a 5' or 3' single-strand exonuclease generates essentially only fragments with a single strand at one end; (2) with partial denaturation, the number of fragments with identifiable single-strand hairpin loops increases (to about 20%); (3) after S1 nuclease digestion, greater than 95% can be fully heat denatured. Cot analysis indicates that these fragments are derived from dispersed sites throughout the genome. Up to 25% of DNA released from lymphocytes during growth similarly resists denaturation, and released DNA and IR-DNA are both enriched in the same set of repetitive sequences. Thus at least a portion of IR-DNA appears to be unstable.