Multiple Initiation of Bacteriophage T4 DNA Replication: Delaying Effect of Bromodeoxyuridine

Karin Carlson
1973 Journal of Virology  
Effects of bromodeoxyuridine (BUdR) substitutions in phage T4 DNA on the initial stages of DNA replication were investigated. Electron microscope studies of partially replicated, light (thymidine-containing) T4 DNA revealed the presence of multiple loops and forks. These DNA preparations had no BUdR in either parental or newly synthesized DNA, and the observations thus show that multiple initiation of DNA replication is a normal event in T4 development and is not caused by the presence of BUdR.
more » ... A comparison of early replicative stages of light and heavy (BUdR-containing) DNA in cells mixedly infected with light and heavy T4 phage showed that early DNA synthesis occurs preferentially on the light template. Heavy and light parental DNA became associated with the protein complex of replicative DNA with equal efficiency, and there was no effect of BUdR on the net rate of DNA synthesis after infection. Newly synthesized DNA from heavy templates sedimented more slowly through alkaline sucrose gradients than did newly synthesized DNA from light templates and appeared to represent fewer replicative regions per molecule. These data indicate that BUdR substitutions in the DNA caused a slight delay in initiation but that replication of heavy DNA proceeded normally once initiated. Several lines of evidence indicate that the initiation of bacteriophage T4 DNA replication takes place at multiple sites along a molecule. Utilizing bromodeoxyuridine (BUdR) to separate replicative and nonreplicative DNA moieties, Kozinski and Kozinski (11) observed that partially replicated DNA molecules (PRM), upon shearing to a size smaller than the total length of replicated DNA, did not yield fragments of either parental or hybrid density. (Hybrid DNA is defined as DNA which has completed the first round of replication. In a density-labeled system, hybrid DNA consists of one thymidine [TdR]-containing [light] strand and one BUdR-containing [heavy] strand, and bands between fully light and fully heavy DNA at equal distance from either in a density gradient.) Later investigations by Delius et al. (2) and by Howe et al. (7) gave similar results whether BUdR was present in the parental DNA or in the newly synthesized progeny DNA, I Present address:
doi:10.1128/jvi.12.2.349-359.1973 fatcat:ebqmcnwghja6nl43zwlhvfhziu