Size distribution of short-chain replication intermediates in HeLa cells

When HeLa cells are pulse-labeled with [ 3H]thymidine under steady-state conditions (HAT or MEMT medium, see Materials and Methods, section (a)), essentially all incorporated label is found in very short DNA chains. In contrast, HeLa cells that are grown in the absence of thymidine (Eagle's Minimal Essential Medium) prior to labeling incorporate [ 3H]thymidine into both short and long DNA chains. This change in the pattern of thymidine incorporation may be the result of a change in the intracellular nucleotide pools ( Kuebbing & Werner, 1975; Werner, 1971 b). Analysis of the size distribution of short DNA chains in HeLa cells by gel filtration on Sepharose 2B reveals that the fraction of short DNA chains greater than a particular size decreases exponentially with increasing size. The distribution curve is biphasic with a change in the slope at chain lengths of about 500 nucleotides. The similarity of these results to those obtained with bacteria ( Diaz et al., 1975 ) suggests a replication mechanism in which larger DNA chains arise through the joining of many extremely short chains with most of the actual polymerization of nucleotides occurring at the level of the shortest chains.