| Abstract: | The ability of EAT cells to initiate DNA synthesis in the presence of high doses of hydroxyurea was examined using the recently developed method for crosslinking DNA in vivo. Since crosslinking blocks elongation but has little effect on initiation (Russev and Vassilev (1982) J. Mol. Biol. 161, 77-87), this approach permits a separate study of the two stages of the DNA replication. We found out that hydroxyurea did not greatly affect the initiation of DNA replication but strongly inhibited the elongation of the already initiated new DNA chains. This resulted in the formation of short fragments enriched in sequences synthesized at and around the sites where DNA initiation began. These fragments were not ligated to the high molecular weight chromosomal DNA and could be released under denaturing conditions in single-stranded form. The reassociation and electrophoretic analysis showed that they contained about 200 nucleotides long interspersed DNA sequences repeated approx. 10(4) times per haploid genome, that probably served as replication origins. |
