DNA replication intermediates in whole HeLa cells and isolated nuclei

Abstract

Replicative intermediates have been studied in intact HeLa cells and in nuclei isolated from such cells. In whole cells the smallest DNA (primary DNA pieces) observed after pulse labelling with [ 3H]thymidine were 90–160 nucleotides long, and the size of the molecules in this class of DNA did not increase with increasing pulse length. Some increase in size was, however, observed when cells were pulse labelled at 25°C instead of 37°C. Chase experiments using nuclei from pulse-labelled cells suggested that the primary DNA pieces could be chased rapidly into DNA of high molecular weight (30–70 S, corresponding to a molecular weight of 0.7 · 10 7–6.4 · 10 7). Longer chases showed that the label eventually accumulated in DNA with s values > 150 S. In isolated nuclei the primary DNA pieces after a 1 min pulse at 37°C were approximately 200 nucleotides long. Primary pieces of this size were also rapidly chased into the 30–70 S region. However, during longer pulses in vitro a fraction of the primary DNA pieces grew beyond their normal size to reach a size of up to 2000–3000 nucleotides before being attached to the 30–70 S molecules.