Integrity of parental DNA during replication

Abstract

The integrity of parental DNA as assessed by thymine, phosphate, or phosphodiester bond oxygen replacement was studied during replication of Escherichia coli 15T −. Cells containing density labeled ( 13C, 15N) parental DNA were allowed to replicate for one generation in media ( 12C, 14N) containing [ 3H]thymine and 32PO 4, or [ 3H]thymine and H 2 18O, or 32PO 4 and H 2 18O. After DNA isolation, the single-stranded parental strands were purified by equilibrium centrifugation on sequential sodium iodide density gradients. Measurements were made of 3H and 32P in the DNA and of 18O in phosphate derived from the DNA phosphodiester linkages. The amount of 3H and 32P detected in the parental DNA was used to set upper limits on thymine (1 per 10,000 to 20,000 thymine residues) and phosphate (1 per 7000 bases) replacement in each parental DNA strand. The phosphate from parental DNA contained more 18O following replication in 18O-enriched H 2O than before the exposure. This increased 18O content is consistent with frequent (1 per 100 to 500 bases) hydrolytic cleavage of the phosphodiester backbone of each parental DNA strand per round of replication. Such nicks could act as multiple swivels and facilitate unwinding of parental DNA strands during replication and transcription.