The cytogenetic observation that homologous chromatid interchange occurs in Bloom's syndrome more often than normal prompted an investigation of DNA replication in that rare genetic disorder. Using DNA fiber autoradiography, an estimation was made of the rate of one component of ongoing DNA replication, DNA chain growth. The rate in Bloom's syndrome dermal fibroblasts in tissue culture was found to be significantly slower than that in normal control cells. (The rate was found to be normal in Fanconi's anemia cells.) The explanation for the retarded chain growth may be either that an enzyme concerned directly with semiconservative DNA replication is defective or that a defective enzyme not itself concerned directly with replication results in disturbed cellular metabolism, which in turn affects replication.
The regulation of DNA replication at a subchromosomal level in mammalian cells has been investigated. DNA fiber autoradiographs were prepared from mouse L-929 cells pulse labeled with [SH]thymidine. Initiation events and subsequent chain growth occurring over short stretches (up to three replication units in length) of chromosomal DNA were analyzed. The results show that adjacent units usually initiate replication synchronously and that this synchrony is related to the proximity of initiation sites. In addition, adjacent units are of similar size and the rates of replication fork progression within units and on adjacent units are similar. The rate of fork progression increases with increasing replication unit size. Finally, no evidence for fixed termination sites for the units has been found. These observations suggest that despite large variations in size of replication units, timing of initiation events, and rates of fork progression found in chromosomal DNA as a whole, these processes are closely regulated within subchromosomal clusters of active replication units.
Using pulse labeling techniques with [3H]thymidine or [3H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [3H]thymidine of high specific activity, followed by [3H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.
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