Replication of repeated DNA in human cells

Abstract

The replication pattern of the repeated sequence families of human DNA has been studied by means of DNA reassociation curves. Early- and late-replicating DNA fractions were obtained from synchronized cultures of KB cells by labeling cells with bromodeoxyuridine (BUdR) early or late in the DNA synthesis period and isolating the BUdR-containing DNA by CsCl density-gradient centrifugation. Highly repeated and moderately repeated sequence classes labeled with 14 C-deoxycytidine either early or late in the DNA synthesis period were also prepared. The effect of the isolated early- or late-replicating BUdR-DNA on the rate of reassociation of the 14 C-labeled repeated sequences was then tested. Increasing concentrations of early- or late-replicating BUdR-DNA were added to a constant amount of either 14 C-labeled early- or late-replicating repeated sequences, and the fraction of label in double-stranded DNA was determined. Analysis of the DNA reassociation curves so obtained indicates that some repeated sequence families are replicated throughout the DNA synthesis period whereas others are replicated primarily in the second half. This is true for both the highly-repeated and moderately-repeated sequence classes.