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Decreased initiation of DNA synthesis in a temperature-sensitive mutant of hamster cells

✍ Scribed by Eric Eilen; Dr. Roger Hand; Claudio Basilico

Publisher
John Wiley and Sons
Year
1980
Tongue
English
Weight
654 KB
Volume
105
Category
Article
ISSN
0021-9541

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✦ Synopsis

Abstract

We have analyzed ongoing DNA replication in ts BN‐2, a dna^−^ mutant of BHK‐21 cells (Nishimoto et al. '78). At the non‐permissive temperature of 39.5°C, inhibition of ^3^H‐thymidine into acid‐precipitable material begins 1 to 2 h after the cells are released from a block at the start of the S‐phase. The fraction of nuclei incorporating ^3^H‐thymidine is similar to that of wild‐type cells through the synchronized S‐phase of 8 h. Alkaline sucrose gradient analysis shows tht pulse‐labeled DNA from mutant cells is incorporated into high molecular weight material after 3 h at eithr the permissive or non‐permissive temperature. DNA fiber autoradiograms reveal that, at 39.5°C, the rate of replication fork movement is about 30% increased in the mutant as compared to wild‐type cells. In the mutant cells, however, the interval between adjacent initiation sites is increased and the relative frequency of initiation events is decreased at the restrictive temperature. The results indicate that there is a block to ongoing replication in ts BN‐2 at the level of initiation of synthesis on individual replication units; elongation of nascent chains is not inhibited.

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