Estimation of DNA fork displacement rates from isolated mammalian chromosomes

Chromosomes were isolated from Chinese hamster ovary cells that had been pulse-labeled with 3H-thymidine and bromodeoxyuridine and allowed to proceed to metaphase in the presence of colcemid. The chromosomes were then irradiated with 313 nm light and the length of the pulse-labeled DNA segments was estimated by the distribution of 3H-DNA in alkaline sucrose gradients. The average rate of fork displacement, calculated from this measurement, was 0.9 micrometer/min, which was in excellent agreement with measurements from interphase cells. By the same method, hydroxyurea was shown to retard DNA chain growth by 50--60% in both isolated chromosomes and interphase cells.