Unusual base-pairing of newly synthesized DNA in HeLa cells

Abstract

A fraction of DNA in which both newly replicated strands are base-paired to each other has been detected in DNA extracted from HeLa cells synchronized in S phase. This fraction was observed by neutral CsCl gradient analysis of sheared DNA labeled with BrdUrd and [ 3H]dC. A significant proportion of the label was found in DNA substituted in both strands with BrdUrd. The formation of this HH DNA † † Abbreviations used: HH DNA, DNA substituted in both strands with BrdUrd; HL DNA, DNA substituted in one strand with BrdUrd; LL DNA, unsubstituted DNA. is not the result of synchronization artifacts. HH DNA is found in highest proportion in early S phase, but small amounts can be detected throughout S phase. The proportion of HH DNA averages 48% of the extracted radioactive DNA when cells are briefly labeled in the presence of 1 m m-hydroxyurea, an inhibitor of DNA synthesis. Other inhibitors, like cycloheximide or arabinosyl cytosine, do not enhance the proportion of HH DNA. Pulse-chase experiments show that HH DNA is quantitatively chased into singly substituted DNA. HH DNA has a size of between 5500 and 9000 base-pairs and does not contain a high proportion of either palindromic or reiterated sequences. In vivo crosslinking experiments with trimethylpsoralen indicate that HH DNA is formed by branch migration during DNA isolation. This branch migration does not occur at replication forks and is not due to the presence of BrdUrd in newly synthesized DNA. Furthermore, HH DNA is not formed as a result of extrachromosomal replication or strand displacement from the replication bubble. We suggest that HH DNA is formed from DNA involved in the initiation of synthesis at the individual replicon level and suggest a possible model for its formation.