Deoxyribonucleic acid chain growth and organization of replicating units in HeLa cells

Abstract

A method for studying DNA chain growth and chromosomal organization of replicons in HeLa cells was developed. DNA replication was initiated with bromodeoxyuridine followed by pulse labeling of active replicons with [3H]thymidine and growth of the chains for finite intervals in unlabeled thymidine. Photolysis of the bromodeoxyuridine-DNA leader with 313-nm light releases the newly replicated chains that were then analyzed by sedimentation in alkaline sucrose gradients. This method provided data on the rate of chain growth, the bidirectionality of replication and the distribution of the active replicons at specific intervals in the S period. Applying this method to cells caused synthesis of DNA at a lowered temperature (27.degree. C) or with protein synthesis restricted by cycloheximide revealed that the immediate reduction in the rate of DNA replication in both instances was due to a decreased rate of chain growth without derangement of the overall process.